Alpha-1 Antitrypsin Antisense Oligonucleotide Modulates Protease-Antiprotease Imbalance without Further Aggravating Smoke-induced Lung Injury

Alpha-1 antitrypsin (AAT) is a serum protease inhibitor that prevents lung injury from protease production during cigarette smoking but causes severe liver disease once mutated. A custom AAT antisense oligonucleotide (ASO) was found to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knock-down of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups for each of the injury models, smoking (inhale for 3 months at 150mg/m3) and smoke-flu (inhale for 2 weeks and intranasal influenza virus). The ASO and control (No-ASO) were injected subcutaneously at 10ml/kg of body weight, starting with smoking or four days prior to influenza infection weekly at 50mg/kg. ASO treatment during a 3 month smoke exposure significantly increased the expression of Cela1 mRNA and decreased the serum and lung AAT expression. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly affected by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure alone. With the smoke-flu model, similarly the major differences were found between smoke-flu and room air control, with no additional effect with ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment was not robust enough to lead to lung injury. The result also suggest that the AAT ASO approach for treating liver disease is relatively safe at the specified dose as it did not lead to detrimental outcomes in the lung. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Introduction Alpha-1 antitrypsin (AAT) is a serum protease inhibitor that targets the proteases produced 48 during injury that, in the lung, are ultimately responsible for structural destruction. The protease-49 antiprotease paradigm has long been recognized and earlier research has identified neutrophil 50 elastase as one of the most potent proteases that mediates destruction in the lung [1, 2]. Alpha-1 51 antitrypsin inhibits neutrophil elastase activity [3] and prevents the degradation of elastin [4], 52 which helps to maintain the integrity of extra-cellular matrix in the lung. Imbalances between 53 tissue damaging proteases and their inhibitors such as AAT leads to lung destruction and ultimately 54 the development of chronic obstructive pulmonary disease (COPD) [5].
79 With the custom designed mouse AAT ASO, we can understand if AAT ASO inhibits the 80 expression of AAT in the lung similar to that in the liver. In addition, we can determine if AAT 81 modulation affects the outcome of smoking-induced lung injury and acute COPD exacerbation in 82 assessing the overall feasibility for using AAT ASO for treatment of liver disease in patients with 83 a pre-disposition to proteolytic lung destruction. 86 Mouse AAT ASO and control ASO solution were prepared by Ionis Pharmaceutical in 87 collaboration with Keith Blomenkamp and Dr. Jeff Teckman in Saint Louis University with the 88 method described previously [11]. Two filter flasks were aliquoted upon arrival at 5mg/ml 89 concentration and administered via subcutaneous injection with a dose of 10ml/kg body weight 90 once a week, to make up a final weekly concentration of 50mg/kg. 143 The AAT protein expression in the lung was assessed with western blotting (alpha-1 144 antitrypsin antibody, #16382-1-AP, rabbit polyclonal, Proteintech, Rosemont, IL). Serum level of 145 AAT was evaluated with Mouse Alpha 1 Antitrypsin ELISA Kit (Product: ab205088, Let Number: 146 GR3272327). PCR probe for Cela1 (chymotrypsin-like elastase family, member 1) was purchased 147 from ThermoFisher (Applied Biosystems, Foster City, CA; Assay ID: Mm00712898_m1; Cat 148 #4331182). Cela1 was implicated with mouse lung development and human AATD [12,20].

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Statistical analysis 150 Simple pairwise comparisons were performed with Students t-test or Wilcoxon-test (non-151 parametric test for the survival curve) assuming unequal variance. Additional tests were performed 152 after grouping both ASO control and treated mice, for example, overall flu (FN&FA) vs control 153 (AN&AA), when ASO treatment did not make any significant difference. All error bars indicate 154 mean ± SEM. 158 Endogenous AAT protein expression in the serum (Figure 2A, P<0.005; N=8~10) and the 159 lung ( Figure 2B, P<0.001; N=6, from 3 western blots, Figure S2) and were significantly reduced 160 in mice injected with ASO. The injection also significantly increased the Cela1 expression for the 161 air-exposed mice (AA vs AN, P=0.004) but not for the smoke-exposed mice (SA vs SN, P=0.8; 162 Figure 2C). 165 Lung function testing results measured by flexiVent showed an overall significantly 166 increased inspiratory capacity for mice exposed to smoke (SN&SA vs AN&AA, P=0.01, Figure  167 3A). ASO treatment did not contribute to any changes in inspiratory capacity, resistance, or 168 compliance regardless of the exposure conditions ( Figure 3B).

Figure Legends
317 Figure 1. Experimental design. Alpha-1 antitrypsin antisense oligonucleotide treatment 318 was tested on two sets of lung injury models, smoke exposure for chronic obstructive pulmonary 319 disease (COPD) and additional flu infection for COPD exacerbation. Each experiment was 320 randomly divided into 4 groups, first letter indicating the lung injury model, second letter whether 321 treated with ASO.
322 Figure 2. Alpha-1 antitrypsin (AAT) expression was significantly decreased by the 323 antisense oligonucleotide treatment. Although the treatment lead to decreased AAT in the serum 324 (A; P<0.005) and the lung (B; P<0.001) and increased Cela1 elastase expression (C; P=0.014), 325 Cela1 was highly expressed in smoke exposed mice and did not increase further by oligonucleotide 326 treatment (P=0.80).  Figure S2. Alpha-1 antitrypsin antisense oligonucleotide treatment resulted in decreased 343 AAT expression in the lung, one representative western blot (N=2) contributing to Figure 2B.