Emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease

Review

10.1517/13543780903179294 © 2009 Informa UK Ltd ISSN 1354-3784 1505All rights reserved: reproduction in whole or in part not permitted

EmergingoligonucleotidetherapiesforasthmaandchronicobstructivepulmonarydiseaseRosanne M Séguin† & Nicolay FerrariTopigen Pharmaceuticals, Inc., Immunology and Development Support, 2901 Rachel East Street, Room 13, Montreal, Quebec, H1W 4A4, Canada

Chronic respiratory diseases such as asthma and chronic obstructive pulmo-nary disease (COPD) are disorders of the airways largely related to the pres-ence of persistent inflammation. The approval of inhaled corticosteroids in the early 1970s pioneered a new age of therapy in treating chronic inflam-matory airway diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in air-ways and the associated obstruction, inflammation and hyper-responsiveness. Fast forward 40 years: corticosteroids are still an important therapeutic intervention; however, they exhibit limited use in moderate to severe asthma and COPD. Oligonucleotide therapies are an emerging class which include the antisense, the RNAi (siRNA and miRNA), the immunomodulatory, the aptamer and the decoy approaches. As these approaches are rather recent in the respiratory field, most are still early in development. Nevertheless, with limitations of current small molecule therapies and the hurdles faced with biologics, the use of oligonucleotides is relevant and the door is open to the development of this category of therapeutics. This review focuses on the major classes of oligonucleotides that are currently in late stage pre-clinical or clinical development for the treatment of asthma and COPD, and discusses the implications for their use as therapies for respiratory diseases.

Keywords: antisense, asthma, COPD, oligonucleotides, RNAi

Expert Opin. Investig. Drugs (2009) 18(10):1505-1517

1. Introduction

The notion of using DNA antisense oligonucleotide (AON) molecules, which modulate expression of a target gene by binding to its mRNA and preventing translation, was first proposed by Zamecnik and Stephenson in 1978 [1]. This was the first evidence that oligonucleotides might be used for therapeutic intervention and since then AON technology has been developed for a wide array of therapeu-tic purposes. From this original conception, an array of options for RNA-targeting approaches in mammalian cells has expanded to include siRNA [2], ribozymes [3] and micro RNA (miRNA) [4]. The growing interest of such approaches has risen from the fact that several copies of a protein can be produced by each mRNA molecule. It is, therefore, potentially a more efficient approach to target the mRNA rather than the protein itself. Moreover, compared to small molecules and biologics, the array of targets for RNA-targeting drugs is not limited to enzymes, receptors or other cell surface proteins.

Another mechanism by which oligonucleotides can be used as therapeutics, besides targeting the RNA, is through protein binding. Oligonucleotides containing unmethylated cytosine-phosphate-guanosine (CpG) motifs (CpG DNA) bind to toll-like receptor 9 (TLR9) resulting in activation of intracellular signaling which

1. Introduction

2. AONs for respiratory disease

3. Immunomodulating

oligonucleotides (CpG ODN)

4. siRNA

5. DNA decoys

6. Conclusion: future use of

oligonucleotide therapies in

respiratory diseases

7. Expert opinion

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Emergingoligonucleotidetherapiesforasthmaandchronicobstructivepulmonarydisease

1506 ExpertOpin.Investig.Drugs(2009) 18(10)

leads to an immune response [5,6]. Aptamers, also termed as ‘chemical antibodies,’ are short DNA or RNA oligonu-cleotides or peptides that assume a specific and stable 3D shape in vivo, thereby, providing specific tight binding to secreted protein targets to inhibit function [7]. Finally, decoy oligonucleotides bearing the consensus binding sequence of a specific transcription factor can bind the transcription factor and titrate its activity resulting in gene expression changes [8].

Asthma is a chronic inflammatory disease characterized by eosinophilia, mast cell infiltration, and activation of T-helper (TH) cells that express IL -4, -5 and -13 (TH2 type), struc-tural changes including goblet cell hyperplasia, airway smooth muscle cell hypertrophy and subepithelial fibrosis. Together, these structural changes and inflammation are believed to contribute to the reversible airflow obstruction and airway hyper-responsiveness (AHR) which are hallmarks of asthma. Asthma affects an estimated 300 million people worldwide, and is expected to rise to 400 million over the next 15 – 20 years [9]. Of those patients with disease, the moderate to severe often fail to completely respond to con-ventional therapy and it is these patients who account for > 50% of the total healthcare costs associated with asthma [10]. Current asthma therapies can be divided into two general types of medications: bronchodilators and anti-inflammatory. Bronchodilators include the short- and long-acting β2-agonists salbutamol, formoterol and salmeterol. Anti-inflammatory medications include inhaled corticosteroids (ICS) which improve symptoms and decrease exacerbations with a rela-tively safe track record, especially when used at low doses. ICS, however, do not cure asthma as asthma symptoms and inflammation recur on discontinuation of treatment [11], nor do they adequately control asthma in all individuals. While ICS even at low doses are effective at controlling mild asthma, in moderate and severe asthma ICS have important therapeutic limitations. In these patients, therapies can be stepped up to include higher doses of ICS, long-acting β2-agonists or leukotriene receptor antagonists (montelukast sodium, zafirlukast) and 5-lipoxygenase inhibitors (zileuton). In patients who are still uncontrolled, the oral bronchodila-tor theophylline or a biological therapy based on an anti-body targeting IgE of asthmatic patients (omalizumab) can be used along with oral corticosteroids during exacerbations or when everything else is ineffective.

In spite of all these therapies, asthma remains uncon-trolled in most patients, especially those with moderate and severe disease [12] and is associated with direct costs of $6.1 billion a year.

COPD is a collective term used to describe a chronic lung disease usually induced by tobacco smoking or chronic exposure to irritants that is characterized by progressive obstruction of airflow, increased shortness of breath with chronic cough and phlegm production in most patients [13]. Patients with this disease either have chronic obstructive bronchitis, emphysema or a combination of both. Systemic

and local airway inflammation have been implicated in the pathogenesis of COPD [14]. COPD is projected to be the fourth commonest single cause of death worldwide by 2027 by the WHO [15]. Current interventions that have been shown to improve mortality in COPD are cessation of smoking [16] and delivery of supplemental oxygen when hypoxemia is present [17].

Although no medication has been shown to improve sur-vival in COPD, several classes of drugs are used to improve symptoms, quality of life and decrease the rate of exacerba-tions. The first three classes of drugs that will be given to COPD patients are bronchodilators although COPD is characterized by incompletely reversible airway obstruction. Indeed, in symptomatic patients, it is recommended to pre-scribe anticholinergics (ipratroprium, tiotroprium), short and long-acting β2-agonists or a combination of these. If patients remain symptomatic, xanthines (theophylline) can be added to the three other therapeutic classes and ICS are reserved as fourth or fifth line therapy in patients who remain symptomatic and/or have several exacerbations. ICS are the only anti-inflammatory therapy that is approved for COPD in most countries.

ICS are not very effective in COPD because they do not improve survival and have been associated with an increase in the rate of pneumonias [18]. Over the past 20 years, com-panies have been searching for drugs that would alter the long-term prognosis of COPD without success. A promising new class is the PDE4 inhibitors (roflumilast and cilomilast). However, the current PDE4 inhibitors are limited by their low therapeutic:toxicity ratio [19].

It is thus clear that there is an unmet need for new medi-cations that can treat and potentially improve the long-term prognosis of moderate to severe asthma and COPD. Oligo-nucleotide therapies have a huge potential for the treatment of respiratory diseases. Until recently, the majority of strate-gies for the development of oligonucleotides have focused on systemic administration of oligonucleotides for the therapy of cancer or metabolic and cardiovascular diseases. It is inter-esting to note that the only oligonucleotide approved is Vitravene® (Novartis, New York, NY, USA) for cytomegalovirus retinitis which is directly administered to the site of disease (intravitreal). The airways and lung represent an ideal target site for oligonucleotides because of direct access using inhaled delivery, the persistence of oligonucleotides within the lungs for a prolonged time permitting daily or potentially weekly administration, and the degradation of oligonucleotides within the lungs with little systemic delivery, thus avoiding the systemic toxicity that occurs with oligonucleotides or systemic knock down of potential targets [20,21]. While recently many reports have described the use of oligonucle-otides to inhibit promising targets (reviewed in [22]), this review focuses on the evolving strategy as well as the chal-lenges of using oligonucleotide approaches for the treatment of chronic respiratory diseases such as asthma and COPD (Tables 1 and 2).

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Séguin&Ferrari

ExpertOpin.Investig.Drugs(2009) 18(10) 1507

Table1.OligonucleotidetherapeuticpipelineforasthmaandCOPD.

Drug Type Target Indication Company Status

CYT003-QbG10 CpG TLR9 Asthma Cytos Phase II

TPI ASM8 Antisense CCR3 and βchain of IL-3IL-5/GM-CSF R

Asthma Topigen Phase II

AVT-01 Decoy STAT-1 Asthma Avontec Phase II

EPI 2010 Antisense Adenosine A1R Asthma Epigenesis Phase II*

1018 CpG TLR9 Asthma Dynavax Phase II

AVE-7279 CpG TLR9 Asthma Coley/Sanofi-Aventis Phase II

AIR645 Antisense IL-4Rα Asthma Altair/ISIS Phase I

TPI 1100 Antisense PDE4/PDE7 COPD Topigen Phase I

AVE-0675 CpG TLR9 Asthma Coley/Sanofi-Aventis Phase I

SAR21609 CpG TLR9 Asthma Coley/Sanofi-Aventis Phase I

QAX395 CpG TLR9 Asthma Idera/Novartis Phase I

Excellair siRNA Syk kinase Asthma ZaBeCor Phase I

ATL 1102 Antisense VLA-4 Asthma Antisense Therapeutics/Teva Preclinical

AZD1419 CpG TLR9 Asthma/COPD Dynavax/Astra Zeneca Preclinical

*Discontinued programs.

COPD: Chronic obstructive pulmonary disease; CpG: Cytosine-phosphate-guanosine; TLR: Toll-like receptor.

Table2.Generalcharacteristicsofoligonucleotideapproaches.

Type Target Features Modifications Mechanismofaction

Antisense mRNA DNA-likesingle-stranded15 – 25 nt in length

Altered phosphate backbone (phosphorothioate, phosphoroamidate)Analogues with unnatural bases (LNA, FANA, PNA, Morpholino)Modified sugars (OMe, MOE)

RNase H-mediated mRNA degradationSteric blockade of the ribosome (translation arrest) or splicing machinery (splicing arrest)

CpG TLR9 DNA-likesingle-stranded18 – 25 nt in length

Altered phosphate backboneAnalogues with unnatural basesModified sugars (ANA)

TLR9-mediated immune responseA-class: activation of pDC and NK cellsB-class: activation of B cellsC-class: intermediate effects of both A- and B-class

siRNA mRNA RNA-likedouble-stranded19 – 21 nt in length

Altered phosphate backboneModified sugars (OMe, MOE)

RISC-mediated mRNA degradationSteric blockade of the ribosome (translation arrest)

Decoy Protein DNA-likedouble-stranded10 – 20 nt in length

Altered phosphate backbone Target protein binding; titration of protein activity

CpG: Cytosine-phosphate-guanosine; RISC: RNA-induced silencing complex; TLR: Toll-like receptor.

2. AONsforrespiratorydisease

Antisense approaches have proven to be valuable tools for func-tional genomics, target validation and therapeutic purposes [23,24]. The mechanism by which AON can prevent translation can involve enzyme-mediated mRNA degradation and physical block-ade of the translation process. There are two major mechanisms of action of AON (for a review see [25]). The first is that most

AONs activate RNase H, which cleaves the RNA moiety of a DNA–RNA heteroduplex and, therefore, leads to degradation of the target mRNA (Figure 1A). Additionally, AONs that do not elicit RNase H cleavage can be used to inhibit translation by steric blockade of the ribosome (Figure 1B) [26]. When the AONs are targeted to the 5′-terminus, binding and assembly of the translation machinery can be prevented. Finally, AONs can be used to correct aberrant splicing [27,28].

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.



The first program that assessed whether inhaled AON could be effective in humans for the therapy of chronic respiratory diseases were performed with a respirable AON (termed EPI-2010, Epigenesis Pharmaceuticals) that targeted the adenosine A1 receptor mRNA. Adenosine regulates tis-sue function by activating four G-protein Coupled Receptors (GPCRs): A1, A2A, A2B and A3 [29]. Both the concentration of adenosine [30] and expression of adenosine receptors on immune cells are elevated in asthmatic patients [29]. Inhaled adenosine can induce bronchoconstriction in asthma patients and adenosine receptor blockade can prevent this

bronchoconstrictive response [31]. EPI-2010, a 21-mer phos-phorothioate DNA (PS-DNA) targeting adenosine A1 receptor mRNA, was designed to decrease levels of the receptor and subsequently limit the responses to the adenosine. Preclinical efficacy of EPI-2010 was demonstrated using the dust-mite conditioned allergic rabbit model of asthma. Aerolized delivery of EPI-2010 desensitized rabbits to a subsequent challenge to either adenosine or dust-mite allergen [32]. In a similar model in monkeys, a single 5 mg dose of aerosolized EPI-2010 reduced airway sensitivity to adenosine, increasing the PC30 to adenosine up to 245-fold [33].

Transcription Translation

Translation

Translation

Translation

Decoy

Transcriptionfactor Transcription

Steric blockade

Splicingmachinery

Protein

Protein

Protein

Protein

RNase H/RISC mRNAdegradation

mRNA processing

Pre-mRNA mRNADNA

siRNAAON

AON

A.

B.

C.

Figure1.Mechanismoftranslationprevention. The process of mRNA transcription from DNA, its splicing and subsequent translation into protein is outlined at the top. A. AON and siRNA can bind to the corresponding target in the coding sequence of the pre- or mature mRNA triggering RNaseH- or RISC-mediated cleavage and degradation of the mRNA, respectively. B. AON and siRNA can bind to the target sequence in non-coding regions (promoter, intron/exon junctions, 5′ or 3′ UTRs) preventing by steric blockade transcription or mRNA maturation processes by the splicing machinery. C. Decoys containing binding elements can bind to transcription factors resulting in transcriptional gene silencing.AON: Antisense oligonucleotide; RISC: RNA-induced silencing complex.

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Séguin&Ferrari


In clinical studies, EPI-2010 was shown to be well toler-ated in normal subjects and in patients with mild asthma [25]. In patients with mild asthma, a single inhaled dose of EPI-2010 (50 μg/kg) produced a significant decrease in the requirement for rescue bronchodilator and reduced asthma symptom scores [34]. Despite this early success, although being well tolerated at doses up to 9 mg inhaled twice a week for 3 months, EPI-2010 failed to demonstrate efficacy in patients with moderate asthma that was not well con-trolled with ICS and the product development was discon-tinued [33]. The reasons for the lack of success of EPI-2010 are not known but could be related to dose of the drug, dosing frequency and the fact that the other adenosine receptors were not inhibited. Indeed, there is recent evi-dence to suggest that the other three adenosine receptors may also be important in asthma [35].

A crucial process in the development of asthma is the recruitment and persistence of inflammatory cells into the airways, a process regulated by a complex interplay of pro-inflammatory cytokines and chemokines. Targeting either key cytokines and chemokines or their receptors has been shown to be disease-modifying in numerous preclinical models of asthma but knocking down one mediator or one receptor has not been successful in the clinic [36-38]. The lack of efficacy in the clinic may be due to the redundancy of the immune response and the cross talk that occurs between many pro-inflammatory pathways. For example, it has been shown that many mediators acting through the CC chemokine receptor 3 (CCR3) receptor are present and increased in the lungs of patients with asthma and account for at least 50% of the eosinophil chemotactic activity in asthmatic bronchoal-veolar lavage (BAL) [39]. Interestingly, IL-5 has similar effects and increases the response of eosinophils and their progenitors to the chemokines that act through the CCR3 receptor. TPI ASM8 (Topigen Pharmaceuticals) is an AON drug candi-date which contains two PS-DNA AONs; one AON targets common β chain (βc) of IL-5, IL-3 and GM-CSF receptors, and the other CCR3 [40]. Preclinical studies using a rat asthma model demonstrated that blocking βc or CCR3 using specific AONs reduced AHR, reduced influx of eosinophils in response to allergen challenge, and reduced target mRNA and protein expression in lung [41,42]. Furthermore, combination of the two AONs was more effective at a lower dose when compared to each AON alone [40]. In both AONs of the TPI ASM8 product, adenosine residues have been replaced with 2-amino-2′deoxyadenosine: the rationale behind this modification relates to the property of adenosine as a signaling nucleoside that can elicit many physiological responses, including bronchospasm and inflammation particularly in patients with asthma. Elevated levels of adenosine have been found in BAL, blood and exhaled breath condensate of patients with asthma. In addition, inhaled adenosine-5′monophosphate induces bronchoconstriction in asthmatics [43]. Indeed, it was demonstrated that the lung resistance in the animals increased when increasing amounts of adenosine were administered

to the lung, whereas 2-amino-2′deoxyadenosine did not significantly affect lung resistance [40].

In a series of single- and multiple-dose studies in normal subjects and patients with mild asthma, TPI ASM8 was well tolerated following administration by inhalation at doses up to 6 mg and for up to 14 days [44,45]. In a 4-day allergen challenge Phase IIa trial in mild asthmatics, a 1.5 mg single daily dose of TPI ASM8 for 3 days reduced both the early allergic response (EAR) and late allergic response (LAR). Furthermore, TPI ASM8 inhibited eosinophil influx by 46% in the induced sputum of patients and there was a decrease in the induction of target genes (βc and CCR3) in response to the inhaled allergen [46]. To understand the dose response, safety and maximal therapeutic potential of TPI ASM8, another Phase IIa study assessing the efficacy and safety of four esca-lating doses regimens is currently being performed in patients with allergic asthma receiving an allergen challenge [47].

With the intent of targeting more than one mediator as per TPI ASM8, AIR645 (Altair Therapeutics) has also been designed to target two key cytokines receptors, IL-4 and IL-13. These cytokines are important in maintaining the TH2 phenotype and the modulation of eosinophilic inflammation and airway smooth muscle hyperplasia, respectively [48]. AIR645 is a second generation 2′-methoxyethyl AON targeting the common α chain of IL-4 and IL-13 receptors with the potential to be given once weekly because of the lung half-life of its chemistry. Administration by inhalation of an AON targeting the α chain of IL-4/IL-13 receptors in a mouse model of asthma attenuated antigen-induced airway eosinophilia and neutrophilia, mucus production and AHR, and reduced target protein expression on pulmonary antigen presenting cells and epithelial cells [49]. Results from a recently completed escalating single (0.03 – 30 mg) and multiple dose (0.3 – 20 mg) Phase I study with AIR645 in 72 normal volunteers as well as in 8 subjects with controlled asthma demonstrated that nebulized AIR645 was well tolerated and confirmed the low systemic exposure of AON following delivery to the lung as plasma levels were only detected after the 30 mg dose [50]. AIR645 was calculated to have a half-life in the sputum of ∼ 5 days independent of dose level, reaffirming the possibility of one weekly treatment for asthma [50].

Finally, also currently in preclinical development for asthma is ATL1102 (Antisense Therapeutics), which is a second generation AON that targets CD49d, a subunit of the adhesion molecule VLA-4 (very late antigen-4). Adhe-sion molecules are implicated in several facets of asthma, from leukocyte migration, exocytosis, cytokine production and respiratory burst, and VLA-4 is involved in the recruit-ment of eosinophils and T cells [51]. Preclinical studies in an asthma model in mice demonstrated that VLA-4 knock down reduced AHR, eosinophil recruitment and inflam-mation and mucus production [52]. To our knowledge, this drug has not been studied in patients with asthma but is currently in Phase II clinical trials as a treatment for multiple sclerosis.

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.



As discussed earlier, therapeutic interventions in the field of COPD have also focused on reducing the underlying inflammation through selective inhibition of the PDE4 iso-form. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well docu-mented, their low therapeutic:toxicity ratio and the presence of dose-dependent systemic side effects have limited their clinical utility. TPI 1100 (Topigen Pharmaceuticals, Inc.) is a dual PDE4/PDE7 inhibitor comprised of two AONs target-ing the mRNA for the PDE4B/4D and 7A isoforms. The advantages of TPI 1100 would include more potency by adding PDE7 knock down to PDE4 inhibition and low sys-temic delivery of the AON by degradation within the lungs, thus, avoiding the systemic toxicity that is limiting the devel-opment of PDE4 antagonists. In a mouse model of cigarette smoke exposure, which mimics the neutrophilic arm of the inflammatory response in COPD, TPI 1100 exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast [53], a small molecule inhibitor of PDE4 currently in Phase III of devel-opment for COPD [54]. TPI 1100 has received regulatory approval to initiate Phase I clinical trials in humans.

3. Immunomodulatingoligonucleotides(CpGODN)

Unmethylated CpG motifs, originally found in bacterial DNA, have immunostimulatory effects in animals and humans [6]. The observation that oligonucleotides containing selective CpG motifs (CpG ODN) could mediate a similar effect has offered new perspectives in the treatment of many clinical conditions, including allergy and asthma [5]. Based initially on observations that CpG ODN induced TH1-type patterns of immune responses, it was proposed that CpG ODNs could suppress the TH2-type responses that cause many of the mani-festations of allergic diseases such as asthma [55,56]. Evidence supporting this hypothesis came from the work of Kline et al. which, using a model of murine asthma, showed that airway eosinophilia, TH2 cytokine induction, IgE production and AHR were prevented by co-administration of CpG ODN with the antigen [57].

Different classes of immune stimulatory CpG ODN with distinct structural and biological characteristics have been described. Certain CpG motifs (A-class) are especially potent at activating NK cells and inducing IFN-α production by plasma dendritic cells, while other motifs (B-class) are potent B-cell activators [58]. Typically, CpG ODN bind to TLR9 in the endosomal compartment and initiate a signaling cascade that leads to activation of pro-inflammatory transcription factors such as NF-κB (Figure 2) [59].

In preclinical proof-of-concept studies also using a mouse model of asthma, an optimized B-class phosphorothioate CpG ODN, termed 1018 ISS (Dynavax Technologies), adminis-tered alone was shown to be effective at inhibiting eosinophilic airway inflammation when delivered either systemically (i.p.),

or mucosally (i.e., intra-nasally or intra-tracheally) [60]. A single dose of 1018 ISS inhibited airway eosinophilia as effectively as daily injections of corticosteroids for 7 days. Moreover, while both 1018 ISS and corticosteroids inhibited IL-5 generation, only 1018 ISS was able to induce allergen-specific IFN-γ production and redirect the immune system toward a TH1 response. In monkeys with experimentally-induced allergic airways disease, treatment twice a week with seven doses (12.5 mg) of 1018 ISS periodically for 33 weeks attenuated the magnitude of AHR, inflammation and airways remodeling after allergen challenge [61].

In a Phase II clinical study in patients with atopic asthma, inhalation of 1018 ISS (36 mg weekly for 4 weeks) before allergen challenge led to an increase in expression of IFN-γ and IFN-γ-associated genes in sputum cells without any toxicity or increase in adverse events when compared to placebo adminis-tration [62]. Unfortunately, results from this clinical trial could not reproduce the animal data as no attenuation of the EAR and LAR nor any reduction in allergen-induced sputum eosinophils or TH2 related gene expression could be measured in patients. It may be that once the allergic immune response is established in humans, immune stimulation would be ineffective at turning off this abnormal immune response. Further studies are needed to assess whether immune stimulation with CpG ODN can prevent the development of the allergic immune response and/or asthma in humans.

An interesting approach to immunostimulation uses encap-sulated CpG ODN within virus-like particles [63]. This technol-ogy uses the virus-like particle Qb, a recombinantly produced empty virus shell to deliver an A-class CpG ODN termed CYT003-QbG10 (Cytos Biotechnology). CpGs packaged into virus-like particles were shown to have increased stability and in contrast to free CpGs, packaged CpGs prevented splenomegaly, normally associated with immunostimulation in mice, without affecting their immunostimulatory capacity.

Early Phase I/II clinical results in allergic patients with mild asthma showed that CYT003-QbG10 administered subcuta-neously was well tolerated and resulted in almost complete tolerance to the allergen with symptoms of rhinitis and allergic asthma significantly reduced [64]. In a subsequent Phase II study in which patients received six injections of ascending doses of CYT003-QbG10 (300 – 900 μg), a 66% lower aver-age combined asthma symptom and medication score (ACAS) was observed in patients treated with CYT003-QbG10 compared to placebo [65].

The activity of CpG ODN has been shown to be signifi-cantly reduced when the 5′-end of the ODN is not accessi-ble, rather than the 3′-end, suggesting that the 5′-end plays a critical role in immunostimulatory activity [66]. Based on this observation, 3′-3′-linked CpG ODNs that contained two or more identical CpG DNA segments, called immunomodula-tory oligonucleotides (IMOs), were designed and synthesized. In vitro and in vivo studies showed an enhanced immuno-stimulatory activity when compared with linear CpG ODN [67]. In a mouse model of asthma, co-administration of

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Séguin&Ferrari


IMOs during allergen-sensitization abrogated EAR and LAR, AHR as well as release of TH2-type cytokines [68]. Similar results were found in mice with established allergic responses [69] suggesting IMO treatment not only prevented antigen-induced TH2 immune responses but could also reverse established allergic responses. Moreover, once weekly treatment by inhala-tion with 1.5 mg/kg of IMO QAX935 (Idera/Novartis) in allergic monkeys was shown to effectively attenuate inflamma-tory responses in the lung [70]. QAX935 is currently in Phase I studies.

4. siRNA

Double-stranded RNA can also inhibit gene expression through a multi-step process. Once inside the cell, double-stranded RNA is cleaved by the enzyme Dicer, yield-ing fragments of between 21 and 23 nucleotides in length [71], which are siRNA [72]. These siRNA are loaded into Argonaute 2 and the RNA-induced silencing complex.

Endosomalmembrane

TLR9

CpG ODN

Intracellular signaling

NF-κB MAP kinases

Nuclearmembrane

AP-1

Immune/inflammatory response

Production ofinflammatory

cytokines

Induction of gene expression

Figure2.TLR9-mediatedinductionoftheimmuneresponse. Following internalization into an endosomal compartment, CpG-containing oligonucleotides recognize and bind to TLR9 leading to rapid activation of a series of downstream signaling pathways including activation of MAP kinases with subsequent activation of transcription factors (e.g., AP-1) and nuclear translocation of NF-κB, key regulators of many inflammatory response pathways.CpG: Cytosine-phosphate-guanosine; ODN: Oligodeoxynucleotide;

TLR: Toll-like receptor.

Argonaute 2 cleaves the siRNA into an antisense (guide) strand, which recognizes a complementary sequence of tar-get mRNA. The exonuclease and endonuclease activities of the complex then degrade the cellular mRNA, thereby, inhibiting translation into functional protein (Figure 1A) [73]. Direct delivery of siRNA into the lung offers the same ben-efits as with AON: possible decrease in dose required com-pared to systemic delivery, potential reduction of undesired systemic side effects and direct access to target cells.

Among the targets of siRNA being investigated for clinical development is an early and essential signaling molecule in the network that leads to inflammation, Syk kinase. Evidence for the role of Syk kinase was provided in the rat model of pulmonary inflammation where AON targeting Syk kinase in a liposome carrier was delivered by an aerosol. Besides decrease in Syk mRNA expression in alveolar macrophages, suppres-sion of Fc-γ receptor-induced lung inflammation was observed as measured by a decrease in the release of NO, TNF-α and macrophage numbers in BAL [74]. Further studies delivering AON targeting Syk delivered in a liposome aerosol carrier in a rat model of OVA-induced allergic asthma resulted in inhi-bition of the inflammatory cell infiltrate (eosinophils and neutrophils) into the bronchoalveolar space [75]. Inhibition of inflammatory mediators was also shown in studies using siRNA targeting Syk in respiratory epithelial cells, which resulted in decreased release of IL-6 and decreased expression of intercellular adhesion molecule-1 [76,77].

Excellair™ (ZaBeCor, Bala Cynwyd, PA, USA) is an inhaled siRNA drug candidate designed to silence Syk which has received regulatory approval to initiate Phase I clinical trials in humans [78]. The current status of this program is unknown.

5. DNAdecoys

In this novel therapeutic concept, double-stranded DNA oligonucleotides are used to act as decoys for transcription factors. These decoys imitate a promoter consensus DNA-binding site for transcription and effectively prohibit binding of the transcription factor to its natural gene con-trol regions in a competitive manner (Figure 1C). AVT-01 (Avontec), is a short double-stranded oligonucleotide ‘decoy’ which targets STAT-1, a transcription factor shown to be strongly involved in inflammation. OVA-sensitized and chal-lenged mice administered intra-nasally decoy oligonucleotide targeting STAT-1 exhibited significant reduction in numbers of eosinophils and lymphocytes and IL-5 in BAL when compared to controls [79]. In treated mice, there was a reduction in CD40 expression in peri-bronchial infiltrates and in vascular cell adhesion molecule 1 on vascular endothe-lial cells, and the development of AHR after allergen chal-lenge was abolished. In clinical studies, AVT-01 was shown to be safe and well tolerated when administered inhaled as a single dose of 3 and 10 mg in healthy volunteers. In a pilot clinical trial performed in asthmatic patients, a single dose

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.



of AVT-01 attenuated AHR following AMP challenge [80]. The follow-up multiple dosing Phase IIa clinical study in which AVT-01 was delivered once a day for 7 days showed that AVT-01 was safe and well tolerated with a trend but no statistical effect on AHR [81].

6. Conclusion:futureuseofoligonucleotidetherapiesinrespiratorydiseases

Since the first studies that have shown the potential of using AON to modulate target gene expression in the 1970s [1], oligonucleotides have moved from the concept of a tool for possible drug target identification to an effective therapeutic class. Airways and lung diseases are ideal for the use of therapeutic oligonucleotides. Direct administration to the site of action by inhalation permits oligonucleotides to reach and enter its target cells; intra-pulmonary degradation of oligonucleotides with little systemic delivery after inhalation is an advantage over small molecules that avoids systemic toxicity; a long half-life of oligonucleotides in the lungs will lead to daily or even longer chronic dosing and improve compliance in patients; finally, several oligonucleotides (two or more) can be combined into one drug product and have broader albeit specific targeted effects. As therapeutics for respiratory disease, AON and CpG ODN are the most advanced in clinical development with examples of siRNA and decoys moving through preclinical development.

Recently, there have been very few new drug classes approved for asthma and COPD. Approved drugs have con-sisted mostly in improvements in older classes of medica-tions, through the use of combination therapy, longer duration of action or improved delivery devices. This rela-tive lack of advancement and the slow pace of innovation to identify new drug products can be indicative of the compli-cated nature of these chronic diseases as well as a potential limited number of targets for conventional small molecule drugs and biologics. There are aspects of lung diseases which can be considered conducive to oligonucleotide therapy. Oli-gonucleotides can be used to target the specific mediators that are involved in disease and thus avoid the toxicity of a broader approach that is used for example with corticoster-oids. The discovery of many new human genes with the human genome project has increased exponentially the num-ber of potential therapeutic targets for oligonucleotide based approaches. With oligonucleotide therapy, it is possible to combine several oligonucleotides (two or more) into one drug product and have broader yet specific targeted effects as has been performed with TPI ASM8.

With regard to the target organ itself, the lung is an attractive target as it permits direct delivery to the site through inhalation or instillation, enabling oligonucleotides to reach and enter target cells thus potentially reducing total dose. Along with the benefit of reduced dose, the risk of systemic toxicity is also improved as intra-pulmonary administration would limit systemic exposure of the drug. Chemical

modifications which function to extend the half-life of oligonucleotides in the lungs could lead to daily or even longer chronic dosing and improve compliance in patients. That being said, there are several challenges facing oligonu-cleotide therapies attributed mainly to the shortcomings in the properties of the oligonucleotides including their stabil-ity in vivo, delivery to and uptake by the target cells, and possible undesirable secondary side effects. In pulmonary/respiratory diseases, toxicity associated with the administra-tion of therapeutic nucleic acids includes immune stimula-tion, inflammation and possibly hypersensitivity and bronchoconstriction of the airways. The Phase IIa studies that have been performed until now have not shown any of this potential toxicity but longer term studies are needed to confirm these results.

During the past decade, much has been learnt about the medicinal chemistry and the pharmacologic, pharmacoki-netic and toxicologic properties of oligonucleotide mole-cules [24,82-86]. In general, three types of modifications of oligonucleotides can be distinguished: use of nucleotide ana-logues with unnatural bases, use of nucleotides with modi-fied sugars (especially at the 2′ position of the ribose) or altered phosphate backbones [24]. Medicinal chemistry, for example, has allowed the development of new generations of AON with potencies comparable to siRNA [82] and with potentially improved bio-distribution, pharmacokinetics and toxicologic properties over other RNA-targeting approaches. Chemical modifications of siRNA have improved their nuclease stability, decreased the likelihood of triggering an innate immune response, lowered the incidence of off-target effects and improved pharmacodynamics [87]. Finally, modi-fications of CpG ODN have also provided molecules with increased stability and improved immune activation [59].

A major challenge for oligonucleotide-based therapy has been delivery and uptake of oligonucleotides by target cells, in particular for siRNA and decoys which are rather large molecules (∼ 13 kDa compared to ∼ 6 kDa for AON and CpG ODN). While some preclinical data have demonstrated delivery to lung tissue through the systemic route of admin-istration [88], the pulmonary route represents an attractive option which delivers appreciable amounts of oligonucleotides in animals and in humans [21,46,89,90]. In the lung, uptake of oligonucleotides has been observed in immune cells, mainly macrophages, as well as in alveolar epithelial and in airway epithelium cells [91,92]. In chronic lung diseases such as asthma and COPD, remodeling of the structural cells may also contribute to severity of the disease and, as such, these cell types should also include future candidate targets for therapy.

Perhaps the most important issue concerns the effective intracellular delivery of oligonucleotides to their respective site of action in the nucleus or cytoplasm. In studies of cells in culture, delivery agents such as cationic lipids or polymers are required to attain significant antisense or RNAi effects. However, the large size and/or considerable toxicity [93] of

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Séguin&Ferrari


cationic lipid particles and cationic polymers may render them problematic candidates for in vivo utilization. The exact mechanism by which oligonucleotides enter the cells is not well understood but in almost all instances, oligonucleotides (and their various delivery agents) are taken up by some form of endocytosis [93]. While CpG ODN will bind to TLR9 in the endosomal compartment, the AON, siRNA and decoys must exit from the endosome to reach its site of action in the cytosol or nucleus. In fact, the nuclear distribution of AON and cytosolic localization of siRNA following in vitro transfection in epithelial cells are consistent with the presumed sites of action of each type of oligonucleotide to inhibit gene expression [91]. Until now, most inhaled oligonucleotide products have focused on the use of non-viral carriers and have demonstrated functional effects in humans with simple solutions that do not contain any carriers. Because oligonu-cleotides are freely soluble and hygroscopic, the most common dosage form consists of aerosolization of a simple aqueous solution. Besides nebulizers and portable soft mist inhalers, however, other possible delivery options for oligonucleotides include metered dose inhalers and dry powder inhalers.

In the future, engineered viral-mediated delivery may be a technique used, especially for the advancement of siRNA as therapeutics. Another possible option would be the use of nanoparticles bearing antibodies specific for receptors expressed by a select subset of cells, targeting the oligonucleotides to the cells of choice and optimizing their uptake by endo-cytosis. Extensive research by many in the field is being focused to enhance and improve oligonucleotide delivery, which will advance the development of this class of molecules as pharmacotherapy.

7. Expertopinion

Asthma and COPD are chronic respiratory diseases with an unmet need for pharmaco-therapeutic intervention. As we improve our understanding of the key molecules and media-tors involved in the pathophysiology of these diseases, respi-rable oligonucleotide therapies are poised to play a significant role in the treatment. Because a single RNA molecule may be the platform for the synthesis of several copies of a pro-tein (∼ 5000 copies), there is a clear advantage in targeting the mRNA rather than the protein as a more efficient approach to block protein function [94]. Another benefit of the oligonucleotide approach is the relatively short preclini-cal development period required for identification and vali-dation of an oligonucleotide product as compared to small molecules. The delay between identification of targets in the laboratory and the definition of an oligonucleotide product is brief, often < 2 years. There is also the flexibility in the oligonucleotide approach that allows the combination of

several oligonucleotides to different targets in one single drug product. This approach is especially important because of the redundancy of inflammatory pathways and as new important targets are identified this may increase the need for oligonucleotides against several genes in one product. For diseases such as asthma and COPD in which the etiol-ogy is still currently incompletely defined and in which the disease progresses, there may be a need for constant modifi-cations to the therapeutic approach to keep the therapy in synchronicity with disease progression. Oligonucleotides lend themselves to this approach. In the not so distant future, doctors may be able to tailor the therapy regimen to specifically suit the patient on an individual basis through a combination of specific oligonucleotide therapies.

In addition to the rapid development process for newly identified targets, the field of oligonucleotide therapies is advancing quickly, with the development of new approaches consisting of aptamers, ribozymes, RNAi and even more recently miRNAs. MiRNAs are interesting in that they could be used either as therapeutic to modulate mRNA (e.g., using miRNA mimics), but could also themselves be the target for oligonucleotide therapy (e.g., using antagomirs). This scien-tific field seems to be advancing at a significant rate such that in 5 years entire new classes of oligonucleotide therapies could not only have been identified but also already be assessed in clinical development.

Current research is also assessing modifications to oligo-nucleotide chemistry that will improve the efficacy and the safety with little effects on the relatively low costs in production when compared to other biologics.

The inhaled approach for oligonucleotide therapy leads to direct delivery to the site of action in contrast to oral or intravenoous administration and thus reduces systemic expo-sure while decreasing the total dose required for efficacy [89]. Enhanced safety is currently being achieved through an increase in potency and a longer half-life of second and third generation chemistries. However, for chronic diseases such as asthma and COPD which need long-term therapy, the long-term safety of this therapeutic approach will need to be monitored closely.

In summary, this class of therapeutics is, therefore, poised to expand in the upcoming decades because of its advan-tages, especially with topical lung administration and it is notable that industrial product development and commit-ment is evident from all major pharmaceutical companies engaged in respiratory medicine.

Declarationofinterest

The authors are employees of Topigen Pharmaceuticals Inc., and have received no payment in preparation of this manuscript.

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.



BibliographyPapers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.1. Zamecnik PC, Stephenson ML.

Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA 1978;75(1):280-4

• Firstpaperdescribingthepotentialuse ofoligonucleotidesastherapyasdemonstratedbyreductionofvirusproductionfollowingtransfection ofantisensetargetingRoussarcoma virus35SRNA.

2. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411(6836):494-8

3. Lima WF, Crooke ST. Highly efficient endonucleolytic cleavage of RNA by a Cys2His2 zinc-finger peptide. Proc Natl Acad Sci USA 1999;96(18):10010-5

4. Grimson A, Farh KK, Johnston WK, et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007;27(1):91-105

5. Krieg A, Matson S, Fisher E. Oligodeoxynucleotide modifications determine the magnitude of B cell stimulation by CpG motifs. Antisense Nucleic Acid Drug Dev 1996;6(2):133-9

• Alongwith[6]below.

6. Krieg AM, Yi A-K, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995;374(6522):546-9

• Togetherwith[5],excellentreviewarticlesonthemechanismofactionofCpGmotif-containingoligonucleotides.

7. Jayasena SD. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 1999;45(9):1628-50

8. Mann M, Dzau V. Therapeutic applications of transcription factor decoy oligonucleotides. J Clin Invest 2000;106(9):1071-5

9. Masoli M, Beasley R. Asthma exacerbations and inhaled corticosteroids. Lancet 2004;363(9416):1236

10. Chung KF, Godard P, Adelroth E, et al. Difficult/therapy-resistant asthma: the need for an integrated approach to define clinical

phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. ERS task force on difficult/therapy-resistant asthma. European Respiratory Society. Eur Respir J 1999;13(5):1198-208

11. Rabe KF, Adachi M, Lai CK, et al. Worldwide severity and control of asthma in children and adults: the global asthma insights and reality surveys. J Allergy Clin Immunol 2004;114(1):40-7

12. Bateman ED, Bousquet J, Keech ML, et al. The correlation between asthma control and health status: the GOAL study. Eur Respir J 2007;29(1):56-62

13. Drummond MB, Dasenbrook EC, Pitz MW, et al. Inhaled corticosteroids in patients with stable chronic obstructive pulmonary disease: a systematic review and meta-analysis. JAMA 2008;300(20):2407-16

14. Falk JA, Minai OA, Mosenifar Z. Inhaled and systemic corticosteroids in chronic obstructive pulmonary disease. Proc Am Thorac Soc 2008;5(4):506-12

15. Murray CJL, Lopez AD, Mathers CD, Stein CA. The global burden of disease 2000 project; aims, methods, and data sources. In: Organisation WH, editor, Global programme on evidence for health policy discussion paper. Geneva [governmental document] 2001. p. 1-57

16. Anthonisen NR, Skeans MA, Wise RA, et al. The effects of a smoking cessation intervention on 14.5-year mortality: a randomized clinical trial. Ann Intern Med 2005;142(4):233-9

17. Long term domiciliary oxygen therapy in chronic hypoxic cor pulmonale complicating chronic bronchitis and emphysema. Report of the Medical Research Council Working Party. Lancet 1981;1(8222):681-6

18. Celli BR, Thomas NE, Anderson JA, et al. Effect of pharmacotherapy on rate of decline of lung function in chronic obstructive pulmonary disease: results from the TORCH study. Am J Respir Crit Care Med 2008;178(4):332-8

19. Lipworth BJ. Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease. Lancet 2005;365(9454):167-75

20. Templin M, Levin A, Graham M, et al. Pharmacokinetic and toxicity profile of a

phosphorothioate oligonucleotide following inhalation delivery to lung in mice. Antisense Nucleic Acid Drug Dev 2000;10(5):359-68

• See[21].

21. Ali S, Leonard SA, Kukoly CA, et al. Absorption, distribution, metabolism, and excretion of a respirable antisense oligonucleotide for asthma. Am J Respir Crit Care Med 2001;163(4):989-93

• Togetherwith[20],tworesearcharticlessummarizingthepharmacokineticsofinhaledantisenseoligonucleotidesanddemonstratingthelowsystemicbioavailabilityofthedrugproducts.

22. Moschos SA, Spinks K, Williams AE, Lindsay MA. Targeting the lung using siRNA and antisense based oligonucleotides. Curr Pharm Des 2008;14(34):3620-7

• Reviewarticlewhichincludednewlyidentifiedtargets,notyetnecessarily indevelopment.

23. Jason TLH, Koropatnick J, Berg RW. Toxicology of antisense therapeutics. Toxicol Appl Pharmacol 2004;201(1):66-83

24. Kurreck J. Antisense technologies: improvement through novel chemical modifications. Eur J Biochem 2003;270(8):1628-44

25. Crooke ST, editor, Antisense drug technology: principles, strategies and applications. 2nd edition. Boca Raton FL: CRC Press 2008.

•• Firstchaptersinanexcellentbookonantisensedrugtechnologywhichreviewsthedifferenttypesofoligonucleotidetherapies,advancesinchemicalmodificationsofoligonucleotidesanddeliverysystemsofoligonucleotidesaswellasareviewofthevariousindicationsfortherapyincludingcancer,metabolicdiseases,neurologicaldisordersandinflammatorydiseases

26. Ghosh C, Iversen PL. Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers. Antisense Nucleic Acid Drug Development 2000;10(4):263-74

27. Popplewell LJ, Trollet C, Dickson G, Graham IR. Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene. Mol Ther 2009;17(3):554-61

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Séguin&Ferrari


28. Aartsma-Rus A, van Ommen G-JB. Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications. RNA 2007;13(10):1609-24

29. Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov 2008;7(9):759-70

30. Driver AG, Kukoly CA, Ali S, Mustafa SJ. Adenosine in bronchoalveolar lavage fluid in asthma. Am Rev Respir Dis 1993;148(1):91-7

31. Polosa R, Holgate ST. Adenosine receptors as promising therapeutic targets for drug development in chronic airway inflammation. Curr Drug Targets 2006;7(6):699-706

32. Nyce JW, Metzger WJ. DNA antisense therapy for asthma in an animal model. Nature 1997;385(6618):721-5

•• Oneofthefirstpapersdemonstratinginvivoefficacyofinhaledantisenseoligonucleotidesinmonkeysaspotentialtherapyforasthma.

33. Ball HA, Van Scott MR, Robinson CB. Sense and antisense: therapeutic potential of oligonucleotides and interference RNA in asthma and allergic disorders. Clin Rev Allergy Immunol 2004;27(3):207-17

34. Ball H, Sandrasagra A, Tang L, et al. Clinical potential of respirable antisense oligonucleotides (RASONs) in asthma. Am J Pharmacogenomics 2003;3:97-106

35. Wilson CN. Adenosine receptors and asthma in humans. Br J Pharmacol 2008;155(4):475-86

36. Nair P, Pizzichini MMM, Kjarsgaard M, et al. Mepolizumab for prednisone-dependent asthma with sputum eosinophilia. N Engl J Med 2009;360(10):985-93

37. Haldar P, Brightling CE, Hargadon B, et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med 2009;360(10):973-84

38. Hanania NA. Targeting airway inflammation in asthma. Chest 2008;133(4):989-98

39. Lamkhioued B, Renzi PM, Abi-Younes S, et al. Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation. J Immunol 1997;159(9):4593-601

40. Allakhverdi Z, Allam M, Guimond A, et al. Multitargeted approach using antisense oligonucleotides for the treatment of asthma. Ann NY Acad Sci 2006;1082:62-73

•• Oneofthefirstresearcharticlesdemonstratingadditiveefficacyofcombiningtwoantisenseoligonucleotidesinapreclinicalratmodelofasthma.

41. Allakhverdi Z, Lamkhioued B, Olivenstein R, et al. CD8 depletion-induced late airway response is characterized by eosinophilia, increased eotaxin, and decreased IFN-gamma expression in rats. Am J Respir Crit Care Med 2000;162(3):1123-31

42. Fortin M, Ferrari N, Higgins M-E, et al. Effects of antisense oligodeoxynucleotides targeting CCR3 on the airway response to antigen in rats. Oligonucleotides 2006;16:203-12

43. Spicuzza L, Di Maria G, Polosa R. Adenosine in the airways: implications and applications. Eur J Pharmacol 2006;533(1-3):77-88

44. Multiple dose ASM8 in mild asthmatics. September 11, 2007; cited 13 April 2009. Available from: http://clincialtrials.gov/ ct2/show/NCT00402948

45. Study to evaluate the safety and efficacy of TPI ASM8 in subjects with asthma. 2008. Available from: http://clinicaltrials.gov/ct2/show/NCT00550797

46. Gauvreau GM, Boulet LP, Cockcroft DW, et al. Antisense therapy against CCR3 and the common beta chain attenuates allergen-induced eosinophilic responses. Am J Respir Crit Care Med 2008;177:952-8

•• Oneofthefirstresearcharticlesdemonstratingclinicalefficacyofinhaledantisenseoligonucleotidedrugproductasatreatmentforasthma.

47. Efficacy and safety of four escalating dose regimens of TPI ASM8 in patients with allergic asthma. January 14 2009 cited April 13 2009. Available from: http://clinicaltrials.gov/ct2/show/NCT00822861

48. Nelms K, Keegan AD, Zamorano J, et al. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999;17(1):701-38

49. Karras JG, Crosby JR, Guha M, et al. Anti-inflammatory activity of inhaled IL-4 receptor-{alpha} antisense oligonucleotide

in mice. Am J Respir Cell Mol 2007;36:276-85

• Similarto[41],thisresearcharticledemonstratesthepreclinicalefficacyofaninhaledantisenseoligonucleotidetargetingseveralreceptortargets.

50. Hodges MR, Castelloe E, Chen A, et al. Randomized, double-blind, placebo controlled first in human study of inhaled AIR645, an IL-4Ralpha oligonucleotide, in healthy volunteers. In: Medicine AJoRCC, editor, American Thoracic Society, San Diego; 2009. p. A3640

51. Medina-Tato D, Watson M, Ward S. Leukocyte navigation mechanisms as targets in airway diseases. Drug Discov Today 2006;11(19-20):866-79

52. Lofthouse S, Crosby J, Tung D, et al. Aerosol delivery of VLA-4 specific antisense oligonucleotides inhibit airway inflammation and hyperresponsiveness in mice. Respirology 2005;10(Suppl 1):A11

53. Fortin M, Higgins M, Aubé P, et al. TPI 1100: an inhaled antisense oligonucleotide (AON) against phosphodiesterase (PDE) 4 and 7 with significant anti-inflammatory effects in a mouse model for COPD. [Abstract A653] Am J Respir Crit Care Med 2008;177

54. Martorana PA, Beume R, Lucattelli M, et al. Roflumilast fully prevents emphysema in mice chronically exposed to cigarette smoke. Am J Respir Crit Care Med 2005;172(7):848-53

55. Kline JN. Eat dirt: CpG DNA and immunomodulation of asthma. Proc Am Thorac Soc 2007;4(3):283-8

56. Fonseca D, Kline J. Use of CpG oligonucleotides in treatment of asthma and allergic disease. Adv Drug Deliv Rev 2009;61(3):256-62

57. Kline JN, Waldschmidt TJ, Businga TR, et al. Cutting edge: modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 1998;160(6):2555-9

• ResearcharticleoutliningthepotentialefficacyofCpGoligonucleotidesinpreclinicalmousemodelofasthma.

58. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002;20(1):709-60

59. Krieg AM. Therapeutic potential of toll-like receptor 9 activation. Nat Rev Drug Discov 2006;5(6):471-84

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.



60. Broide D, Schwarze J, Tighe H, et al. Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice. J Immunol 1998;161(12):7054-62

61. Fanucchi MV, Schelegle ES, Baker GL, et al. Immunostimulatory oligonucleotides attenuate airways remodeling in allergic monkeys. Am J Respir Crit Care Med 2004;170(11):1153-7

62. Gauvreau GM, Hessel EM, Boulet L-P, et al. Immunostimulatory sequences regulate interferon-inducible genes but not allergic airway responses. Am J Respir Crit Care Med 2006;174(1):15-20

• Researcharticleofclinicalresponse andsafetyofCpGmotifcontainingoligonucleotides.

63. Storni T, Ruedl C, Schwarz K, et al. Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects. J Immunol 2004;172(3):1777-85

64. Senti G, Johansen P, Haug S, et al. Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial. Clin Exp Allergy 2009;39(4):562-70

• ResearcharticledemonstratingclinicalefficacyandsafetyofencapsulatedCpGmotifcontainingoligonucleotides.

65. Cytos Biotechnology presents analysis of patient diaries including six month follow-up data from Phase II study with CYT003-QbG10 monotherapy for the treatment of allergic diseases. January 13, 2009 cited 13 April 2009. Available from: http://www.cytos.com/userfiles/file/ Cytos_Press_E_090113.pdf

66. Yu D, Zhao Q, Kandimalla ER, Agrawal S. Accessible 5′-end of CpG-containing phosphorothioate oligodeoxynucleotides is essential for immunostimulatory activity. Bioorg Med Chem Lett 2000;10(23):2585-8

67. Yu D, Kandimalla ER, Bhagat L, et al. ‘Immunomers’–novel 3′-3′-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents. Nucleic Acids Res 2002;30(20):4460-9

• Researcharticledemonstratingpreclinicalefficacyofimmunomers,modifiedCpGoligonucleotides.

68. Agrawal DK, Edwan J, Kandimalla ER, et al. Novel immunomodulatory oligonucleotides prevent development of allergic airway inflammation and airway hyperresponsiveness in asthma. Int Immunopharmacol 2004;4(1):127-38

69. Zhu FG, Kandimalla ER, Yu D, et al. Modulation of ovalbumin-induced Th2 responses by second-generation immunomodulatory oligonucleotides in mice. Int Immunopharmacol 2004;4(7):851-62

70. Dubois G, Simmons J, Martin E, et al. Effects of a novel synthetic TLR9 agonist on repeated allergen challenge in allergic monkeys. Recent Advances in Pattern Recognition Toll 2008. [Abstract p18]

71. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000;101(1):25-33

• See[73].

72. de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007;6(6):443-53

• Togetherwith[73],excellentreviewarticlesexplainingthemechanismofactionofsiRNA.

73. Zeng Y, Yi R, Cullen BR. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 2003;100(17):9779-84

74. Stenton GR, Kim M-K, Nohara O, et al. Aerosolized Syk antisense suppresses Syk expression, mediator release from macrophages, and pulmonary inflammation. J Immunol 2000;164(7):3790-7

75. Stenton GR, Ulanova M, Dery RE, et al. Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase. J Immunol 2002;169(2):1028-36

76. Ulanova M, Puttagunta L, Marcet-Palacios M, et al. Syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 2005;288(3):L497-507

77. Ulanova M, Asfaha S, Stenton G, et al. Involvement of Syk protein tyrosine kinase in LPS-induced responses in macrophages. J Endotoxin Res 2007;13(2):117-25

78. ZaBeCor’s investigational new drug application approved by the FDA. November 12, 2008 cited 13 April 2009. Available from: http://www.zabecor.com/news/news030409.php

79. Quarcoo D, Weixer S, Groneberg, D,et al. Inhibition of signal transducer and activator of transcription 1 attenuates allergen-induced airway inflammation and hyperreactivity. J Allergy Clin Immunol 2004;114(2):288-95

80. Kerstjens HA, Oosterhuis B, van Marle SP et al. Single dose decoy oligonucleotide directed against STAT-1 attenuates bronchial hyperresponsiveness to AMP. Proc Am Thorac Soc 2006;3:A825 [abstract]

• Demonstrationofclinicalefficacyofdecoyoligonucleotidesforrespiratorymedicine.

81. AVONTEC announce results of a multiple dose clinical proof of concept study with its drug candidate AVT-01 decoy ODN in asthmatic patients. February 28, 2008 cited 13 April 2009. Available from: http://avontec.com/pdf/PR_asthma_phase_IIa_2008_02_28.pdf

82. Ferrari N, Bergeron D, Tedeschi A-L, et al. Characterization of antisense oligonucleotides comprising 2′-Deoxy-2′-Fluoro-beta-D-Arabinonucleic Acid (FANA): specificity, potency, and duration of activity. Ann NY Acad Sci 2006;1082:91-102

83. McKay RA, Miraglia LJ, Cummins LL, et al. Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression. J Biol Chem 1999;274(3):1715-22

84. Sternberger M, Schmiedeknecht A, Kretschmer A, et al. Geneblocs are powerful tools to study and delineate signal transduction processes that regulate cell growth and transformation. Antisense Nucleic Acid Drug Dev 2002;12(3):131-43

85. Wahlestedt C, Salmi P, Good L, et al. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. PNAS 2000;97(10):5633-8

86. Yoo BH, Bochkareva E, Bochkarev A, et al. 2′-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro. Nucleic Acids Res 2004;32(6):2008-16

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Séguin&Ferrari


87. Behlke MA. Chemical modification of siRNAs for in vivo use. Oligonucleotides 2008;18(4):305-20

88. Geary R, Yu R, Watanabe T, et al. Pharmacokinetics of a tumor necrosis factor-{alpha} phosphorothioate 2′-o-(2-methoxyethyl) modified antisense oligonucleotide: comparison across species. Drug Metab Dispos 2003;31(11):1419-28

89. Guimond A, Viau E, Aube P, et al. Advantageous toxicity profile of inhaled antisense oligonucleotides following chronic dosing in non-human primates. Pulm Pharmacol Ther 2008;21(2):845-54

90. Duan W, Chan JH, McKay K, et al. Inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide

attenuates asthma in mice. Am J Respir Crit Care Med 2005;171(6):571-8

91. Griesenbach U, Kitson C, Escudero Garcia S, et al. Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo. Respir Res 2006;7:26-41

92. Zhang X, Shan P, Jiang D, et al. Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis. J Biol Chem 2004;279(11):10677-84

93. Juliano R, Alam MR, Dixit V, Kang H. Mechanisms and strategies for effective delivery of antisense and siRNA

oligonucleotides. Nucleic Acids Res 2008;36(12):4158-71

•• ExcellentsummaryarticlereviewingthevariousdeliverystrategiesandchallengesforantisenseandsiRNAoligonucleotides.

94. Popescu F-D. Antisense- and RNA interference-based therapeutic strategies in allergy. J Cell Mol Med 2005;9(4):840-53

AffiliationRosanne M Séguin† & Nicolay Ferrari†Author for correspondenceTopigen Pharmaceuticals, Inc., Immunology and Development Support, 2901 Rachel East Street, Suite 13, Montréal, Québec, H1W 4A4, Canada Tel: +514 868 0077; Fax: +514 868 0011; E-mail: [email protected]

Exp

ert O

pin.

Inv

estig

. Dru

gs D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cGill

Uni

vers

ity o

n 09

/24/

13Fo

r pe

rson

al u

se o

nly.

Documents

Emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease