Gene Therapy for Immunoglobulin E, Complement-Mediated, and Eosinophilic Disorders

Abstract

Immunoglobulin E, complement, and eosinophils play an important role in host defense, but dysfunction of each of these components can lead to a variety of human disorders. In this review, we summarize how investigators have adapted gene therapy and antisense technology to modulate immunoglobulin E, complement, and/or eosinophil levels to treat these disorders.

INTRODUCTION

Immunoglobulin E (IgE), the complement system, and eosinophils are components of immune/inflammatory host defense that function along with T cells, B cells, natural killer, and macrophages to protect against environmental insults.¹ IgE represents a class of antibodies important in host defense against parasites,^2–4 components of the complement system function to enhance the ability of antibodies and phagocytes to clear microbes and damaged cells,^5–7 and eosinophils are a form of white blood cells that function in host defense against parasites and other infectious agents.⁸ While IgE, complement, and eosinophils play important roles in host defense, each of these immune/inflammatory components, when dysfunctional, excessive, or deficient, can contribute to the pathogenesis of human disease.¹

Elevated levels of IgE directed against an environmental antigen play a central role in the pathogenesis of allergic asthma, conjunctivitis, and allergic rhinitis.^9–11 Mutations in components of the complement system, either in excess or deficiency, are associated with a variety of disorders,^12–14 and excess levels of eosinophils play a role in the pathogenesis of allergic disorders as well as disorders where excess eosinophils cause organ injury.^15–17 Despite a variety of therapies focused on treating the allergic, complement, and eosinophilic disorders, many of these therapies are aimed at symptoms only, and because most of these disorders are chronic, therapy requires repetitive administration.

In the context of an unmet medical need for novel therapies for these disorders, gene therapists have begun to assess whether gene therapy technologies may be useful to treat IgE-, complement-, and eosinophilic-related disorders. This review is focused on summarizing the state of the art of gene therapy for these disorders.

GENE THERAPY FOR FOOD ALLERGIES

Food allergies are defined as an adverse health effect arising from a specific immune response that occurs, reproducibly, on exposure to a given food.¹⁸ The immune response to food may be IgE-mediated (immediate reactions), non-IgE-mediated (delayed reactions), or mixed.¹⁸ IgE-mediated food allergies are common, with increasing prevalence in the past decades.^19–22 The central event in the pathogenesis of food allergy is the increased sustained production of antigen/food-specific IgE antibodies, which are bound by high-affinity Fcɛ receptors on mast cells and basophils.^23–25 On cross-linking with the allergen, this triggers the release of histamine and other proinflammatory mediators, which lead to the classic symptoms of type I hypersensitivities, including systemic reactions such as anaphylaxis,^23–25 characterized by life-threatening respiratory and/or circulatory failure associated with skin and mucosal changes.²⁶ Approximately 32 million people in the United States have food allergies: 5.6 million children (nearly 8%) and >26 million (11%) adults.²² Greater than 170 foods have been reported to cause allergic reactions with the leading foods including shellfish, milk, peanut, tree nuts, egg, fin fish, wheat, soy, and sesame.^22,27,28

While some food allergies (milk, egg, wheat, and soy) typically have a high rate of resolution in childhood and adolescence, others, such as peanut, tree nut, fish, and shellfish allergies, tend to be lifelong or rarely resolved.²⁹ Food allergies pose a significant burden on affected individuals, resulting in dietary and social restrictions, fear of accidental reactions, high levels of anxiety related to risk of severe reactions, fatalities, and, as a consequence, reduced quality of life.³⁰ Diet adherence and self-management of anaphylactic reactions have low compliance, especially in the adolescent population.³¹

Although strides have been made in food allergy awareness, there is no satisfactory therapy, which places a significant economic burden on the health care system.³⁰ Several allergen-specific and nonspecific therapies, aiming to acquire persistent food tolerance, are under investigation as potential treatments. However, to date, immunotherapy is the most promising therapeutic approach for food allergy treatment.^29,32–34 With the absence of a definitive cure, current effective management of an IgE-mediated food allergy is based on patient and family education, strict allergen avoidance, and prompt recognition and treatment of allergic reactions.^18,29

Two gene therapy approaches for the management of food allergies have emerged, which aim to either prevent or treat the development of food allergy and to restore or induce immune tolerance against food allergens (Table 1).^35,36

Table 1.

Preclinical gene therapy for food allergy

Vector	Dose (Route)	Experimental Model	Results	References
Plasmid
pCMVArah2 chitosan nanoparticle	50 μg (oral)	Mice (AKR/J)	Increased serum IgG2a, fecal IgA Decreased/delayed anaphylaxis Decreased plasma histamine and vascular permeability	35
AAV
AAVrh.10anti-hIgE	1 × 1011 gc (intravenous)	Mice NSG	Reduction in total, peanut specific and free serum IgE Reduction in histamine, and anaphylaxis score Abrogation of passive cutaneous anaphylaxis and clinical anaphylaxis, reduced mortality (secondary to anaphylaxis)	36

AAV, adeno-associated virus; AAVrh.10anti-hIgE, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE monoclonal antibody, omalizumab (xolair); Arah2, dominant peanut allergen; gc, genome copies; IgE, immunoglobulin E; NSG, NOD-scid IL-2Rγ^null.

Roy et al.³⁵ addressed whether mucosal immunoregulatory mechanisms can be exploited by DNA vaccination to modulate food-specific IgE hypersensitivity reactions. These investigators used a murine model of peanut allergy to assess the efficacy of oral immunization with chitosan-DNA nanoparticles carrying the gene for the molecular determinant of peanut allergy. Peanut-sensitive mice were fed either chitosan-DNA nanoparticles containing the LacZ gene (bacterial β-galactosidase) or “naked” plasmid DNA-LacZ to assess the expression and distribution of transduced genes. Induction of mucosal immune responses was confirmed in immunized mice with increased levels of secretory IgA against Arah2 (a major peanut allergen) in fecal extracts. Elevated serum anti-Arah2 IgG2a was also found in the nanoparticle-treated mice compared with controls, suggesting a Th1 response. Compared with nonimmunized mice or “naked” DNA-treated mice, mice immunized with nanoparticles had a substantial reduction in anaphylaxis associated with a reduction in IgE levels, plasma histamine, and vascular leakage.

Pagovich et al.³⁶ used a different approach for anti-hIgE gene therapy to treat peanut allergy. These investigators hypothesized that a one-time administration of an AAV gene transfer vector expressing the genetic sequence of omalizumab (anti-IgE) would provide persistent high circulating levels of anti-IgE sufficient to protect against repeated peanut exposure in the peanut allergic host. A humanized model of peanut allergy that reproduces a robust allergic response that mimics the human allergic cascade was developed using NOD-scid IL-2Rγ^null (NSG) mice reconstituted with blood mononuclear cells from peanut allergic individuals with a clinical history of anaphylaxis and laboratory data that supported the diagnosis (elevated total IgE and elevated peanut-specific IgE). These mice had a reconstituted functional immune system that generated a robust molecular (human IgG, IgE, and peanut-specific IgE) and clinical (elevated histamine levels and anaphylaxis score; puffiness around the eyes and mouth, decreased activity, pilar erecti, and labored breathing) phenotype when sensitized and challenged with crude peanut extract.

A single intravenous administration of an AAV serotype rh.10 coding for anti-human IgE (AAVrh.10anti-hIgE) protected from the peanut-induced anaphylaxis through continuous expression of anti-IgE. The therapy was efficacious both before and after peanut sensitization. Translation of this therapy to the clinic has challenges since adverse side effects have been reported with anti-IgE therapy, including anaphylaxis, cardiovascular and cerebrovascular events, and malignancy, and there are theoretical risks associated with absent/low IgE levels.^37–44 With this caveat, the efficacy of AAVrh.10 anti-human IgE treatment is potentially applicable to other IgE-mediated allergic disorders, ushering in a new strategy for allergy therapeutics (Table 2).

Table 2.

Preclinical gene therapy for asthma

Vector	Dose (Route)	Experimental Model	Results	References
ILs
Retrovirus
RV-CMV-IL-4RA	1 × 103 CFU (intranasal)	Mice (Balb/c)	Inhibited AHR Inhibited allergic inflammation Reduced TH2 cytokines (BALF)	83
Adeno-associated virus
AAV2CMV-rASIL-5	5 × 1012 vg (intravenous)	Rats (Sprague-Dawley)	Decreased IgE (serum) Reduced inflammatory responses	73
AAV2CMV-rASIL-5	5 × 1012 vg (intravenous)	Rats (Sprague-Dawley)	Decreased eosinophils, IL-5 (BALF), decreased TGF-β1 and TGF-β2 (peribronchial space) Inhibited increase in bronchial wall area and airway smooth muscle area	74
AAV2CMV-rASIL-4	5 × 1012 vg (intravenous)	Rats (Sprague-Dawley)	Decreased IL-4 and eosinophils (BALF) Attenuated remodeling—reduced collagen deposition and total bronchial wall and airway smooth muscle area	72
AAV2CMV-mIL-4RA	5 × 1011 vg (intratracheal) 1012 (intramuscular)	Mice (Balb/cByJ)	Reduced AHR, airway eosinophils, and mucous production Reduced TH2 cytokines (BALF)	81
Adenovirus
AdPGK-mIL-12 plus AdPGK-mIL-10	1 × 108 PFU (intratracheal)	Mice (Balb/c)	Inhibited AHR Reduced pulmonary eosinophils and neutrophils Reduced TH2 cytokines and chemokines (BALF)	77
Oligonucleotides
IL-4Rα ASO	10–500 μg/kg (inhalation)	Mice (Balb/c)	Reduced TH2 cytokines and chemokines (BALF) Reduced AHR and allergic induced lung inflammation	82
Plasmids
pCMV-mIL-10/ pCMV-mIL-12/ pCMV-mTGF-β plus liposome	10 μg (intratracheal)	Mice (Balb/c)	Reduced antigen-induced airway inflammation Reduced eosinophils and neutrophils (BALF)	75
pCMV-mIL-12	100 μg (intramuscular)	Mice (Balb/c)	Reduced airway hyperreactivity Reduced expression of CD44 and CD49d on pulmonary leukocytes Reduced TH2 cytokines (BALF)	76
pCMV-mIL-4RA plus jetPEI (transfection reagent)	10 μg (intratracheal)	Mice (Balb/c)	Protected from airway inflammation Reduction in eosinophils, increased IL-13 levels, and decreased IFN-γ (BALF) Decreased IL-4 and IL-13 (serum)	179
Lentivirus
LV-SEC4	3 × 105–3 × 106 IFU (intratracheal)	Mice (Balb/c)	Protected from AHR Reduced eotaxin (BALF) Reduced IL-5 mRNA (lungs)	71
Cytokines
pCMV-mIFN-γ plus liposome	10 μg (aspiration)	Mice (AKR/J)	Reduced AHR Increased expression IFN-γ (BALF)	67
pCMV-mIFN-γ plus liposome	10 μg (intravenous/intratracheal)	Mice (Balb/c)	Inhibited AHR (IV/IT) Inhibited airway eosinophilia (IV/IT) and serum IgE (IV)	68
pCMV-mIFN-γ-chitosan nanoparticle	10 μg (intranasal)	Mice (Balb/c)	Increased CD40 and decreased CD80 levels on DCs; reduced allergic immune response	69
Transcription factors
GATA-3 ASO	200 μg/treatment (intranasal)	Mice (Balb/c)	Reduced airway hyperresponsiveness Suppressed TH2 cytokines in BALF and lung interstitium	91
Oligonucleotide
GATA-3 DNAzyme (Gd21)	200 μg (intranasal)	Mice (Balb/c)	Reduced AHR	93
LV-siRNA GATA-3	2.2 × 106 IFU (intratracheal)	Mice (Balb/c)	Suppressed AHR Inhibited Th2 cytokines (BALF; IL-4, IL-5)	92
STAT6 siRNA	100 μg (intransal)	Mice (Balb/c)	Blocked allergic airway inflammation and AHR Diminished IL-4 and IL-13 levels in lung tissue	132
AAV-T-bet	1 × 1010 vg (intranasal)	Mice (Balb/c)-OVA sensitized	Inhibited airway inflammation TH1/TH2 transcription factor and cytokine profile balance Decreased eosinophils (BALF) Decreased IgE (serum) Reduced peribronchial inflammation score	87
Oligonucleotide
NF-κB p65 ASO	20 μg/treatment (intravenous)	Mice (C57BL/6)-OVA sensitized	Reduced AHR Blocked Th2 cytokines (BALF) Reduced eosinophils (BALF) Reduced serum IgE, IgG	108
Binding proteins
Plasmid
pCMV-Gal-3	50 μg (intranasal)	Mice (A/J)	Reduced airway remodeling Improvement in AHR Downregulation of IL-5	88
Adeno-associated virus
rAAV2CMV-shRNA	1 × 1011 gc (intratracheal)	Mice (Balb/cByJNarl)-OVA sensitized	Improvement in AHR Decreased eosinophil infiltration, eotaxin, IL-13 (BALF) Decreased OVA-spec IgE (serum)	109
Plasmid
Signal transduction
pDGKα	100 μg (intramuscular)	Mice (Balb/c)-OVA sensitized	Decreased allergic airway inflammation Decreased infiltration eosinophils (BALF) Decreased IgE (serum)	180
Oligonucleotide
Syk ASO+liposome	500 μg (aerosolized)	Rats (Brown Norway)-OVA sensitized	Suppressed AHR Blocked upregulation of β2 integrins, α4 integrins, and ICAM-1, inhibited TNF, decreased eosinophils (BALF)	107
Co-stimulatory proteins
Oligonucleotide
CD86 ASO	100 μg/kg (aerosol)	Mice (Balb/c)-OVA sensitized	Suppressed AHR eosinophilia and mucus production	181
Oligonucleotide
pPol III-CD40 siRNA	60 μg/treatment (intravenous)	Mice (Balb/c)-OVA sensitized	Inhibited DC and B cell function and generation of regulatory T cells	135
Oligonucleotide
FOXP3 modified mRNA	20 μg (intratracheal)	Mice (Balb/c)-OVA sensitized	Decreased AHR and lung inflammation, BALF Decreased eosinophils (BALF)	182

Ad, adenovirus; ASO, antisense oligonucleotides; BALF, bronchoalveolar lavage fluid; CFU, colony-forming unit; CMV, cytomegalovirus promoter; DC, dendritic cell; pDGKα, phosphorylation of diacylglycerol kinase alpha; Gal-3, galectin-3; IFN-γ, interferon gamma; IL, interleukin; IL-4RA, IL-4 receptor antagonist; LV, lentivirus; mRNA, messenger RNA; OVA, ovalbumin; pPol III-CD40, Pol III promoter-mediated CD40; rASIL-4, antisense rat IL-4 cDNA; rASIL-5, antisense rat IL-5 cDNA; RV, retrovirus; SEC4, siRNA expression cassette targeting 5′UTR region of IL-5 mRNA; shRNA, short hairpin RNA; siRNA, small interfering RNA; STAT6, signal transducer and activator of transcription 6; Syk, spleen tyrosine kinase; T-bet, T-box expressed in T cells; TGF-β, transforming growth factor beta; TNF, tumor necrosis factor; vg, viral genomes.

ASTHMA

Asthma is characterized by symptoms of episodic or chronic cough, wheezing, chest tightness and production of sputum with chronic inflammation of the airways, airway hyperresponsiveness, and variable airflow obstruction.^1,45,46 Although these symptoms are reversible, chronic disease can lead to airway remodeling, including airway wall fibrosis, smooth muscle and mucous-cell hyperplasia, and decline in lung function.^47,48

The development of allergic asthma is determined by genetic predisposition and exposure to allergens, infections, and pollution.^49–51 Extensive studies of the pathophysiology of chronic airway inflammation and reversible increases in airway resistance have uncovered several maladaptive immunologic pathways that might be countered by gene therapy.^52,53

The immediate asthmatic response is mediated by IgE-dependent mast cell release of mediators, such as histamine, leukotrienes, cytokines, and prostaglandins.^25,54,55 The late reaction is a manifestation of eosinophilic airway inflammation associated with the development of airway hyperresponsiveness.⁵⁶ Repeated allergen exposure and inflammatory responses can cause epithelial damage exposing deeper airway structures, and inflammatory and structural cell production of growth factors and pro-fibrogenic cytokines lead to angiogenesis, smooth muscle proliferation, and basement membrane thickening.⁴⁷

Over the past several decades, there have been insights in understanding the immunological mechanisms initiating and mediating allergic airway responses. Despite this progress, mainstream therapy still consists of disease control with anti-inflammatory glucocorticoid-based drugs, bronchodilators, antihistamines, and monoclonal antibody-based biologics that target interleukin (IL)-5, IgE, or anti IL-4Rα (Table 3).^57–65 Although these therapies are often effective at relieving symptoms, they do not cure the disease, require repeated dosing, and depend on patient compliance and coordination.⁵³ In the United States, asthma accounts for more than 4,000 deaths per year (https://wonder.cdc.gov/ucd-icd10.html), and ∼25–30% of asthmatics require long-term therapy with inhaled corticosteroids (https://wonder.cdc.gov/ucd-icd10.html, https://www.cdc.gov/asthma/asthmadata.htm). Thus, development of a safe long-lasting therapeutic agent remains an important goal for effective therapy.

Table 3.

Approved biologics for allergic disorders

Name of Biologic	Trade Name	Dose (Route)	Target	Indication	References
Dupilumab	Dupixent	Weight based: initial loading dose (400/600 mg), followed by 200/300 mg SC Q2wk	IL-4α receptor	Moderate-to-severe eosinophilic or oral steroid-dependent asthma Atopic dermatitis Chronic rhinosinusitis with nasal polyposis Eosinophilic esophagitis Prurigo nodularis	35
Omalizumab	Xolair	Asthma:75–375 mg SC Q2/4wka CRSwNP:75–600 mg SC Q2/4wka CSU: 150 or 300 mg SC Q4wk	IgE (Cɛ3 domain)	Moderate-to-severe persistent allergic asthma CRSwNP CSU allergic asthma
Mepolizumab	Nucala	Asthma: 40–100 mg SC Q4wk CRSwNP: 100 mg SC Q4wk EGPA: 300 mg Q4wk HES: 300 Q4wk	IL-5	Severe eosinophilic asthma CRSwNP EGPA Hypereosinophilic syndrome	36
Benralizumab	Fasenra	30 mg SC Q4wk for the first three doses, followed by Q8wk thereafter	IL-5α receptor	Severe eosinophilic asthma

^a Dose and dosing frequency determined by serum total IgE level (IU/mL).

CRSwNP, chronic rhinosinusitis with nasal polyps; CSU, chronic spontaneous urticaria; EGPA, eosinophilic granulomatosis with polyangiitis; Q, every; SC, subcutaneous; wk, week.

Asthma is primarily mediated by the overproduction of Th2-type cytokines.^25,54,55,65 In contrast, Th1 cytokines, such as interferon gamma (IFN-γ), IL-12, and IL-18, downregulate the Th2 immune responses and dampen the allergic disease process.⁶⁶ Numerous studies of gene transfer to the lung have helped elucidate the mechanisms responsible for asthma and have raised the possibility of effective gene therapy that could overcome the limitations of current therapies.^52,53 Murine models of allergic asthma represent the majority of gene transfer studies involving modulation of airway immunobiology (Table 4).

Table 4.

Preclinical gene therapy for allergic rhinitis

Vector	Dose (Route)	Experimental Model	Results	References
Adenovirus
AdexICAICOSIg	1.5 × 108 PFU (intranasal)	Mice (Balb/c)	Reduced eosinophils and IL-5 levels (nasal mucosa) Reduced OVA-specific IgE (serum)	183
Plasmid
pLVX-ShRNA2-mCCR3	8 μL (intranasal)	Mice (Balb/c)	Reduced nose scratching and sneezing	134
pGAGmIL-12-lipoplex	4 μg/dose (intranasal)	Mice (Balb/c)	Reduced IgE levels (serum) Inhibited eosinophils in nasal mucosa	137
pGEGsTNFR-IgGFc-liposome	5 μg (intranasal)	Mice (Balb/c)	Reduced eosinophils (nasal mucosa)	136
STAT6 siRNA-lipofectamine	1.5 nmol/day (intranasal)	Mice (Balb/c)	Reduced IL-4 and IL-5 production	184

AdexICAICOSIg, adenovirus vector expressing inducible human co-stimulatory and Fc portion of human IgG1; CCR3, CC chemokine receptor 3; PFU, plaque-forming unit; pGAGmIL-12, Epstein–Barr virus-based plasmid vector expressing murine IL-12 from a CAG promoter with p35 and p40 subunits linked by IRES; pGEGsTNFR-IgGFc, Epstein–Barr virus-based plasmid vector expressing sTNFR from a CAG promoter and IgG Fc from an IRES; pLVX-ShRNA2-mCCR3, lentivirus-based vector expressing CCR3 from U6 promoter; sTNFR, soluble murine tumor necrosis factor receptor.

Given the central role of Th2 cells in allergic diseases, the downregulation of Th2 differentiation and activation is a promising approach for the treatment of allergic diseases, especially in asthma phenotypes with high levels of these mediators (e.g., IL-5, IL-13, or IL-4).

Early gene therapy studies targeted IFN-γ to shift the immune response from a Th2 toward a Th1 pathway.^67,68 Ovalbumin (OVA)-sensitized mice treated with adenovirus-mediated expression of IFN-γ (Ad-IFN-γ) exhibit significantly lower levels of Th2 cytokines IL-4 and IL-5, serum OVA-specific IgE, lung eosinophils, and airway hypersensitivity.^67–69 The administration of Ad-IFN-γ to mice with established airway hypersensitivity significantly reduced airway hypersensitivity, Th2 cytokines, and lung inflammation.⁶⁷

IL-5 is the key cytokine in eosinophil growth and differentiation in bone marrow and release into the periphery.⁷⁰ The inhibition or reduction of IL-5 with an antibody is a promising therapeutic strategy in eosinophilic-mediated disorders such as asthma. Lentivirus-delivered small interfering RNA (siRNA) targeting IL-5 efficiently modulated the asthma phenotype of a mouse model, including allergic hyperresponsiveness, cellular infiltration of lung tissues, eotaxin levels in bronchoalveolar lavage fluid, and elevated IL-5 messenger RNA (mRNA) in the lungs.⁷¹ Systemic administration of antisense IL-5 or antisense IL-4, transferred by an adeno-associated virus, reduced IL-5 or IL-4 expression, allergic inflammation, and airway remodeling in a rat model.^72–74

A number of investigators have used gene therapy to express inhibitory or immunosuppressive cytokines, IL-10, IL-12, or transforming growth factor beta (TGF-β).^75–77 Increased levels of IL-12 result in IFN-γ expression, Th1-cell differentiation, and suppression of Th2-cell function. Lee et al.⁷⁸ constructed a single-chain polypeptide consisting of both subunits of IL-12 separated by a proteolytically resistant linker. Liposome-mediated transfer of this plasmid encoding IL-12 to the airway epithelium of mice with allergic airway inflammation led to reduced IL-5, increased IFN-γ, decreased eosinophil infiltration in the lung, and significant reductions in allergen-induced airway hyperresponsiveness. Hogan et al.⁷⁹ used a recombinant vaccinia virus to deliver both subunits of murine IL-12 into the lungs of OVA-sensitized mice before and after aerosol rechallenge with OVA. This suppressed Th2 cytokine production and prevented airway inflammation and airway hyperresponsiveness through an IFN-γ dependent pathway. Reduced levels of antigen-specific IgE and IgG2 were observed with airway hyperresponsiveness sustained for up to 4 weeks following gene transfer.

Walter et al.⁸⁰ administered a recombinant adenovirus expressing murine IL-18 intranasally to mice and examined its effects on OVA-induced airway hyperresponsiveness. They found increased IFN-γ and reduced IL-4, consistent with a shift toward a Th1 response, accompanied by reduced airway eosinophilia and goblet cell hyperplasia. These findings suggest that IL-18 gene transfer obviated airway hyperresponsiveness, reversing the asthma phenotype. Zavorotinskaya et al.⁸¹ reported the effects of AAV-mediated gene transfer of a competitive inhibitor of IL-4R, the common subunit of the IL-4 and IL-13 receptors. This approach attenuated IL-4- and IL-13-mediated inflammation, reduced airway mucous production, and prevented allergen-induced increases in airway hyperresponsiveness. Other investigators targeted IL-4Rα using Moloney murine leukemia retrovirus, plasmid, or naked anti-sense oligonucleotides to the IL-4 receptor in asthmatic mice, with reduction in hallmarks of asthma, including airway hyperresponsiveness, eosinophil infiltration, and Th2 cytokine production.^81–83

Another approach is to target IL-10, which is both immunosuppressive and anti-inflammatory. IL-10 and TGF-β inhibit the expression of numerous proinflammatory cytokines and chemokines, the main inhibitory cytokines produced by Treg cells in allergen immunotherapy.^75,76 Intratracheal administration of adenovirus-expressing IL-10 inhibited airway hypersensitivity and decrease the recruitment of eosinophils and neutrophils in the lungs of OVA-sensitized and challenged mice.⁷⁷ Hansen et al.⁸⁴ used T helper cells genetically modified with a retroviral vector to secrete latent TGF-β, demonstrating that TGF-β secreting T helper cells reduce airway hypersensitivity and airway inflammation in OVA-induced mice. These findings highlight the important roles that IL-10 and TGF-β play in regulating allergic inflammation.

IL-1 is a proinflammatory cytokine required for allergen-specific Th2 cell activation and the development of airway hyperresponsiveness.⁸⁵ IL-1 receptor antagonist (IL-1ra) is a natural inhibitor that binds to IL-1 receptor type I without inducing signal transduction.⁸⁶ Intranasal administration of an adenovirus vector coding for IL-1ra (Ad-IL-1ra) to OVA-sensitized mice showed significantly decreased airway hyperresponsiveness severity and reduced pulmonary infiltration of eosinophils and neutrophils.⁸⁶ In addition, there was suppression of IL-5 and eotaxin with concomitant enhancement of IFN-γ in bronchoalveolar lavage fluid and decreased peribronchial inflammation.

An alternative gene therapy strategy to reduce allergic immune responses is targeting transcription factors that elicit Th2-responses. AAV vector-mediated overexpression of the transcription factor T-bet, the regulator of Th1 versus Th2 lineage commitment, has been studied in a mouse OVA-induced asthma model.⁸⁷ AAV-mediated T-bet overexpression resulted in reduced levels of IL-4 and IL-5 and increased IFN-γ levels in lavage fluid. In addition, there was a reduction of eosinophils in lung lavage fluid, IgE in serum and bronchial inflammation.⁸⁷ Galectin-3 (Gal-3) is an IgE-binding protein that belongs to a family of proteins that bind β-galactosides.⁸⁸ Gal-3 has been implicated in inflammation and allergic pathologies and has been shown to downregulate IL-5 gene expression in a number of cell types including eosinophils, T cell lines, and antigen specific T cells. del Pozo et al.⁸⁹ showed that plasmid-mediated overexpression of Gal-3 after intranasal instillation decreased airway hyperresponsiveness, eosinophilia, and serum IgE levels in OVA-induced asthma.⁸⁸

A number of investigators have studied the therapeutic potential in mouse models of allergic inflammation gene therapy-mediated of blocking GATA-3, the Th2 differentiation transcription factor that regulates the expression of IL-4, IL-5, and IL-13.^90–94 These cytokines are important for isotype class switching of B cells to IgE synthesis (IL-4, IL-13) and recruitment of mast cells (IL-4, IL-9, and IL-13).^95,96 The number of GATA-3-positive cells is increased in the asthmatic lung and in allergic rhinitis and is further increased after allergen challenge.^91–93 Transgenic mice overexpressing GATA-3 in T cells show enhanced airway hypersensitivity, increased subepithelial fibrosis, and muscle hyperplasia after repeated allergen exposure.⁹⁷ Local intranasal administration of GATA-3 antisense oligonucleotides in a murine model resulted in reduced Th2 cytokine production, airway hypersensitivity, and lung inflammation, including eosinophil infiltration.⁹³ The silencing of the GATA-3 gene by administration with GATA-3 siRNA alleviated airway eosinophilia, Th2 cytokine production, and airway hyperresponsiveness.^91,92

An alternative therapeutic target is the use of decoy oligonucleotides. These short double-stranded synthetic oligonucleotides contain binding sites that are recognized by nuclear transcription factors. Binding to decoy oligonucleotides prevents transcription factors from efficiently binding the consensus sequences of their target genes, preventing target gene expression and reduction of the allergic response. Beneficial effects of decoy oligonucleotides blocking NF-κβ, activator protein-1, and signal transducer and activator of transcription 1 (STAT1) on experimental asthma in mice have been demonstrated.^98–100 A decoy oligonucleotide blocking both STAT1 and STAT3 transcription factors reduced allergic inflammation and pulmonary CD40 expression in a rat model of OVA-induced allergic inflammation.¹⁰¹ One challenge in the use of decoys is their short active half-life requiring taking the drugs before each exposure or trigger.

The normal resolution of inflammation in the lung occurs via the regulated removal of unneeded cells by apoptosis.¹⁰² Allergic asthma is associated with reduced apoptosis of inflammatory cells such as eosinophils and lymphocytes in the lung.^45,103 A single administration of adenoviruses-expressing fas ligand (FasL) in OVA-immunized mice significantly alleviated airway hypersensitivity and eosinophilia by inducing the apoptosis of eosinophils and reducing levels of IL-5 and eotaxin.¹⁰⁴ The delivery of dendritic cells genetically engineered to express FasL to OVA-immunized allergic mice decreased airway hyperresponsiveness.¹⁰⁵ An alternative approach by da Silva et al.¹⁰⁶ demonstrated that a thymic nonapeptide, thymulin (serum thymus factor), was capable of preventing cascades of inflammatory and fibrotic responses in a mouse model of allergic asthma.

A single dose of intratracheally administered thymulin-expressing plasmids reversed asthma pathology 20 days after therapy, including chronic inflammation, pulmonary fibrosis, and mechanical dysregulation. Other preclinical studies in murine models of asthma have shown the potential for using small-molecule inhibitors of kinases (e.g., p38 mitogen-activated protein kinase and NF-κβ), which are crucial in regulating the expression of inflammatory genes that are overexpressed in asthma^107,108 as well as blocking acidic mammalian chitinase (AMCase) in allergic airway diseases.¹⁰⁹ Blocking of AMCase and inhibition of kinases have been shown to improve allergic airway inflammation and hyperresponsiveness in murine asthma models.^{105,107,109–111}

A number of asthma gene therapy clinical trials have been carried out. A Phase IIa trial demonstrated that inhalation of a mixture of naked antisense oligonucleotides against CC chemokine receptor 3 (CCR3; the eotaxin receptor) and the common β chain of the IL-3/IL-5/granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (TPI ASM8; a drug containing two modified phosphorothioate antisense oligonucleotides: TOP004 directed against human β_c of IL-3, IL-5, and GM-CSF receptors, and TOP005 directed against human CCR3) reduced both allergen-induced airway eosinophilia and the early asthmatic response in mild atopic asthmatic patients.¹¹² Another Phase IIa study investigated the efficacy and safety of four escalating dose regimens of TPI ASM8 in patients with allergic asthma and showed a reduction of the late asthmatic response.¹¹³

Krug et al.¹¹⁴ evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme), that is able to cleave and inactivate GATA-3 mRNA naked DNAzyme in asthmatic patients by inhalation. Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma, and biomarker analysis showed an attenuation of Th2-regulated inflammatory responses.¹¹⁴ Overall, the therapy was found to be safe and well tolerated.

In summary, gene therapy for asthma has the potential to improve patient quality of life and significantly impact short- and long-term clinical outcomes in affected individuals. However, while there are many interesting asthma-related gene therapy preclinical approaches, and promising early-stage clinical trials, there is, at present, no gene therapy for asthma that is close to approval for patients.

ALLERGIC RHINITIS

Allergic rhinitis is defined as symptoms of sneezing, nasal pruritus, airflow obstruction, and clear nasal discharge.^115,116 Allergens that induce allergic rhinitis include seasonal pollens and molds, and perennial indoor allergens, such as dust mites, pets, pests, and molds.¹¹⁷ Allergic rhinitis is the most common type of chronic rhinitis, affecting up to 40% of the population, and the frequency and prevalence of sensitization to inhalant allergens are increasing.^118,119 Severe allergic rhinitis is associated with significant impairments in quality of life including missed or unproductive time at work and school, sleep disturbances, and decreased involvement in outdoor activities.^117,120 The presence of seasonal perennial allergic rhinitis significantly increases the probability of asthma: up to 40% of people with allergic rhinitis have or will have asthma^121,122 and atopic eczema frequently precedes allergic rhinitis.^123,124

The pathophysiology of allergic rhinitis induces numerous inflammatory cells, including mast cells, CD4-positive T cells, B cells, macrophages, and eosinophils.^125,126 In allergic individuals, upon exposure to an inciting allergen, predominantly Th2 T cells infiltrate the nasal mucosa and release cytokines, including IL-3, IL-4, IL-5, and IL-13 that promote IgE production by plasma cells.^125,126 Cross-linking of IgE bound to mast cells by allergens, in turn, triggers the release of mediators, such as histamine and leukotrienes, that are responsible for increased vascular permeability, pruritus, rhinorrhea, mucous secretion, and airway smooth muscle contraction.^125,126 The mediators and cytokines released during the early phase of an immune response to an inciting allergen trigger a further cellular inflammatory response over the next 4–8 h (late-phase inflammatory response), which results in recurrent symptoms, usually nasal congestion, that often persists.^125,126

The diagnosis of allergic rhinitis is made clinically on the basis of characteristic symptoms and response to empirical treatment with antihistamines or nasal glucocorticoids.^117,126,127 Formal diagnosis of allergic rhinitis is based on evidence of sensitization, measured either by the presence of allergen-specific IgE in the serum or by positive epicutaneous skin tests (i.e., wheal and flare responses to allergen extracts) and a history of symptoms that correspond with exposure to the sensitizing allergen.¹¹⁷ Mainstay therapeutic options include avoidance of inciting allergens, nasal saline irrigation, oral antihistamines, intranasal corticosteroids, combination intranasal corticosteroid/antihistamine sprays, leukotriene receptor antagonists, and allergen immunotherapy.¹¹⁷ Allergen immunotherapy, which induces immunological tolerance through repeated exposure to low levels of allergens, has gained interest, despite the long treatment period and risk for anaphylaxis. Other current therapeutic efforts include specific inhibitors or blocking antibodies to target specific mediators (i.e., IgE and various cytokines) that are implicated in allergic rhinitis.^110,117,128

A number of murine models of allergic rhinitis have been studied as a therapeutic platform for gene therapy. Chatila et al.¹²⁹ investigated targeting the STAT family. These transcription factors play an important role in T cell differentiation by activating T cell transcription factors. Activation of STAT6 is critical for the differentiation of naive T cells into Th2 effector cells.¹³⁰ During allergic pulmonary inflammation, STAT6 is required for Th2 lymphocyte and eosinophil homing to the airways and expression drives mucus production and airway hyperresponsiveness.^130,131 In a murine model of allergen sensitization, intranasal administration of an siRNA against STAT6 significantly inhibited the development of allergen-induced airway inflammation, goblet cell hyperplasia, and airway hyperresponsiveness¹³² and reduced sneezing and nasal rubbing.¹³²

Wu et al.¹³³ targeted CCR3 expressed by eosinophils, mast cells, and Th2 cells. A lentiviral vector expressing a short hairpin RNA (shRNA) against CCR3 (CCr3shRNA) was tested by intranasal administration in a mouse model of allergic rhinitis.¹³³ CCr3shRNA reduced nasal symptoms, the level of OVA-specific IgE, inflammatory, and mast cell infiltration in the nasal cavity, and relieved the histopathological changes of nasal mucosa.¹³³ This was accompanied by reduced levels of histamine, tryptase, and PGD2 in bone marrow, peripheral blood, and nasal mucosa. Similarly, a lentiviral vector expressing siRNA against CCR3 administered intranasally to a mouse model of allergic rhinitis resulted in lower levels of eosinophils in nasal lavage, blood, and bone marrow.¹³⁴

Using gene silencing with siRNA, Suzuki et al.¹³⁵ evaluated the therapeutic effects of CD40 siRNA on inhibition of the allergic response in an OVA model of allergic rhinitis. To inhibit the interaction of antigen-presenting cells and T cells, CD40 siRNAs were administered to OVA-immunized mice.¹³⁵ Treated mice showed substantially reduced nasal symptoms and reduced local eosinophil accumulation, anti-OVA-specific IgE antibodies, and increased levels of IL-4 and IL-5.¹³⁵

Other promising gene therapy strategies to abrogate allergic rhinitis include intranasal instillation of an Epstein–Barr virus (EBV)-based plasmid expressing a decoy tumor necrosis factor alpha receptor-IgGFc.¹³⁶ Treated mice showed decreased sneezing and itching of the nose and decreased infiltration of eosinophils, mast cells, and IL-5⁺ cells in the nasal mucosa. Similarly, intranasal administration of an EBV-based plasmid expressing IL-12, which promotes differentiation to a Th1 response, resulted in lower serum levels of IgE (Table 5).¹³⁷

Table 5.

Preclinical gene therapy studies for atopic dermatitis

Vector	Dose/Route	Experimental Model	Result	References
CD86siRNA	Topical	Mice (NC/Nga)a	Decreased contact sensitivity Decreased DC in skin Decreased IgE (serum)	141

^a Atopic dermatitis prone mice.

GENE THERAPY FOR ATOPIC DERMATITIS

Atopic dermatitis is a common chronic inflammatory skin disease affecting up to 20% of children and 10% of adults in industrialized countries and the prevalence is increasing.¹³⁸ Clinical features include erythema, edema, lichenification, excoriations, oozing, and crusting.¹³⁹ Pruritus is a crucial and dominant feature of atopic dermatitis and impacts quality of life with associated comorbidities such as sleep loss and psychological distress.¹³⁹ The pathogenesis of atopic dermatitis is not clearly elucidated, although skin barrier defects and altered immune responses are postulated as key components in disease development.¹⁴⁰

The main therapeutic objectives of treatment of atopic dermatitis are reductions in pruritus and skin inflammation and prevention of flares, while minimizing side effects. Management can be difficult and time-consuming, requiring a multidimensional approach that includes patient/parent education, elimination of exacerbating factors, and restoration of epidermal and skin barrier functions, combined with various pharmacological therapies depending on disease severity.¹³⁹ Current management strategies of atopic dermatitis rely on patient education, emollient therapy, and topical corticosteroids and trigger avoidance.¹³⁹

The skin is an attractive target for gene therapy as it is an easily accessible organ. Ritprajak et al.¹⁴¹ developed an siRNA-based therapy for allergic skin disease targeting CD86, an important co-stimulatory ligand on dendritic cells. Blockade of the CD86 pathway in mice interferes with antigen-specific T cell responses in murine Th2-mediated allergic disease models.^142–144 A therapeutic approach for treating allergic skin disease using cream-emulsified CD86 siRNA to target cutaneous dendritic cells inhibited murine contact hypersensitivity and markedly decreased the number of dendritic cells in the skin.¹⁴¹ CD86 siRNA treatment suppressed the clinical manifestations of atopic dermatitis and reduced the antigen-specific production of IL4 and serum IgE and IgG1antibodies (Table 6).¹⁴¹

Table 6.

Preclinical gene therapy for complement-mediated disorders

Vector	Dose (Route)	Experimental Model	Results	References
Hereditary angioedema
AAVrh.10hC1EI	1 × 1011 gc (intravenous)	Mice (heterozygote C1EI model; CRISPR/Cas9)	Sustained C1EI activity levels Correction of vascular leak (skin and organs)	164
AAV5. SERPING1	Dose response intravenous	Mice (Rag2−/−) and Cynomolgus macaques	Stable levels of C1INH	165
AAV8-anti-kallikrein	1 × 1013	Mice (C57bl/6)	Inhibited kallikrein (murine and human) Reduced carrageenan induced paw swelling	168
C2 deficiency
AAV.hC2	1 × 1010, 3 × 1010, 1 × 1011 (intravenous)	Mice (C57bl/6)	Sustained levels of C2	154
NTLA-2002		NHP/mice	Lowers kallikrein activity >90% × 6 months	Intellia tx (https://www.intelliatx.com)

AAvrh.10hC1EI, adeno-associated virus vector expressing human C1 esterase inhibitor (a serine protease inhibitor that normally inhibits proteases in the contact, complement, and fibrinolytic systems) coding sequence; C1EI/C1INH, C1-esterase inhibitor; hC2, human complement component2; NHP, nonhuman primate; NTLA-2002, CRIPS/Cas9 gene editing technology targeting kallikrein B1 (KLKB1) gene; SERPING1, gene provides instructions for making C1 esterase inhibitor, a type of serine protease inhibitor.

GENE THERAPY FOR COMPLEMENT-MEDIATED DISORDERS

The complement system is an integral part of the innate immune response and acts as a bridge between innate and acquired immunity. It consists of a series of proteins that are mostly synthesized in the liver and are present in the plasma and on cell surfaces as inactive precursors.^7,145 The primary function of the complement system is to protect from infection, to remove particulates such as damaged or dying cells, microbes, or immune complexes, and to modulate adaptive immune responses.^7,145 Inherited deficiencies of nearly all complement components and regulators have been identified and found to be associated with severe diseases including primary immune deficiency, autoimmunity, chronic renal disease, as well as severe recurrent infections with gram-negative and gram-positive microbes.^7,146

There is increasing evidence that complement activation contributes to the pathogenesis of inflammatory diseases. Attempts to efficiently inhibit complement include the application of endogenous soluble complement inhibitors (C1-inhibitor, recombinant soluble complement receptor 1) and the administration of antibodies, blocking key proteins of the complement cascade (e.g., C3, C5), neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium.¹⁴⁷

Deficiencies of complement components of the classical activation pathway, C1, C2, and C4, lead to increased susceptibility to bacterial infections and increased risk of developing autoimmune disease, particularly systemic lupus erythematosus.^146,148,149 Complement component C2 functions as a key regulator in the early activation phase of the classical pathway and participates in the formation of the classical pathway C3 convertase C4b2a.⁷ C2 is also a critical component of the lectin pathway. When mannose-binding lectin (MBL) or ficolins in complex with MBL-associated serine protease (MASP) molecules bind to relevant carbohydrate molecules, this leads to the activation of MASP-2, which then may cleave both C2 and C4, thereby forming the same C3 convertase as in classical pathway activation.⁷ Thus, C2 is an important component of both the classical and the lectin pathways of complement activation and is involved in the first-line defense against microbial infection essential for detection and clearance of invading pathogens.⁷

Complement C2 deficiency is the most common genetically determined complete complement deficiency with a prevalence of 1:20,000 individuals of Caucasian ancestry.^150,151 The deficiency is, in the majority of cases, caused by homozygosity for C2 genes having deletions in exon 6, resulting in complete absence of C2, or in some cases caused by other C2 gene mutations.^152,153 In the absence of C2, C3 is, in many situations, not efficiently cleaved resulting in a limited deposition of C3 fragments on immune complexes and on the surface of apoptotic cells. Circulating apoptotic cells become a source of self-antigen for autoantibodies that participate in the formation of immune complexes. The immune complexes are deposited throughout the body, potentially causing localized inflammatory reactions in the joints and kidneys, and ultimately leading to renal disease from chronic activation of the complement system.⁷ Currently, there are no specific treatments for complement deficiencies. Infection prevention and appropriate treatment of infections with antibiotics, when they do occur, is central to the care of patients with these deficiencies.

A number of gene therapy approaches have been developed to treat the complement disorders (Table 7). Rakhe et al.¹⁵⁴ explored the feasibility of using AAV gene therapy to deliver complement component C2. AAV.hC2 intravenously administered to wild-type mice had circulating C2 levels of ∼40 μg/mL (human reference range; 20–40 μg/mL; 1 × 10¹⁰) 30 days post-administration. At higher doses, increased expression of hC2 was detectable early (day 3), subsequently declined at day 10, rebounded at day 14, and remained stable to day 30. This early transient decline may indicate complement consumption due to classical pathway activation in the first week following vector transduction.

Table 7.

Preclinical gene therapy studies for eosinophilic disorders

Vector	Dose/Route	Experimental Model	Results	References
AAVrh.10anti-Siglec-F (AAVrh.10anti-Eos)	1 × 1011 gc (intravenous)	Mouse (NSG); IL-5 induced eosinophilia (with AAV vector)	Induced eosinophil apoptosis Reduced eosinophils in the blood and organs Reduced mortality	185,186
AAVrh.10anti-Siglec-F (AAVrh.10anti-Eos)	1 × 1011 gc (intravenous)	Mouse (Balb/c)	Induced eosinophil apoptosis Reduced eosinophils in the blood and esophagus Alleviated food impaction	187

AAvrh.10anti-Eos; adeno-associated virus vector codes for a monoclonal antibody against an eosinophil receptor (Siglec-F [mouse]) that induces eosinophil apoptosis.

Hereditary angioedema (HAE) is a potentially life-threatening autosomal dominant deficiency characterized by recurrent swelling of cutaneous tissue, gastrointestinal tract, and respiratory tract.^14,155–157 Edema of the airway mucosa may cause dysphagia, stridor, and respiratory compromise leading to death by asphyxiation.^14,155–157 More than 99% of HAE cases are caused by deficiency in plasma functional C1-esterase inhibitor (C1-INH, a serine protease inhibitor), which regulates the contact, complement, and fibrinolytic systems.^158,159 Mutations of the SERPING1 gene result in a functional deficiency of C1-INH, leading to the uncontrolled, spontaneous activation of C1 and consumption of C2 and C4.^14,157 The decreased functional activity of C1-INH activates the contact system on endothelial surfaces, which undergo autoactivation triggering further activation of coagulation factors that convert prekallikrein into plasma kallikrein.^158,160 Kallikrein cleaves and activates bradykinin, causing vasodilation, increased vascular permeability and plasma leakage into the extracellular space, leading to edema formation.^161–163

Current therapy of HAE involves treatment of acute attacks by parenteral administration of purified C1-INH, kallikrein inhibitor, or inhibitor of the bradykinin receptor.¹⁴ Prophylactic treatment involves the administration of C1-INH concentrate, synthetic androgens, or antifibrinolytics.¹⁴ Because of the recurrent and unpredictable nature of HAE attacks, the risk of death by asphyxiation, and the high costs associated with protein therapy, restoring functional levels of C1 esterase inhibitor in patients by providing a copy of the human C1-INH cDNA by gene therapy is a promising therapeutic approach.

Qui et al.¹⁶⁴ hypothesized that a one-time administration of an AAV gene transfer vector expressing the genetic sequence of C1 esterase-inhibitor would provide sustained C1EI activity levels in plasma, sufficient to prevent angioedema episodes. In a novel CRISPR/Cas9 murine model of HAE that has decreased C1EI and C4 plasma levels and increased vascular permeability, the administration of AAVrh.10hC1EI (AAV serotype rh.10 coding for human C1-INH) resulted in sustained human C1E levels above the predicted therapeutic levels. Strikingly, AAVrh10.hC1EI-treated mice displayed a marked decrease in dye extravasation in paws and organs, compared with untreated littermates.

These results demonstrate that a single treatment with AAVrh.10hC1EI has the potential to provide long-term protection from angioedema attacks in the affected population. In an alternative approach, Woloszynek et al.¹⁶⁵ evaluated an AAV5 SERPING1 gene therapy vector (BMN331) to augment functional C1-INH levels via continuous production of C1-INH from the liver. Intravenous administration of BMN331 in Rag2^−/− mice produced stable levels of functional human C1-INH, without detected liver abnormalities. Treatment of cynomolgus macaques with BMN331 produced significant circulating levels of C1-INH without reported adverse effects.¹⁶⁵

Another therapeutic approach for HAE uses CRISPR/Cas9 technology to knockout the kallikrein B1 (KLKB1) gene. Knockout of KLKB1 in nonhuman primate and rodent studies showed a 90% reduction in kallikrein activity, a level that translates to a therapeutically meaningful impact on HAE attack rates.¹⁶⁶ This approach is now in the clinic. A single dose of NTLA-2002 led to 65% and 92% mean plasma kallikrein reductions at 25 and 75 mg doses, respectively, at week 8. HAE attacks were reduced by 91% in the 25 mg dose cohort through week 16; two of three patients remain attack free since treatment with third patient attack free since week 10 through latest follow-up.¹⁶⁷

Similarly, targeting the kallikrein pathway, Bruder et al.¹⁶⁸ delivered an AAV8 vector expressing an anti-kallikrein antibody to wild-type mice, resulting in high and sustained circulating levels of therapeutic antibody. Serum from vector injected mice effectively inhibited human kallikrein activity in vitro and inhibited endogenous mouse kallikrein ex vivo, demonstrating the functionality of the therapeutic. Clinical efficacy of this vector was demonstrated in mice by protection from swelling in a mouse model of carrageenan-induced paw edema.

One potential adverse effect of gene therapy targeting the complement system is activation of the complement pathway in response to gene therapy. Strategies to downregulate the complement pathway could minimize the undesired consequences that compromise the safety and efficacy of gene delivery vectors.

GENE THERAPY FOR HYPEREOSINOPHILIC DISORDERS

There are a variety of primary and secondary hypereosinophilic disorders characterized by chronic elevation of blood eosinophil levels, invasion of organs with eosinophils, and associated organ damage.¹⁶⁹ Eosinophils are highly specialized bone marrow-derived granulocytic cells that play a role in combating parasites and other pathogens.¹⁷⁰ In normal individuals, eosinophils represent <5% of white blood cells, with an absolute count of 300–500/μL.^169,171 Eosinophils normally persist in the circulation 8–12 h and survive in tissues 8–12 days.^170,172 Eosinophils carry a variety of cytotoxic mediators in cytoplasmic granules, including major basic protein, cationic protein, peroxidase, and neurotoxin, and can release reactive oxygen species, lipid mediators, destructive enzymes, and a variety of cytokines.¹⁷⁰ If eosinophils invade tissues in sufficient numbers, they are capable of causing significant organ damage and dysfunction.^169,173

Chronic eosinophilic leukemia-not otherwise specified (CEL-NOS), a subtype adult CEL characterized by persistent elevation of blood eosinophils (>1.5 × 10³/μL) of unknown cause, by dysfunction of organs infiltrated with eosinophils is unresponsive to any therapy.^169,174,175 The disease is characterized clinically by weight loss, cough, weakness, diarrhea, splenomegaly, hepatomegaly, and cardiac and lung dysfunction, and survival of ∼2 years.^{169,174–176} Pagovich et al.¹⁷⁷ developed a one-time gene therapy for CEL-NOS using an AAVrh.10 vector coding for an anti-eosinophil monoclonal antibody (AAVrh.10anti-Eos) that induced eosinophil apoptosis and provided sustained expression of the transgene in a murine model of CEL-NOS. The CEL-NOS mouse model using NSG mice was created using a vector expressing the cytokine IL-5 (AAVrh.10mIL-5), which stimulated the bone marrow to persistently generate high blood levels of eosinophils leading to tissue eosinophil infiltration and eventually death. The administration of AAVrh.10anti-Eos (10¹¹ genome copies) induced apoptosis of circulating and tissue eosinophils and reduced mortality when compared with untreated mice.

Camilleri et al.¹⁷⁸ also developed a novel gene therapy for eosinophilic esophagitis, which is characterized by persistent blood eosinophilia, infiltration of eosinophils into the esophagus, and release of eosinophil mediators that damage the tissue, resulting in upper gastrointestinal morbidity, food impaction, and dysphagia.¹⁷⁸ A novel mouse model of eosinophilic esophagitis was established that mimics the human disease by sensitization and challenge with peanut extract, which induced elevated levels of blood eosinophils, elevated IgE levels, infiltration of eosinophils in the esophagus, and food impaction. After treatment with a single administration of AAVrh.10mAnti-Eos, mice had persistent, high levels of anti-Siglec-F antibody expression, reduced peripheral and esophageal eosinophil numbers, and alleviation of food impaction when compared with untreated mice. These results suggest that a single treatment with AAVrh.10m.anti-Eos has the potential to provide substantial therapeutic benefit to patients with a number of hypereosinophilic disorders with unmet therapeutic need (Table 7).

FUTURE OF GENE THERAPY FOR IMMUNOGLOBIN E, COMPLEMENT-MEDIATED, AND EOSINOPHILIC DISORDERS

Given the severity, prevalence, and frequently lifelong persistence of allergic diseases, there remains an urgent need for improved therapeutic options. Many allergic diseases are chronic, and therefore, long-term gene expression that is robust enough to provide a therapeutic level of protein is required to provide adequate treatment. Allergen-specific and allergen-nonspecific strategies to date have shown only partial rates of response and have been further complicated by an increased frequency of adverse reactions. Furthermore, a main obstacle to these therapies is the limited effectiveness and duration of treatment and the high cost.

The therapeutic approaches highlighted in this review demonstrate the potential value of gene therapy technology in not only improving our understanding of allergic diseases but also highlight the potential to modulate the immune response with a single administration and to transform the management and treatment of allergic diseases.

Recent advances in the field of gene therapy for allergic disorders are expected to bring this therapeutic platform closer to the clinic. With a better understanding of the pathogenesis of IgE-mediated, complement, and eosinophilic disorders, coupled to improvements in vector design and safety profiles, it is likely that gene therapy will become a reality for some allergic disease entities. The critical question is whether the technology is sufficiently robust to provide sufficient levels of the therapeutic protein or monoclonal to effectively and safely treat these disorders. While gene therapy has the ability to provide persistent, effective therapy with a single administration, there is also the safety issue of not being able to shut off the therapy in the context of an adverse event. If this issue can be solved, gene therapy should be applicable not only to IgE, complement, and eosinophilic disorders but also to a number of other chronic immune/inflammatory disorders.

AUTHOR DISCLOSURE

No competing financial interests exist.

FUNDING INFORMATION

This study was supported, in part, by the National Institutes of Health AI122040.