WO2008101354A1 - Gpr65 as a therapeutic target in allergic airway inflammation - Google Patents
Gpr65 as a therapeutic target in allergic airway inflammation Download PDFInfo
- Publication number
- WO2008101354A1 WO2008101354A1 PCT/CA2008/000350 CA2008000350W WO2008101354A1 WO 2008101354 A1 WO2008101354 A1 WO 2008101354A1 CA 2008000350 W CA2008000350 W CA 2008000350W WO 2008101354 A1 WO2008101354 A1 WO 2008101354A1
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- WO
- WIPO (PCT)
- Prior art keywords
- gpr65
- expression
- mice
- inflammation
- allergic
- Prior art date
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Definitions
- GPR65 AS A THERAPEUTIC TARGET IN ALLERGIC AIRWAY INFLAMMATION
- Asthma is a very common disease and represents a major medical burden in the United States and abroad, accounting for US$12.7 billion of health care cost per year in the United States (Weiss and Sullivan, 2001).
- the disease is characterized by a decline in Forced Expiratory Volume in the first second (FEVl), chronic lung inflammation, characterized by an infiltration of eosinophils mast cells and Th2 CD4+ lymphocytes.
- FEVl Forced Expiratory Volume in the first second
- IL-4, 5 and 13 Key mediators such as IL-4, 5 and 13 are produced by these cells leading to airway constriction, mucus hypersecretion and airway remodeling, resulting in a decrease in lung function.
- GPR65 as a protein target involved in the pathophysiology of allergic inflammation and describes a method for treating allergic inflammation by attenuating GPR65 expression or activity.
- GPR65 also called gpr65, tdag ⁇ , g protein coupled receptor 65, htdag ⁇ , and t-cell death associated gene 8
- expression is shown to be significantly upregulated in a mouse model of allergic airway inflammation, and GPR65-deficient mice are demonstrated to display attenuated eosinophilia and DL- 13 levels in response to an allergic challenge.
- the application further discloses that delivery of GPR65-specific antisense oligonucleotides, that are capable of reducing GPR65 expression in the lung, attenuate airway inflammation in response to allergic challenge.
- the disclosure provides a method for ameliorating inflammation is a subject comprising administering a therapeutic agent which attenuates GPR65 expression or activity.
- Suitable therapeutic agents for use in the methods of the invention include agents that are capable of attenuating (e.g., inhibiting) expression of the GPR65 gene including, but not limited to GPR65-specific oligonucleotides or siRNA.
- patients afflicted with respiratory diseases characterized by an inflammatory component including but not limited to asthma, chronic obstructive pulmonary disease, cystic fibrosis, and bronchiectasis could benefit from treatment with a therapeutic agent which attenuates GPR65 expression or activity.
- the therapeutic agents are delivered directly to an individual's airway surfaces.
- a standard approach for such delivery is the use of an inhaled aerosol administered by a nebulizer (for aqueous formulations comprising the therapeutic agent) or metered dose inhaler (for the delivery of a dry powder formulation comprising the therapeutic agent).
- small molecules capable of specifically inhibiting (e.g., blocking) GPR65 activity will also have utility as regulators of the pathophysiology of allergic inflammation. It is further contemplated that a therapeutic agent which attenuates GPR65 expression or activity could be administered in combination with an agent selected from the group consiting of a corticosteroid, an anticholinergic agent, a leukotriene receptor antagonist, an inhibitor of leukotriene synthesis and a beta-adrenergic receptor agonist.
- FIG. 1 Effect of GPR65 deletion on OVA-induced pulmonary phenotype.
- OVA-sensitized wild type or GPR65 knockout mice were subjected to PBS or OVA aerosol.
- BAL and lungs were harvested and analyzed as described in "Methods”.
- A Bronchoalveolar lavage (BAL) total airway cellular inflammation.
- B BAL airway eosinophil inflammation.
- C BAL IL-13 levels.
- FIG. 3 Inflammatory potential of LNA gapmers.
- FIG. 4 Knockdown of GPR65 expression levels using sequence-specific ASO. Relative GPR65 expression levels in BALF cells (A-B) or right lung (C). GPR65 expression levels were determined using assays located at either position 58, at the exon 1-2 boundary (A,C), or at position 1836 (B) of the GPR65 consensus full-length transcript.
- A in vivo ASO intratracheal administration paradigm.
- B GPR65 mRNA expression in mouse lung harvested from animals intratracheally administered GPR65 ASO and subsequently exposed to PBS or OVA aerosol challenge. *p ⁇ 0.05 vs OVA-challenged animals.
- FIG. 6 Effect of nucleic acid-mediated knockdown of GPR65 mRNA in mouse lung: pulmonary phenotype.
- OVA-sensitized wild type mice intratracheally administered GPR65.16, control ASO, or vehicle were subjected to PBS or OVA aerosol.
- BAL and lungs were harvested and analyzed as described in "Materials and Methods".
- A BAL total airway cellular inflammation.
- B BAL airway eosinophil inflammation.
- C BAL IL- 13 levels. *p ⁇ 0.05 vs OVA-challenged animals.
- GPR65 has been recently cloned as a gene upregulated in response to glucocorticoids in the thymus.
- GPR65 has been shown to be highly expressed in many hematopoetic lineages including T- cells, B-cells, Neutrophils, and Macrophages, with lower level expression in lung tissue reported (Im et al., 2001; Radu et al., 2005).
- Over expression of GPR65 both in vitro and in vivo have implicated this receptor in GC mediated T-CeIl death.
- GPR65 has also been reported to be a receptor for the lysophopholid psychosine.
- GACC Galactosyl ceramidase
- GPR65 In addition to its potential role as a receptor for psychosine, GPR65 has been shown to be acid sensing (Ishii et al., 2005; Wang et al., 2004). Cells overexpressing GPR65 can sense low pH conditions leading to cAMP accumulation. Interestingly psychosine has been shown to antagonize the pH induced cAMP accumulation. Knockout mice for GPR65 have been generated which are normal healthy and fertile, and display no baseline immune defects (Radu et al. 2006). Despite its reported role as a psychosine receptor and a mediator of GC induced T-cell death, both GC and psychosine responses are unaffected in the absence of GPR65 in mice.
- mice deficient for GPR65 are unable to display pH induced cyclic-AMP accumulation in peripheral T-cells and splenocytes, thus confirming a pH sensing role for this receptor in vivo (Radu et al., 2005).
- Example 1 In an effort to gain a better understanding of the pathophysiology of allergic airway disease and in doing so identify novel therapeutics for asthma, we and others have conducted gene expression profiling experiments in an ovalbumin antigen challenge mouse model of allergic airway disease.
- the data provided in Example 1 identifies GPR65 as a gene upregulated in mouse models of asthma.
- GPR65 As a gene implicated in respiratory disease, we subjected ovalbumin-sensitized GPR65-deficient mice to an antigenic challenge with OVA. As shown in Example 2, GPR65 -deficient mice display a dramatic attenuation of OVA induced eosinophilia and IL- 13 levels. More specifically, GPR65 deficient mice were protected from developing OVA-induced airway inflammation, as determined by a significant reduction in total cell and eosinophil numbers comparable to that observed with glucocorticoid (Dexamethasone) treatment ( Figures 2A, 2B).
- Sensitized wild-type mice subjected to OVA displayed a significant increase in detectable BAL DL- 13 levels. As shown in Figure 2C a reduction in OVA-induced EL- 13 levels was also observed in mice lacking GPR65, correlating with a reduction in pulmonary inflammation.
- the role of GPR65 in the pathophysiology of allergic airway inflammation was further validated by evaluating the efficacy of GPR65 -specific antisense oligonucleotides (ASO) on the pulmonary phenotype of an antigenic challenge response.
- ASO GPR65 -specific antisense oligonucleotides
- Example 4 describes the effects of delivering GPR65 -specific ASOs to selectively knockdown lung GPR65 mRNA expression in sensitized and OVA- aerosolized wild type B ALB/c mice.
- mice Male TDAG8xBalb/c mice (GPR65 knockout mice; 7-1 lwks; 20-25g) were generously provided by Dr. Owen Witte (HHMI, UCLA, LA, USA) and maintained at Mispro Biotech Services Inc. (Montreal, Canada). TDAG8xBalb/c mice were confirmed null for TDAG8 employing previously published protocols (Radu CG et al. 2006. MoL Cell. Biol. 26: 668-677). Sex and age-matched stock BALB/c mice were purchased from Charles River (Canada). Animal protocols were approved by the animal care committees at Merck Frosst Canada Ltd. and Mispro Biotech Services, Inc.
- OVA ovalbumin
- Al(OH) 3 aluminum hydroxide
- OVA- immunized GPR65 KO or wild type control mice were randomized and groups of mice subjected to a 0.5X PBS (Mediatech) or 5% OVA (in 0.5X PBS) nebulized aerosol challenge (DeVilbiss; nebulization rate ⁇ 0.15mL/min) for 20 min via whole body exposure (Buxco) for three consecutive days.
- Dexamethasone sodium phosphate (Vetoquinol USA; lmg/kg; dosing volume 5mL/kg) was administered by i.p. injection 24 hours prior to beginning and 30 minutes prior to each of the three aerosol challenges.
- mice were immunized and challenged with ovalbumin aerosol as indicated above. Twenty four hours after the last aerosol challenge, mice were deeply anesthetised via i.p. administration of euthanol (120mg/kg i.p.). Animals were tracheostomized and bronchoalveolar lavage (BAL) was performed using 0.5mL IX PBS (Mediatech). Lungs were immediately excised, rinsed in phosphate buffered saline, and snap frozen in liquid nitrogen. Sample RNA was amplified and labeled using a custom automated version of the aminoallyl MessageAmp II kit (Ambion).
- mice were deeply anesthetised via i.p. administration of euthanol (120mg/kg i.p.). Animals were tracheostomized and bronchoalveolar lavage (BAL) was performed using 0.5mL IX PBS (Mediatech). Total cell numbers in the recovered volume (-70-80% recovery) were determined via automated cell count (Celldyn3500). Cytospin slides of BAL cells were prepared using a cytocentrifuge (Cytospin; Shandon, Pittsburgh, PA) and stained with May- Grunwald-Giemsa stain. Differential cell counts were determined by light microscopy from a count of 100 cells. Remaining BAL supernatant was frozen individually in aliquots and stored at -80 0 C.
- ASOs antisense oligonucleotides targeting both the 5' and 3' UTR and the coding region of GPR65 were designed using the EDT ASO design tool and applying rule set chimeric 20-mer (http://www.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx) (see Table 1).
- ASO sequences were evaluated for their potential off-target activity by aligning each sequence against the Mus musculus transcriptome (Blast). When possible, ASO sequences displaying a max score that is less or equal to 28.2 were selected. Table 1 provides a list of the GPR65-specific ASO that were designed and evaluated in vivo. The ASOs were designed as 5-10-5 LNA gapmers on a phosphorothioate backbone.
- CNTRLl and CNTRL2 Two distinct ASO controls were evaluated (CNTRLl and CNTRL2).
- ASO were ordered as LNA-phosphorothioate 5-10-5 gapmers and supplied by Integrated DNA technologies (Coralville, Iowa, USA) or Medicorp (Montreal, Quebec, Canada). All oligonucleotides were purified by HPLC and supplied as lyophilized stocks. ASO stocks were reconstituted in sterile D'PBS at a concentration of 4 mg/ml and frozen at -80oC until use.
- GPR65.1 5'-+C*+A*+T*+C*+A*C*T*T*C*T*T*A*G*G*C*+T*+G*+T*+C*+A-3'
- GPR65.9 5'-+C*+A*+G*+A*+G*G*G*T*A*T*T*T*G*T*C*+A*+T*+A*+G*+C-3'
- GPR65.16 5'-+G*+A*+T*+T*+G*G*A*G*A*T*T*G*G*T*C*+G*+T*+G*+C-3'
- GPR65.17 5'-+C*+A*+C*+C*+G*C*C*T*C*T*T*T*+G*+C*+C*+T-3'
- GPR65.1 (SEQ ID NO.: 1), GPR65.5 (SEQ ID NO.:2 ), GPR65.6 (SEQ ID NO.: 3), GPR65.8 (SEQ ID NO.:4), GPR65.9 (SEQ ID NO.:5 ), GPR65.10 (SEQ ID NO.: 6), GPR65.12 (SEQ ID NO.:7 ), GPR65.13 (SEQ ID NO.:8 ), GPR65.14 (SEQ ID NO.:9 ), GPR65.15 (SEQ ID NO.:10 ), GPR65.16 (SEQ ID NO.: 1 1 ), GPR65.17 (SEQ ID NO.: 12), GPR65.18 (SEQ ID NO.: 13), GPR65.19 (SEQ ID NO.: 14), CNTRL.l (SEQ ID NO.: 15), and CNTRL.2 (SEQ ID NO.:16 ).
- mice were aerosolized directly into the lungs of 10 week old Balb/c males using a microsprayer (PennCentury, Philadelphia, USA). Briefly, mice were anesthetised by intraperitoneal injection of Ketamine / Domitor at 40 / 1 mpk. Anesthetised mice were maintained in a supine position suspended from the upper incisors using a rubber band strapped to an angled rodent workstand (Highland Medical Equipment, Temecula, California, USA).
- mice were maintained in a supine hanging position for 2 minutes following aerosolization. Following this, mice were removed form the platform and injected with antisedan subcutaneous Iy at 1 mpk to reverse the anesthesia. During all manipulations, mice were maintained on a heating blanket to mitigate the possibility of hypothermia upon anesthesia.
- mice OVA-immunized BALB/c control mice were anesthetized, and then subjected to five non-surgical intratracheally administrations of GPR65 ASO (50 ⁇ g GPR65.16/ 50 ⁇ L instillation delivered on days 7, 10, 14, 17, 19) or irrelevant control ASO.
- ASO-instilled mice were subjected to PBS or OVA aerosol challenge as per above on days 17-19. Animals were tracheostomized and BAL harvested, and whole lungs processed for mRNA extraction as indicated below.
- RNA extraction RNA extraction. cDNA conversion, and Real-time PCR
- Taqman primers for the internal control (Ppib) and GPR65 (2 independent assays used Mm00433695_ml Mm02619732_sl) were purchase from Applied Biosystems.
- the PCR reactions were denatured for 2 min at 5O 0 C followed by 2 min at 95°C and then subjected to 50 cycles of amplification (94°C for 1 s followed by 20 s at 6O 0 C).
- GPR65 expression levels were normalized to the endogenous control (Ppib) using the ⁇ Ct method and expressed as a proportion relative to the levels observed in vehicle treated animals (100%).
- EXAMPLE 1 GPR65 IS UPREGULATED IN LUNG TISSUE OF MICE EXHIBITING AN ALLERGIC RESPONSE
- Results of the expression profiling experiment provided in Figure 1 illustrates that several thousand genes are significantly regulated in this model compared to control animals (Figure IA). Consistent with this model being representative of airway inflammation in humans, several genes were upregulated that have been implicated in human airway disease (eg. Mmpl2, IL-13, Chia, Muc5B) (Table 1). Functional annotation of these upregulated genes shows that there is a predominant inflammatory gene signature (Figure IB), hi this profiling experiment, GPR65 was shown to be upregulated 4.9 fold in this model thus implicating GPR65 in the pathophysiology of allergic inflammation. Previous profiling studies in a similar model of allergic airway inflammation have also shown GPR65 to be up-regulated (Zimmerman et al., 2004).
- GPR65 in the pathophysiology of allergic inflammation, and identifies GPR65 as a new protein target for the development of novel therapeutic agents that are capable of ameliorating allergic airway inflammation.
- mice were protected from developing OVA-induced airway inflammation, as determined by a significant reduction in total cell and eosinophil numbers comparable to that observed with glucocorticoid (Dexamethasone) treatment ( Figures 2 A, 2B).
- ASO antisense oligonulceotide
- mice were treated with 5 doses of aerosolized ASO or D'PBS over the course of 12 days and sacrificed 24 hours following the fifth dose, on day 13. Overall, the instillation of LNA-based ASO did not result in overt morbidity such as weight loss and mortality with the exception of mice treated with GPR65.1 (1 dead) and GPR65.9 (2 dead).
- CNTRL.1 was initially characterized using 2'0Me gapmers on a phosphorothiate backbone and was shown to be relatively innocuous following its administration to mice. Unexpectedly, treatment with CNTRL.1 as a LNA gapmer resulted in a potent cellular inflammation response when compared to the PBS control group ( Figure 3A).
- mice treated with GPR65.16 and GPR65.17 demonstrated moderate increases in monocytes and neutrophils compared to PBS-treated animals ( Figure 3B).
- GPR65.16 was selected as a lead ASO to determine the impact of GPR65 gene expression knockdown on the response to ovalbumin challenge in OVA-sensitized animals.
- antisense oligonucleotide (ASO) technology was employed to selectively knockdown lung GPR65 mRNA expression in sensitized and OVA-aerosolized wild type BALB/c mice.
- OVA-sensitized mice were intratracheally instilled with GPR65.16, control ASO, or vehicle ( Figure 5A).
- GPR65.16 intratracheal administration significantly and selectively reduced GPR65 mRNA expression in mouse lung ( Figure 5B).
- T cell death-associated gene 8 (TD AG8) as a novel acid sensing G-protein-coupled receptor. J. Biol. Chem. 280: 9083-9087.
- TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J. Biol. Chem. 279: 45626-45633.
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Abstract
The disclosure identifies GPR65 as a protein target involved in the pathophysiology of allergic inflammation and describes a method for treating allergic inflammation by attenuating GPR65 expression or activity. GPR65 expression is shown to be significantly upregulated in a mouse model of allergic airway inflammation, and GPR65-deficient mice are demonstrated to display attenuated eosinophilia and IL-13 levels in response to an allergic challenge. The application further discloses that delivery of GPR65-specific antisense oligonucleotides, that are capable of reducing GPR65 expression in the lung, attenuate airway inflammation in response to allergic challenge.
Description
TITLE OF THE INVENTION
GPR65 AS A THERAPEUTIC TARGET IN ALLERGIC AIRWAY INFLAMMATION
BACKGROUND OF THE INVENTION
Asthma is a very common disease and represents a major medical burden in the United States and abroad, accounting for US$12.7 billion of health care cost per year in the United States (Weiss and Sullivan, 2001). The disease is characterized by a decline in Forced Expiratory Volume in the first second (FEVl), chronic lung inflammation, characterized by an infiltration of eosinophils mast cells and Th2 CD4+ lymphocytes. Key mediators such as IL-4, 5 and 13 are produced by these cells leading to airway constriction, mucus hypersecretion and airway remodeling, resulting in a decrease in lung function.
Current therapeutics for mild to moderate asthma consists of long and short acting Beta agonists, inhaled corticosteroids and leukotrienes antagonists. While these agents are effective at controlling symptoms such as acute bronchoconstriction and inflammation, many asthmatics do not have effective symptom control, either due to lack of efficacy of the therapeutic or lack of compliance due to difficulty of administration in the case of inhaled products and or unwanted side effects. In addition, none of these current therapeutics lead to disease modification and they are ineffective at controlling mucus hypersecretion. As a result of this the need for effective and safe asthma therapeutics remains high.
SUMMARY OF THE INVENTION
The disclosure identifies GPR65 as a protein target involved in the pathophysiology of allergic inflammation and describes a method for treating allergic inflammation by attenuating GPR65 expression or activity. GPR65 (also called gpr65, tdagδ, g protein coupled receptor 65, htdagδ, and t-cell death associated gene 8) expression is shown to be significantly upregulated in a mouse model of allergic airway inflammation, and GPR65-deficient mice are demonstrated to display attenuated eosinophilia and DL- 13 levels in response to an allergic challenge. The application further discloses that delivery of GPR65-specific antisense oligonucleotides, that are capable of reducing GPR65 expression in the lung, attenuate airway inflammation in response to allergic challenge.
The disclosure provides a method for ameliorating inflammation is a subject comprising administering a therapeutic agent which attenuates GPR65 expression or activity. Suitable therapeutic agents for use in the methods of the invention include agents that are capable of attenuating (e.g., inhibiting) expression of the GPR65 gene including, but not limited to GPR65-specific oligonucleotides or siRNA.
Based on the data provided in this disclosure patients afflicted with respiratory diseases characterized by an inflammatory component, including but not limited to asthma, chronic obstructive
pulmonary disease, cystic fibrosis, and bronchiectasis could benefit from treatment with a therapeutic agent which attenuates GPR65 expression or activity.
In one aspect of the invention, the therapeutic agents are delivered directly to an individual's airway surfaces. A standard approach for such delivery is the use of an inhaled aerosol administered by a nebulizer (for aqueous formulations comprising the therapeutic agent) or metered dose inhaler (for the delivery of a dry powder formulation comprising the therapeutic agent).
Based on the data provided herein it is also contemplated that small molecules capable of specifically inhibiting (e.g., blocking) GPR65 activity will also have utility as regulators of the pathophysiology of allergic inflammation. It is further contemplated that a therapeutic agent which attenuates GPR65 expression or activity could be administered in combination with an agent selected from the group consiting of a corticosteroid, an anticholinergic agent, a leukotriene receptor antagonist, an inhibitor of leukotriene synthesis and a beta-adrenergic receptor agonist.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Expression of GPR65 in the mouse OVA model: Data shown here is the result of combining 5 OVA sensitized and challenged mice. OVA stimulation results in a robust signature with 3,280 sequences up- and 3,939 sequences down-regulated at p<=1.0E-4. GPR65 is up-regulated 4.9 fold with a p value < 1.0E-30.
Figure 2 Effect of GPR65 deletion on OVA-induced pulmonary phenotype. OVA-sensitized wild type or GPR65 knockout mice were subjected to PBS or OVA aerosol. BAL and lungs were harvested and analyzed as described in "Methods". A, Bronchoalveolar lavage (BAL) total airway cellular inflammation. B, BAL airway eosinophil inflammation. C, BAL IL-13 levels.
Figure 3 Inflammatory potential of LNA gapmers. Total (A) and differential (B) cell counts in BALF derived from ASO treated animals and vehicle control. Shown are averages with error bars indicating standard deviation. 4 animals used per group except GPR65.1 (n=3) and GPR65.9 (n=2).
Figure 4 Knockdown of GPR65 expression levels using sequence-specific ASO. Relative GPR65 expression levels in BALF cells (A-B) or right lung (C). GPR65 expression levels were determined using assays located at either position 58, at the exon 1-2 boundary (A,C), or at position 1836 (B) of the GPR65 consensus full-length transcript.
Figure 5 Antisense oligonucleotide (ASO) directed against GPR65 significantly decreases GPR65 mMRA expression in mouse lung. OVA-sensitized wild type mice intratracheally administered GPR65.16, control ASO, or vehicle were subjected to PBS or OVA aerosol. Mouse whole lungs were harvested and mRNA extracted as described in "Methods". A, in vivo ASO intratracheal administration
paradigm. B, GPR65 mRNA expression in mouse lung harvested from animals intratracheally administered GPR65 ASO and subsequently exposed to PBS or OVA aerosol challenge. *p<0.05 vs OVA-challenged animals.
Figure 6 Effect of nucleic acid-mediated knockdown of GPR65 mRNA in mouse lung: pulmonary phenotype. OVA-sensitized wild type mice intratracheally administered GPR65.16, control ASO, or vehicle were subjected to PBS or OVA aerosol. BAL and lungs were harvested and analyzed as described in "Materials and Methods". A, BAL total airway cellular inflammation. B, BAL airway eosinophil inflammation. C, BAL IL- 13 levels. *p<0.05 vs OVA-challenged animals.
DETAILED DESCRIPTION OF THE INVENTION
Recently it has been reported that the airways of asthmatics as well as patients with COPD are acidic compared to healthy individuals, as measured by inhaled breath condensate (Hunt et al, 2000; Kostikas et al., 2002). This is most clearly noted during acute asthmatic exacerbations where exhaled breath condensate has been shown to reach pH 5.5. Interestingly, pH approaches normal levels upon treatment with inhaled corticosteroids. A causal role for acidic conditions in asthma is supported by the strong correlation between acid reflux disease and asthma noted in several studies (Mathew et. al., 2004; Jiang and Huang, 2005). Acidic pH has been postulated to contribute to the host response upon pathogen challenge. It is hypothesized that this in turn contributes to increased oxidative stress as well as eosinophil necrosis leading to an enhanced acute exacerbation. In addition, decreased airway pH has been shown to impair mucociliary clearance by attenuating ciliary beat function and increasing mucus viscosity (Holma and Hegg, 1989; Luk and Dulfano, 1983).
While many of the pathophysiological responses to pH may be non-specific, several proteins have been identified that directly respond to protons. The acid-sensing ion channel family is the best example which has been implicated in several disease most notably neuropathic pain (Wemmie et al., 2006). In addition, a family of G-protein coupled receptors have been shown to sense protons (Tomura et al., 2005). Thus it is reasonable to hypothesize that in the lung some of the effects induced by low pH may be mediated by specific protein targets.
One member of the proton sensing GPCR family known as TDAG8, or GPR65 has been recently cloned as a gene upregulated in response to glucocorticoids in the thymus. In addition to thymic expression, GPR65 has been shown to be highly expressed in many hematopoetic lineages including T- cells, B-cells, Neutrophils, and Macrophages, with lower level expression in lung tissue reported (Im et al., 2001; Radu et al., 2005). Over expression of GPR65 both in vitro and in vivo have implicated this receptor in GC mediated T-CeIl death. GPR65 has also been reported to be a receptor for the lysophopholid psychosine.
Psychosine is normally present in very low levels in the brain, but high levels of psychosine accumulate in the absence of Galactosyl ceramidase (GALC) which is found in the condition known as Globoid cell leukodystrophy or Krabbe's (Svennerholm et al., 1980) This in-born error of metabolism leads to
multinucleated cells in the white matter and neuronal demyelination. It has been shown in vitro that GPR65 can mediate psychosine signaling inhibiting forskolin induced cAMP accumulation and leading to a calcium flux, as well as mediating a defect in cytokenisis leading to a globoid cell phenotype in vitro. Despite the published data, however, the direct effect of phychosine on GPR65 is controversial and several groups have been unable to reproduce these data.
In addition to its potential role as a receptor for psychosine, GPR65 has been shown to be acid sensing (Ishii et al., 2005; Wang et al., 2004). Cells overexpressing GPR65 can sense low pH conditions leading to cAMP accumulation. Interestingly psychosine has been shown to antagonize the pH induced cAMP accumulation. Knockout mice for GPR65 have been generated which are normal healthy and fertile, and display no baseline immune defects (Radu et al. 2006). Despite its reported role as a psychosine receptor and a mediator of GC induced T-cell death, both GC and psychosine responses are unaffected in the absence of GPR65 in mice. However, mice deficient for GPR65 are unable to display pH induced cyclic-AMP accumulation in peripheral T-cells and splenocytes, thus confirming a pH sensing role for this receptor in vivo (Radu et al., 2005).
In an effort to gain a better understanding of the pathophysiology of allergic airway disease and in doing so identify novel therapeutics for asthma, we and others have conducted gene expression profiling experiments in an ovalbumin antigen challenge mouse model of allergic airway disease. The data provided in Example 1 identifies GPR65 as a gene upregulated in mouse models of asthma.
To further validate GPR65 as a gene implicated in respiratory disease, we subjected ovalbumin-sensitized GPR65-deficient mice to an antigenic challenge with OVA. As shown in Example 2, GPR65 -deficient mice display a dramatic attenuation of OVA induced eosinophilia and IL- 13 levels. More specifically, GPR65 deficient mice were protected from developing OVA-induced airway inflammation, as determined by a significant reduction in total cell and eosinophil numbers comparable to that observed with glucocorticoid (Dexamethasone) treatment (Figures 2A, 2B).
Sensitized wild-type mice subjected to OVA displayed a significant increase in detectable BAL DL- 13 levels. As shown in Figure 2C a reduction in OVA-induced EL- 13 levels was also observed in mice lacking GPR65, correlating with a reduction in pulmonary inflammation. The role of GPR65 in the pathophysiology of allergic airway inflammation was further validated by evaluating the efficacy of GPR65 -specific antisense oligonucleotides (ASO) on the pulmonary phenotype of an antigenic challenge response. Example 4 describes the effects of delivering GPR65 -specific ASOs to selectively knockdown lung GPR65 mRNA expression in sensitized and OVA- aerosolized wild type B ALB/c mice. As shown herein, the direct intratracheal delivery of anti-sense oligonucleotides (ASO) against GPR65, was associated with a significant attenuation of O VA-dependent total, eosinophil airway inflammation and BAL IL-13 (Figure 6A-C). Thus, implicating GPR65 in the regulationOVA-dependent eosinophilia independent of any developmental defect that may be present in
GPR65 deficient mice. Considered as a whole the data provided herein establishes the utility of a therapeutic entity directed against GPR65 for the treatment of allergic airway disease.
MATERIALS AND METHODS Mice
Male TDAG8xBalb/c mice (GPR65 knockout mice; 7-1 lwks; 20-25g) were generously provided by Dr. Owen Witte (HHMI, UCLA, LA, USA) and maintained at Mispro Biotech Services Inc. (Montreal, Canada). TDAG8xBalb/c mice were confirmed null for TDAG8 employing previously published protocols (Radu CG et al. 2006. MoL Cell. Biol. 26: 668-677). Sex and age-matched stock BALB/c mice were purchased from Charles River (Canada). Animal protocols were approved by the animal care committees at Merck Frosst Canada Ltd. and Mispro Biotech Services, Inc.
Immunization and Antigen Challenge
Animals were systemically sensitized by intraperitoneal (i.p.) administration of 20μg ovalbumin (OVA; Grade V; Sigma-Aldrich) in 0.4mL of a 5mg/mL suspension of aluminum hydroxide (Al(OH)3; Sigma-Aldrich). Seven days later, animals were subjected to a second i.p. sensitization of 1 Oμg OVA in 0.2mL of a 5mg/mL Al(OH)3 suspension. Seven days after the second sensitization, OVA- immunized GPR65 KO or wild type control mice were randomized and groups of mice subjected to a 0.5X PBS (Mediatech) or 5% OVA (in 0.5X PBS) nebulized aerosol challenge (DeVilbiss; nebulization rate ~0.15mL/min) for 20 min via whole body exposure (Buxco) for three consecutive days.
Dexamethasone sodium phosphate (Vetoquinol USA; lmg/kg; dosing volume 5mL/kg) was administered by i.p. injection 24 hours prior to beginning and 30 minutes prior to each of the three aerosol challenges.
Expression Profiling For in vivo gene profiling studies, wild type male BALB/c mice were immunized and challenged with ovalbumin aerosol as indicated above. Twenty four hours after the last aerosol challenge, mice were deeply anesthetised via i.p. administration of euthanol (120mg/kg i.p.). Animals were tracheostomized and bronchoalveolar lavage (BAL) was performed using 0.5mL IX PBS (Mediatech). Lungs were immediately excised, rinsed in phosphate buffered saline, and snap frozen in liquid nitrogen. Sample RNA was amplified and labeled using a custom automated version of the aminoallyl MessageAmp II kit (Ambion). Hybridizations to custom Agilent microarrays were completed as previously described by (Hughes et al. Nat Biotech (2001), 19(4):342-7). Sample amplification, labeling, and microarray processing were completed by the Rosetta Inpharmatics Gene Expression Laboratory.
Bronchoalveolar lavage
Twenty four hours after the last aerosol challenge, mice were deeply anesthetised via i.p. administration of euthanol (120mg/kg i.p.). Animals were tracheostomized and bronchoalveolar lavage
(BAL) was performed using 0.5mL IX PBS (Mediatech). Total cell numbers in the recovered volume (-70-80% recovery) were determined via automated cell count (Celldyn3500). Cytospin slides of BAL cells were prepared using a cytocentrifuge (Cytospin; Shandon, Pittsburgh, PA) and stained with May- Grunwald-Giemsa stain. Differential cell counts were determined by light microscopy from a count of 100 cells. Remaining BAL supernatant was frozen individually in aliquots and stored at -800C.
GPR65 -specific Oligonucleotides
Fourteen antisense oligonucleotides (ASOs) targeting both the 5' and 3' UTR and the coding region of GPR65 were designed using the EDT ASO design tool and applying rule set chimeric 20-mer (http://www.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx) (see Table 1).
ASO sequences were evaluated for their potential off-target activity by aligning each sequence against the Mus musculus transcriptome (Blast). When possible, ASO sequences displaying a max score that is less or equal to 28.2 were selected. Table 1 provides a list of the GPR65-specific ASO that were designed and evaluated in vivo. The ASOs were designed as 5-10-5 LNA gapmers on a phosphorothioate backbone.
Two distinct ASO controls were evaluated (CNTRLl and CNTRL2). ASO were ordered as LNA-phosphorothioate 5-10-5 gapmers and supplied by Integrated DNA technologies (Coralville, Iowa, USA) or Medicorp (Montreal, Quebec, Canada). All oligonucleotides were purified by HPLC and supplied as lyophilized stocks. ASO stocks were reconstituted in sterile D'PBS at a concentration of 4 mg/ml and frozen at -80oC until use.
TABLE 1 GPR65-SPECEFIC OLIGONUCLEOTmES
OLIGO SEQUENCE
GPR65.1 5'-+C*+A*+T*+C*+A*C*T*T*C*T*T*A*G*G*C*+T*+G*+T*+C*+A-3' GPR65.5 5'-+T*+T*+T*+C*+C*T*T*C*T*T*C*G*C*T*T*+G*+C*+A*+G*+A-3'
GPR65.6 5'-+G*+T*+C*+C*+A*G*T*T*G*T*C*T*T*T*A*+T*+T*+C*+C*+A-3'
GPR65.8 5'-+C*+A*+C*+T*+C*G*T*T*T*C*A*T*C*T*T*+T*+C*+C*+A*+C-3'
GPR65.9 5'-+C*+A*+G*+A*+G*G*G*T*A*T*T*T*G*T*C*+A*+T*+A*+G*+C-3'
GPR65.10 5'-+G*+T*+T*+G*+A*G*G*T*T*T*A*T*C*T*G*+C*+C*+A*+T*+T-3' GPR65.12 5'_+G*+A*+T*+C*+C*T*T*C*T*C*T*T*C*T*C*+G*+C*+T*+G*+T-3'
GPR65.13 5'-+C*+T*+T*+G*+G*T*G*T*C*T*A*T*T*G*T*+G*+T*+T*+T*+C-3'
GPR65.14 5'-+G*+C*+C*+T*^*C*A*C*C*T*C*T*T*A*G*+T*+C*+T*+A*+T-3'
GPR65.15 5'_+G*+T*+G*+T*+C*A*T*G*T*T*T*C*C*C*T*+G*+T*+T*+T*+C-3'
GPR65.16 5'-+G*+A*+T*+T*+G*G*A*G*A*T*T*G*G*T*C*+G*+G*+T*+G*+C-3' GPR65.17 5'-+C*+A*+C*+C*+G*C*C*T*C*T*T*G*C*T*T*+G*+C*+C*+C*+T-3'
GPR65.18 5i-+T*+T*+c*+T*+T*c*T*c*T*c*T*T*A*G*G*+G*+T*+T*+T*+c.3.
GPR65.19 5'-+A*+G*+T*+C*+C*C*G*C*G*T*G*C*T*T*C*+T*+T*+A*+G*+G-3'
CNTRL.1 5'-+C*+T*+C*+T*+T*T*C*T*C*T*A*T*C*C*T*+C*+T*+C*+A*+C-3'
CNTRL.2 5'-+C*+C*+T*+T*+C*C*C*T*G*A*A*G*G*T*T*+C*+C*+T*+C*+C-3'
(+=LNA, *=phosphorothioate bond)
GPR65.1 (SEQ ID NO.: 1), GPR65.5 (SEQ ID NO.:2 ), GPR65.6 (SEQ ID NO.: 3), GPR65.8 (SEQ ID NO.:4), GPR65.9 (SEQ ID NO.:5 ), GPR65.10 (SEQ ID NO.: 6), GPR65.12 (SEQ ID NO.:7 ), GPR65.13 (SEQ ID NO.:8 ), GPR65.14 (SEQ ID NO.:9 ), GPR65.15 (SEQ ID NO.:10 ), GPR65.16 (SEQ ID NO.: 1 1 ), GPR65.17 (SEQ ID NO.: 12), GPR65.18 (SEQ ID NO.: 13), GPR65.19 (SEQ ID NO.: 14), CNTRL.l (SEQ ID NO.: 15), and CNTRL.2 (SEQ ID NO.:16 ).
Intratracheal instillation of ASO
ASO were aerosolized directly into the lungs of 10 week old Balb/c males using a microsprayer (PennCentury, Philadelphia, USA). Briefly, mice were anesthetised by intraperitoneal injection of Ketamine / Domitor at 40 / 1 mpk. Anesthetised mice were maintained in a supine position suspended from the upper incisors using a rubber band strapped to an angled rodent workstand (Highland Medical Equipment, Temecula, California, USA). Holding the tongue up against the mandible portion of the jaw to allow direct visualization of the tracheal opening and epiglottis, a Jelco 2OG catheter was gently inserted in the trachea with its opening located a few millimeters away from the carina. Following this, the needle of the microsparyer was inserted in the catheter with the tip of the microspayer needle resting approximately 1 millimeter away from the catheter opening. Rapidly, a 50 μL volume was directly aerosolized in the lungs. To ensure complete distribution of the reagent, mice were maintained in a supine hanging position for 2 minutes following aerosolization. Following this, mice were removed form the platform and injected with antisedan subcutaneous Iy at 1 mpk to reverse the anesthesia. During all manipulations, mice were maintained on a heating blanket to mitigate the possibility of hypothermia upon anesthesia.
In vivo Antisense GPR65 knockdown
For in vivo studies employing antisense oligonucleotide (ASO), OVA-immunized BALB/c control mice were anesthetized, and then subjected to five non-surgical intratracheally administrations of GPR65 ASO (50μg GPR65.16/ 50μL instillation delivered on days 7, 10, 14, 17, 19) or irrelevant control ASO. ASO-instilled mice were subjected to PBS or OVA aerosol challenge as per above on days 17-19. Animals were tracheostomized and BAL harvested, and whole lungs processed for mRNA extraction as indicated below.
IL- 13 elisa
Bronchoalveolar lavage fluid supernatant (50μL of undiluted sample) from each animal was assessed for IL- 13 levels using the Quantikine mouse IL-13 ELISA kit (Cat# M1300CB, R&D systems, Minneapolis, USA) as per the manufacturer's instructions.
RNA extraction. cDNA conversion, and Real-time PCR
Total RNA was extracted from mouse tissues using a total RNA isolation kit (RNeasy kit; Qiagen, Valencia, CA). Samples were treated with DNase on-column (Qiagen, Valencia, CA). RNA isolated from BALF cells and lung tissues was converted into cDNA using the Taqman reverse transcription kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA). 8 μL of cDNA was then amplified using the Taqman Fast universal PCR master mix supplemented with 0.2 units uracil N-glycosylase and reactions ran on a ABI Prism 7900 HT detection system (Applied Biosystems, Foster City, CA). Taqman primers for the internal control (Ppib) and GPR65 (2 independent assays used Mm00433695_ml Mm02619732_sl) were purchase from Applied Biosystems. The PCR reactions were denatured for 2 min at 5O0C followed by 2 min at 95°C and then subjected to 50 cycles of amplification (94°C for 1 s followed by 20 s at 6O0C). GPR65 expression levels were normalized to the endogenous control (Ppib) using the ΔΔCt method and expressed as a proportion relative to the levels observed in vehicle treated animals (100%).
EXAMPLE 1 : GPR65 IS UPREGULATED IN LUNG TISSUE OF MICE EXHIBITING AN ALLERGIC RESPONSE
In an effort to identify genes that are regulated in allergic inflammation gene expression profiling studies were conducted from whole lung tissue from ovalbumin-sensitized mice subjected to an antigenic challenge. This model has been shown to be predictive of clinical response since clinically efficacious therapeutics are effective in this model.
Results of the expression profiling experiment provided in Figure 1 illustrates that several thousand genes are significantly regulated in this model compared to control animals (Figure IA). Consistent with this model being representative of airway inflammation in humans, several genes were upregulated that have been implicated in human airway disease (eg. Mmpl2, IL-13, Chia, Muc5B) (Table 1). Functional annotation of these upregulated genes shows that there is a predominant inflammatory gene signature (Figure IB), hi this profiling experiment, GPR65 was shown to be upregulated 4.9 fold in this model thus implicating GPR65 in the pathophysiology of allergic inflammation. Previous profiling studies in a similar model of allergic airway inflammation have also shown GPR65 to be up-regulated (Zimmerman et al., 2004). This observation implicates GPR65 in the pathophysiology of allergic inflammation, and identifies GPR65 as a new protein target for the development of novel therapeutic agents that are capable of ameliorating allergic airway inflammation.
TABLE 2 LIST OF GENES HIGHLY UP-REGULATED EV THE MOUSE OVA MODEL
1 1 4
EXAMPLE 2: EFFECT OF GPR65 DELETION ON OVA-MDUCED PULMONARY PHENOTYPE
To directly evaluate the functional role GPR65 has in the mouse OVA model, we subjected GPR65 deficient mice (Radu etal, 2006) as well as wild type strain, age and gender matched animals to three consecutive OVA challenges at one day intervals. Total cell and eosinophil numbers in BAL were significantly increased in OVA-challenged versus phosphate buffered saline (PBS)-challenged wild type mice, as detected in bronchoalveolar lavage fluid (BALF). Interestingly, GPR65 deficient mice were protected from developing OVA-induced airway inflammation, as determined by a significant reduction in total cell and eosinophil numbers comparable to that observed with glucocorticoid (Dexamethasone) treatment (Figures 2 A, 2B).
There were no statistically significant differences in BAL macrophage, neutrophil, or lymphocyte numbers. Increases in pro-inflammatory cytokines in the lungs of antigen sensitized and aerosolized animals have been linked to pulmonary inflammation. We therefore measured the changes in BALF EL- 13 levels in these groups of mice. Sensitized wild type mice subjected to OVA displayed a significant increase in detectable BAL EL- 13 levels. A reduction in OVA-induced EL- 13 levels was observed in mice lacking GPR65, correlating with a reduction in pulmonary inflammation (Figure 2C).
EXAMPLE 3 : IDENTIFICATION OF GPR65 -SPECIFIC ANTISENSE OLIGONUCLEITDE
To further confirm the role of GPR65 in allergic inflammation we wanted to test whether attenuation of GPR65 activity with a specific antisense oligonulceotide (ASO) would result in an attenuation of airway inflammation. To identify potent ASOs we tested fourteen distinct GPR65 specific ASO and a control oligo (CNTRLl) in vivo. The ASOs were designed as 5-10-5 LNA gapmers on a phosphorothioate backbone.
Mice were treated with 5 doses of aerosolized ASO or D'PBS over the course of 12 days and sacrificed 24 hours following the fifth dose, on day 13. Overall, the instillation of LNA-based ASO did not result in overt morbidity such as weight loss and mortality with the exception of mice treated with GPR65.1 (1 dead) and GPR65.9 (2 dead).
Cell counts on bronchoalveolar lavage fluid (BALF) to determine whether the instillation of ASO resulted in a cellular inflammatory response. Compared to PBS treated animals, ASO treated animals demonstrated varied cellular responses to treatment with ASO (Figure IA). CNTRL.1 was initially characterized using 2'0Me gapmers on a phosphorothiate backbone and was shown to be relatively innocuous following its administration to mice. Unexpectedly, treatment with CNTRL.1 as a LNA gapmer resulted in a potent cellular inflammation response when compared to the PBS control group (Figure 3A).
Differential analysis of the cellular components of the BALF of CNTRL.1 treated animals demonstrated a dramatic increase in monocytes, neutrophils, and lymphocytes when compared to PBS treated animals (Figure 3B). GPR65 has been reported to be expressed in hematopoietic lineages such as macrophages and neutrophils. Consistently, relative GPR65 expression levels in BALF cells were markedly elevated in several ASO treated animals including those that received CNTRL.1 when compared to levels observed in PBS-treated animals. Nonetheless, we identified two GPR65-specific ASO, GPR65.16 and GPR65.17, that demonstrated >60% knockdown in BALF cells (Figure 4A-B) and lungs (Figure 4C) when compared to PBS-treated animals.
BALF cell counts from mice treated with GPR65.16 and GPR65.17 demonstrated moderate increases in monocytes and neutrophils compared to PBS-treated animals (Figure 3B). GPR65.16 was selected as a lead ASO to determine the impact of GPR65 gene expression knockdown on the response to ovalbumin challenge in OVA-sensitized animals.
EXAMPLE 4: EFFECT OF ASO-MEDIATED GPR65 KNOCKDOWN ON OVA-INDUCED PULMONARY PHENOTYPE
To assess if modulation of GPR65 in a therapeutic paradigm could alter antigen-induced pulmonary inflammation, antisense oligonucleotide (ASO) technology was employed to selectively knockdown lung GPR65 mRNA expression in sensitized and OVA-aerosolized wild type BALB/c mice. OVA-sensitized mice were intratracheally instilled with GPR65.16, control ASO, or vehicle (Figure 5A). In contrast to control ASO or vehicle treatment, GPR65.16 intratracheal administration significantly and selectively reduced GPR65 mRNA expression in mouse lung (Figure 5B). Comparable to observations
in GPR65 knockout animals, GPR65 mRNA knockdown was associated with a significant attenuation of OVA-dependent total, eosinophil airway inflammation and BAL IL- 13 (Figure 6A-C). These findings demonstrate that GPR65 is induced under pulmonary inflammation conditions, and nucleic acid-mediated knockdown of GPR65 in a therapeutic paradigm can ameliorate lung airway inflammation. Importantly this data show that lung specific inhibition of GPR65 is sufficient to ameliorate airway inflammation Overall the data presented in the disclosure identifies GPR65 as a protein target of allergic inflammation. .
REFERENCES
Chen H., Sun J., Buckley S., Chen C, Warburton D., Wang X.F., and Shi W. (2005). Abnormal mouse lung alveolarization caused by Smad3 deficiency is a developmental antecedent of centrilobular emphysema. Am. J. Physiol. Lung Cell. MoI. Physiol. 288: L683-91.
Holma, B., and Hegg, P.O. (1989). pH- and protein-dependent buffer capacity and viscosity of respiratory mucus. Their interrelationships and influence on health. Sci. Total Environ. 84: 71-82.
Hunt, J.F., Fang, K., Malik, R., Snyder, A., Malhotra, N., Platts-Mills, T.A.E., and Gaston, B. (2000). Endogenous Airway Acidification: Implications for asthma pathophysiology. Am. J. Respir. Crit. Care Med. 161 : 694-699.
Im, D.-S., Heise, C.E., Nguyen, T., O'Dowd, B. F., and Lynch, K.R. (2001). Identification of amolecular target of psychosine and its role in globoid cell formation. J. Cell Biol. 153: 429-434.
Ishii, S., Kihara, Y., and Shimizu, T. (2005). Identification of T cell death-associated gene 8 (TD AG8) as a novel acid sensing G-protein-coupled receptor. J. Biol. Chem. 280: 9083-9087.
Jiang, S.P., and Huang, L.W. (2005). Role of gastroesophageal reflux disease in asthmatic patients. Eur. Rev. Med. Pharmacol. Sci. 9: 151-160.
Kostikas, K., Papatheodorou, G., Ganas, K., Psathakis, K., Panagou, P., and Loukides, S. (2002). pH in expired brieath condensate of patients with inflammatory airway diseases. Am. J. Repir. Crit. Care Med. 165: 1364-1370.
Luk, C.K., and Dulfano, M.J. (1983). Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants. Clin. Sci. 64: 449-451.
Mandeville L, Aubin J., LeBlanc M., Lalancette-Hebert M., Janelle M.F., Tremblay G. M., and Jeannotte L. (2006). Impact of the loss of Hoxa5 function on lung alveogenesis. Am. J. Pathol. 169: 1312-27.
Mathew, J.L., Sing, M., and Mittal, S.K. (2004). Gastro-oesophageal reflux and bronchial asthma: current status and future directions. Postgrad. Med. J. 80: 701-705.
Radu, C.G., Nijagal, A., McLaughlin, J., Wang, L., And Witte, O.N. (2005). Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells. PNAS. 102: 1632-1637.
Radu, C. G., Cheng, D., Nijagal, A., Riedinger, M., McLaughlin, J., Yang, L.V., Johnson, J., and Witte, O.N. (2006). Normal immune development and glucocorticoid-induced thymocyte apoptosis in mice deficient for the T-cell death-associated gene 8 receptor. MoI. Cell. Biol. 26: 668-677.
Suga T., Kurabayashi M., Sando Y., Ohyama Y., Maeno T., Maeno Y., Aizawa H., Matsumura Y., Kuwaki T., Kuro-0 M., Nabeshima Y., and Nagai R.. (2000). Disruption of the klotho gene causes pulmonary emphysema in mice. Defect in maintenance of pulmonary integrity during postnatal life. Am. J. Respir. Cell MoI. Biol. 22: 26-33.
Svennerholm, L., Vanier, M.-T., and Mansson, J.-E. (1980). Krabbe disease: a galactosylsphingosine (psychosine) lipidosis. J. Lipid Res. 21: 53-64.
Tomura, H., Mogi, C, Sato, K., and Okajima, F. (2005). Proton-sensing and lysolipid-sensitive G- protein-coupled receptors: A novel type of multi-functional receptors. Cell. Signal. 17: 1466-1476.
Wang, J.Q., Kon, J., Mogi, C, Tobo, M., Damirin, A., Sato, K., Komachi, M., Malchinkhuu, E., Murata, N., Kimura, T., Kuwabara, A., Wakamatsu, K., Koizumi, H., Uede, T., Tsujimoto, G., Kurose, H., Sato, T., Harada, A., Misawa, N., Tomura, H., and Okajima, F. (2004). TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J. Biol. Chem. 279: 45626-45633.
Weiss, K.B., and Sullivan, S.D. (2001). The health economics of asthma and rhinitis: I. Assessing the economic impact. J. Allergy Clin. Immunol. 107: 3-8.
Wemmie, J.A., Price, M.P., and Welsh, MJ. (2006). Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci. 29: 578-586.
Zimmermann N., Mishra, A., King, N.E., Fulkerson, P. C, Doepker, M.P., Nikolaidis, N.M., Kindinger, L. E., Moulton, E.A., Aronow, BJ. , and Rothenberg, M.E. (2004). Transcript signatures in experimental asthma: Identification of STAT6-dependent and -independent pathways. J. Immunol. 172: 1815-1824.
Claims
1. A method for ameliorating inflammation is a subject comprising administering a therapeutic agent which attenuates GPR65 expression or activity.
2. The method according to claim 1 wherein the therapeutic agent comprises an agent which attenuates GPR65 expression.
3. The method of claim 2 wherein the agent is a GPR65-specific oligonucleotide.
4. The method of claim 3 wherein the oligonucleotide is GPR65.16 (SEQ ID NO: 11).
5. The method of claim 1 wherein the therapeutic agent is delivered directly to the subject's airway surfaces.
6. The method of claim 5 wherein the agent is delivered to the airway surfaces by inhalation.
7. The method of claim 2 wherein the therapeutic agent is administered in combination with an agent selected from the group consisting of a corticosteroid, an anti-cholinergic agent, a leukotriene receptor antagonist, an inhibitor of leukotriene synthesis and a beta-adrenergic receptor agonist.
8. The method according to claim 2 wherein the subject is an asthmatic or an individual afflicted with chronic obstructive pulmonary disease, bronchiectasis or cystic fibrosis.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017508457A (en) * | 2014-02-27 | 2017-03-30 | ザ・ブロード・インスティテュート・インコーポレイテッド | T cell balance gene expression, composition and method of use thereof |
WO2017053681A1 (en) * | 2015-09-24 | 2017-03-30 | Wisconsin Alumni Research Foundation | Methods of expanding hematopoietic stem cells, compositions, and methods of use thereof |
WO2021245427A1 (en) * | 2020-06-05 | 2021-12-09 | Pathios Therapeutics Limited | N-phenylaminocarbonyl pyridino-, pyrimidino and benzo-tropanes as modulators of gpr65 |
WO2021245426A1 (en) * | 2020-06-05 | 2021-12-09 | Pathios Therapeutics Limited | N-(phenylaminocarbonyl) tetrahydro-isoquinolines and related compounds as modulators of gpr65 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996027603A1 (en) * | 1995-03-03 | 1996-09-12 | Millennium Pharmaceuticals, Inc. | Compositions and methods for the treatment and diagnosis of immune disorders |
WO1996039442A1 (en) * | 1995-06-06 | 1996-12-12 | Human Genome Sciences, Inc. | G-protein receptor htnad29 |
WO2003065045A2 (en) * | 2002-02-01 | 2003-08-07 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with the gpr65 receptor |
-
2008
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996027603A1 (en) * | 1995-03-03 | 1996-09-12 | Millennium Pharmaceuticals, Inc. | Compositions and methods for the treatment and diagnosis of immune disorders |
WO1996039442A1 (en) * | 1995-06-06 | 1996-12-12 | Human Genome Sciences, Inc. | G-protein receptor htnad29 |
WO2003065045A2 (en) * | 2002-02-01 | 2003-08-07 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with the gpr65 receptor |
Non-Patent Citations (2)
Title |
---|
COURTNEY A.U. ET AL.: "Childhood Asthma: Treatment Update", AM. FAM. PHYSICIAN, vol. 71, 15 May 2005 (2005-05-15), pages 1959 - 1968 * |
RADENNE F. ET AL.: "L'asthem Du Sujet Age (Asthma In The Elderly)", REV. MAL. RESPIR., vol. 20, February 2003 (2003-02-01), pages 95 - 103 * |
Cited By (7)
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JP2017508457A (en) * | 2014-02-27 | 2017-03-30 | ザ・ブロード・インスティテュート・インコーポレイテッド | T cell balance gene expression, composition and method of use thereof |
US11209440B2 (en) | 2014-02-27 | 2021-12-28 | The Broad Institute, Inc. | T cell balance gene expression, compositions of matters and methods of use thereof |
WO2017053681A1 (en) * | 2015-09-24 | 2017-03-30 | Wisconsin Alumni Research Foundation | Methods of expanding hematopoietic stem cells, compositions, and methods of use thereof |
US10584315B2 (en) | 2015-09-24 | 2020-03-10 | Wisconsin Alumni Research Foundation | Methods of expanding hematopoietic stem cells, compositions, and methods of use thereof |
US11053475B2 (en) | 2015-09-24 | 2021-07-06 | Wisconsin Alumni Research Foundation | Methods of expanding hematopoietic stem cells, compositions, and methods of use thereof |
WO2021245427A1 (en) * | 2020-06-05 | 2021-12-09 | Pathios Therapeutics Limited | N-phenylaminocarbonyl pyridino-, pyrimidino and benzo-tropanes as modulators of gpr65 |
WO2021245426A1 (en) * | 2020-06-05 | 2021-12-09 | Pathios Therapeutics Limited | N-(phenylaminocarbonyl) tetrahydro-isoquinolines and related compounds as modulators of gpr65 |
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