AAV9 Gene Replacement Therapy for Respiratory Insufficiency in Very-long Chain Acyl-CoA Dehydrogenase Deficiency

Summary

Very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an autosomal recessive disorder of fatty acid oxidation. Fatty acids are a major source of energy during catabolic stress, so the abscense of VLCAD can result in a metabolic crises and respiratory insufficiency. The etiology of this respiratory insufficiency is unclear. Thus, our aims were: 1) to characterize respiratory pathophysiology in VLCADD mice (VLCAD^−/−), and 2) to determine if AAV9-mediated gene therapy improves respiratory function. For the first aim, VLCAD^−/− and wildtype (WT) mice underwent an exercise/fast “stress protocol” and awake spontaneous breathing was evaluated using whole-body plethysmography (WBP) both at baseline and during a hypercapnic respiratory challenge (FiO₂: 0.21; FiCO₂: 0.07; nitrogen balance). During hypercapnia, VLCAD^−/− mice had a significantly lower frequency, tidal volume, minute ventilation, and peak inspiratory and expiratory flow, all of which indicate respiratory insufficiency. Histologically, the cardiac and respiratory muscles of stressed VLCAD^−/− animals had an accumulation of intramyocellular lipids. For the second aim, a single systemic injection of AAV9-VLCAD gene therapy improved this respiratory pathology by normalizing breathing frequency and enhancing peak inspiratory flow. In addition, following gene therapy, there was a moderate reduction of lipid accumulation in the respiratory muscles. Further, VLCAD protein expression was robust in cardiac and respiratory muscle. This was confirmed by immuno-staining with anti-human VLCAD antibody. In summary, stress with exercise and fasting induces respiratory insufficiency in VLCAD^−/− mice and a single injection with AAV9-VLCAD gene therapy ameliorates breathing.

Synopsis:

Prolonged fasting and exercise in VLCAD knock out mice results in significant respiratory depression; and a single injection with the AAV9-VLCAD gene therapy enhances respiratory output.

INTRODUCTION

Very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disorder caused by mutation in the ACADVL (acyl-coA dehydrogenase, very long chain) gene that encodes VLCAD. The VLCAD enzyme catalyzes the first oxidation step of the long chain fatty acid oxidation cycle, making it vital for the generation of energy from fatty acids in organs and tissue with high metabolic demands. During periods of increased energy demand, such as prolonged fasting or exercise, VLCADD results in rhabdomyolysis, cardiac deficits, and hypoglycemia (Tong et al 2006; Giuliani et al 2013; Yamamoto et al 2013). Hypoglycemia leads to mobilization of free fatty acids which enter the mitochondria via the carnitine cycle for oxidation (Wanders et al 1999). When VLCAD is present, fatty acids in the acyl Co-A form are oxidized to acetyl-CoA. This is converted into ketones that supply energy when glucose levels are low. However, VLCAD deficiency prevents ketone formation and results in the accumulation of fatty acid intermediates. These have toxic effects on the liver, heart, and skeletal muscle, and produces metabolic acidosis (Yamada and Taketani 2018).

VLCADD has a heterogenous clinical picture and is classified into three groups: 1) an early onset severe form that occurs in infancy and results in cardiomyopathy, multi-organ failure, and sudden death 2) a childhood onset intermediate form which presents following periods of illness, fasting, and/or poor feeding, and 3) a later onset form that presents in adolescence or adulthood and results in rhabdomyolysis, myoglobinuria, and exercise induced muscle intolerance (Vianey-Saban et al 1998; Giuliani et al 2013). Symptoms usually present after periods of exercise, illness, or cold exposure when there is an increased energy demand. The impact of VLCADD on respiratory pathology is unclear. However, several case reports have described VLCADD patients who developed respiratory failure after a period of fasting and exercise. In addition, respiratory function assessments following recovery from respiratory failure revealed residual muscle weakness (Pons et al 2000; Tong et al 2006; Giuliani et al 2013; Merritt et al 2014). Thus, the degree of respiratory involvement in these three phenotypes has not been closely examined.. It is unclear whether respiratory insufficiency is due to an overall decrease in energy production or a direct consequence of lipid accumulation and muscle respiratory cycle pathology.

To better understand the mechanisms behind VLCADD respiratory cycle deficits, we characterized the myopathy and pathophysiology of respiratory insufficiency in exercised and fasted VLCADD mice (VLCAD^−/−). VLCAD^−/− mice are an established model for the study of VLCADD because they exhibit cardiomyopathy and have metabolic derangements after stress with fasting and cold (Exil et al 2006; Cox et al 2009; Keeler et al 2012; Xiong et al 2014). However, no study has described the respiratory phenotype in a clinically relevant fast/exercise stress model. This characterization is necessary for the development of therapies that adequately targets respiratory insufficiency in VLCADD. Our second aim was to assess the impact of a single intravenous injection of AAV9-mediated VLCAD gene replacement therapy on respiratory dysfunction in exercise and fasted VLCAD^−/− mice. Our group has previously demonstrated that AAV9 gene therapy significantly improves the biochemical and phenotypical pathology in fasting and cold stress in these VLCAD^−/− (Keeler et al 2012) and our goal here was to see whether it also corrects the respiratory dysfunction.

MATERIALS AND METHODS

Animals.

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts Medical School. VLCAD^−/− mice were generated by breeding VLCAD^−/+ animals creating VLCAD^−/− (thereafter VLCAD^−/−) and VLCAD^+/+ (hereafter WT) controls. See Supplement for details of animals used.

Experimental design.

VLCAD^−/− and WT mice were exercised and fasted to simulate clinically relevant physiological stress. VLCAD^−/− and WT mice, both female and male, (~10 to 12 weeks of age) were divided into 6 groups: Groups 1–2 with no stress control VLCAD^−/− (n=8) and WT mice (n=8), Groups 3–4 with stressed (fasting and exercise) VLCAD^−/− (n=12) and WT mice (n=12), Group 5 with stressed VLCAD^−/− mice (n=8), receiving dextrose solution (immediately following exercise and fasting and prior to respiratory measures, and Group 6 with stressed VLCAD^−/− mice (n=8) treated with (rAAV9)-VLCAD intravenously via the tail vein 4 weeks prior to the start of fasting-exercise challenge.

Exercise protocol:

Using a custom-designed treadmill, all mice in the “stress” groups ran at a speed of 3.5m/min for two-hours, three times a week. Mice were exercised on the first week and on the last week during the three-week period. On the last day of the three week experiment, stressed mice were fasted with access to water for 18 to 24 hours prior to blood glucose measures, breathing assessments, and measurements of pulmonary mechanics. Blood glucose levels were measured using Nova Max Glucose Monitor and Strips (Nova Biomedical, Waltham, MA).

Dextrose administration:

An intravenous (i.v.) infusion of a 10% dextrose solution is first line treatment for an acute metabolic crises in VLCADD patients (Arnold et al 2009). Therefore, in a subgroup of mice, we injected a 10% dextrose solution i.v. at a dose of 0.5 mg/g.

rAAV vectors:

rAAV9 vectors were generated to express human VLCAD (by delivering the hACADVL, hereafter referred to as VLCAD) under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter. rAAV vectors were produced, purified, and titered as previously described (Keeler et al 2012). Mice received a single i.v. injection of 1 × 10¹² vector genomes (vg) at a volume of 140μL.

Genomic DNA extraction and quantitative-PCR.

DNA was extracted and quantified as previously described (Keeler et al 2012; Keeler et al 2018). See Supplement for details.

Histology:

Immunohistochemistry, Oil Red O histochemical staining, and transmission electron microscopy were performed on harvested VLCAD and WT tissue. See Supplement for details.

Pulmonary Physiology.

These studies determined the impact of VLCADD on ventilation. Ventilation was quantified using whole-body plethysmography in unrestrained, unanesthetized mice as previously described (Stoica et al 2017; Keeler et al 2018). See Supplement for details.

Measures of Airway Resistance:

Pulmonary mechanics were performed at the study end point using forced oscillometry (FlexiVent system, SCIREQ, Montreal, Ca) at baseline and in response to incremental doses of methacholine as previously described (Keeler et al 2017). See Supplement for details.

Statistics and Quantification.

All statistics were carried out using Prism 8 Software (GraphPad Software, San Diego, CA). All data are presented as means ± SEM. Significant differences were determined by either two way ANOVA with post-hoc analysis using Bonferroni’s multiple comparison test (for respiratory physiology measures) or unpaired Student’s t-test (for glucose measurements and fiber typing analysis). Differences were considered significant if p < 0.05.

RESULTS

I. Assessment of the respiratory phenotype in a mouse model of VLCAD deficiency

Fast and exercise “stress” induces the VLCADD phenotype of abnormal blood glucose.

We measured blood glucose levels to assess whether the VLCAD^−/− mice developed hypoglycemia following the fast and exercise stress protocol. Under nonstressed conditions, VLCAD^−/− and littermate WT controls show no significant differences in blood glucose levels (178.8±76.2 and 223.3±74.2, respectively, ns) (Fig SI). However following the fasting/exercise stress, VLCAD^−/− mice developed notably significant hypoglycemia as compared to WT controls (77.8±17.4 and 128.7± 23.8, respectively, p=0.0003) (Fig. S1).

VLCAD deficiency resulted in muscle lipidosis (lipid accumulation) and disruption of muscle architecture following fasting and exercise.

To assess the level of lipid accumulation and muscle integrity in respiratory muscles, we performed electron microscopy (EM) on exercised and fasted VLCAD^−/− animal diaphragms and intercostal muscles. Drastic intramuscular lipid droplet accumulation was evident in osmium-philic muscle fiber isotype I in the diaphragm and intercostal muscles in stressed VLCAD^−/− mice compared to those in stressed WT mice (Fig. 1A - C). Furthermore, ultrastructural observations of diaphragm and intercostal muscles in these stressed animals revealed areas of disorganization in muscle fiber morphology, accumulation of lipid droplets, and abnormal and disorganized mitochondrial morphology in VLCAD^−/− mice, but not in WT mice (Fig. 1D – J). To further assess respiratory muscle architecture, we performed immunohistochemical staining for myofiber isotyping but found no significant muscle isotype differences in the intercostal muscles and diaphragm of the VLCAD^−/− and WT mice (Fig. S2).

Figure 1. VLCAD deficiency results in lipid accumulation in respiratory muscles.

Representative micrographs of 1.5 μm thick plastic sections showing osmium-philic slow-twitch oxidative aerobic (dark gray) muscle fibres (myoisotype I) and lightly stained fast-twitch glycolitic muscle fibres (myoisotype II) in the parasternal intercostal muscle of a challenged WT mouse (A). Cross sectional (B) and longitudinal section (C) showing accumulations of osmium-philic lipid droplets (black dots) within the slow-twitch oxidative fibres of the parasternal intercostal muscle in a challenged VLCAD^−/− mouse. Scale bar = 10 μm (A and B), and 75 μm (B). Ultrastructure of respiratory muscles. Transmission electron micrographs showing neat organization of diaphragm (D) and intercostal (F) muscles morphology in a stressed WT mouse. In stressed VLCAD^−/− mice, there are areas of disorganized muscle fiber morphology (circled) (E) accompanied by the accumulation of swollen mitochondria (arrows) and enlarged lipid droplets (asterisks) (G – J) as compared to stressed WT mice. Scale bar = 10 μm (D, E), 1 μm (H), 2 μm (F, G) and 0.2 μm (I, J).

VLCAD deficiency results in significant respiratory depression following fasting and exercise.

To determine if the VLCAD^−/− mouse model had respiratory insufficiency, we performed whole body plethsmography (WBP) measurements in awake, spontaneously breathing mice. There were no significant changes in baseline breathing in VLCAD^−/− and WT mice after stress with fasting and exercise. However, there was a significant difference in respiratory function in response to a hypercapnic respiratory challenge: VLCAD^−/− mice had a significantly lower tidal volume, minute ventilation, peak expiratory flow, and peak inspiratory flow (p<0.05). Moreover, a decrease in breaths per minute was observed in both VLCAD^−/− mice and WT mice compared to respective mice that did not fast and exercise (p<0.05) (Fig. 2A). However, forced oscillometry measurements did not show any significant differences in pulmonary resistance or compliance of stressed VLCAD^−/− mice compared to WT mice (Fig. S3).

Figure 2. WBP measurements of awake unrestrained spontaneous breathing.

Following a respiratory challenge with hypercapnia (7% CO₂, 21% O₂, nitrogen balance), VLCAD^−/− (KO) mice have significantly lower tidal volumes, minute ventilation, peak inspiratory flow, and peak expiratory flow (p<0.05) compared to WT mice. In both systemic AAV9 treatment and dextrose injection groups, there is increased frequency of breathing comparable to that of animals that did not undergo exercise and fasting challenge, and AAV9 treatment improves peak inspiratory flow. However, neither treatment improved tidal volume, minute ventilation or peak expiratory flow. Error bars ± SD; *p<0.05 compared to VLCAD^−/− no challenge, # p<0.05 compared to WT no challenge, ^p<0.05 compared to WT challenge, &p<0.05 compared to VLCAD^−/− AAV9 – treated, +p<0.05 compared to VLCAD^−/− dextrose treated.

II. AAV9-VLCAD gene therapy

AAV9-mediated delivery of the gene for VLCAD ameliorated respiratory insufficiency and reduced lipid accumulation in fast and exercise stressed VLCAD^−/− mice.

To determine if AAV gene therapy could correct the respiratory insufficiency observed in VLCAD^−/− mice, we injected VLCAD^−/− animals with 1×10¹² vg AAV9-VLCAD systemically one month prior to the exercise and fast stress protocol. VLCAD^−/− mice had a significant increase in frequency of breathing compared to untreated animals and were not significantly different from non-stressed VLCAD^−/− or WT animals. Furthermore, the peak inspiratory flow in the AAV9-treated VLCAD^−/− challenged mice was not significantly different from that of the WT challenged group. Peak inspiratory flow is a reflection of respiratory muscle strength. However, the AAV9-VLCAD treated mice continued to have a decreased tidal volume, minute ventilation, and peak expiratory flow compared to non-stressed VLCAD^−/− mice animals (Fig. 2B). When we compared AAV gene therapy to administration of i.v. dextrose (the current rescue therapy for a metabolic crises in VLCADD patients), we found a similar increase in breaths per minute, but no improvement in the peak inspiratory flow within the dextrose group. AAV9-VLCAD treatment also resulted in significant clearance of lipid accumulates (as seen by Oil Red O staining) in cardiac muscle, as well as moderate reduction of lipid accumulation within the respiratory muscles and tongue (Fig. 3).

Figure 3. Oil Red O staining in cardiac muscle, diaphragm, intercostal, and tongue muscles.

Accumulation of intramyocellular lipid droplets is evident in the tongue, diaphragm, intercostal, and cardiac muscles of stressed and non-stressed VLCAD^−/− animals. This accumulation is not observed in WT animals. Significant clearance of lipid accumulates in rAAV9-ACADVL treated stressed VLCAD^−/− mice is evident in cardiac muscle, although reduction of lipid accumulation within the respiratory muscles is less evident. Scale bars = 20 μm (heart), 50 μm (diaphragm and intercostal muscles), and 65 μm (tongue).

Vector Genome Copies and VLCAD immunohistochemistry

To determine the transduction of AAV9-VLCAD in the heart and muscles of respiration, vector genome copies were measured in the heart, diaphragm, intercostals, and tongue. We observed robust transduction in all targeted tissues (Fig. 4A). Outcomes for heart transduction were consistent with our previous findings of AAV9-ACADVL distribution (Keeler, 2012). This study confirms that the vector is also able to transduce to the muscles of respiration. Furthermore, immunohistochemical staining with anti VLCAD antibody revealed that high protein levels were expressed in the heart and diaphragm muscles (Fig. 4B).

Figure 4. Vector genome concentration in rAAV9-ACADVL treated mouse tissues.

(A). Quantitative-PCR for vector genome shows robust transduction in the heart followed by the diagram muscle, with less vector genomes evident in the intercostal and tongue muscles. Error bars ± SEM. Immunohistochemical verification of human VLCAD protein translation in rAAV9-ACADVL treated mouse cardiac muscle, respiratory muscles and tongue (B). Use of anti-human VLCAD antibody shows no cross-reactivity with mouse VLCAD protein. Consistent with vector genome concentration, immunostaining reveals high human protein levels expressed in the heart and diaphragm, and lower expression levels in the intercostal and tongue muscles. Scale bar = 100 μm.

DISCUSSION

This study is the first to characterize the respiratory phenotype of the VLCAD-deficient mouse model. Our most significant findings are that VLCAD deficiency in this model results in respiratory depression following exercise and fasting during a hypercapnic challenge, and that a single systemic injection of AAV9-VLCAD gene therapy ameliorates the respiratory dysfunction.

Impact of VLCADD on Breathing

Although not considered a classical symptom of VLCADD, respiratory insufficiency has been described in patients with VLCADD (Pons et al 2000; Tong et al 2006; Giuliani et al 2013). However, the exact etiology of these breathing deficits were unclear. One patient had progressive muscle weakness during a fasted state resulting in respiratory failure and the need for intubation and mechanical ventilation. After improvement of acute symptoms, chronic subclinical respiratory muscle weakness was evident from pulmonary function testing (Tong et al 2006).

Respiratory insufficiency has never been studied in VLCAD^−/− mouse models despite growing evidence of its importance in VLCADD. Respiratory mechanics were examined in this study to assess the impact of VLCAD deficiency on pulmonary resistance and compliance. Specifically, this provides information on airway resistance and airway mechanics. Previous studies using LCAD knock out mice showed reduced pulmonary compliance in comparison to WT animals (Goetzman et al 2014). It has been postulated that LCAD impacts surfactant production (Goetzman et al 2014). Overall, we did not find a difference in pulmonary mechanics between VLCAD^−/− and WT mice – either at baseline or during stress. To our knowledge, no pathology in the airways and lung parenchyma has been documented in humans with VLCADD. Thus, the normal pulmonary mechanics in VLCAD^−/− mice led us to hypothesize that pulmonary insufficiency is caused by a combination of energy deprivation and respiratory muscle involvement.

Using WBP, we found a distinct phenotype of respiratory insufficiency in awake spontaneously breathingVLCAD^−/− mice. This insufficiency was evident in the fast and exercise stressed mice during a hypercapnic challenge. Hypercapnia sends a chemical signal to stimulate breathing in order to blow off excess CO₂. Defects were noted in the frequency of breaths, tidal volume, and peak inspiratory and expiratory flows. However, the respiratory rate could be corrected to WT levels by providing an alternative energy source, in this case therapeutic treatment with dextrose. The peak inspiratory flow and peak expiratory flow reflect inspiratory and expiratory muscle strength, primarily in the diaphragm and intercostals (ElMallah et al 2015; Stoica et al 2017; Keeler et al 2018). Interestingly, the peak inspiratory and expiratory flows were not improved following dextrose administration. Thus, the normalization of the respiratory rate is most likely because of energy rescue, whereas the lack of response in the peak inspiratory and expiratory flows and tidal volumes is most likely a reflection of persistent muscular pathology.

Impact of VLCADD on Muscle

In light of our our WBP findings on respiratory muscle weakness, we investigated the extent of lipid accumulation in the muscles of respiration. Research in both human and rodent models describes lipotoxicity in skeletal muscle that results in decreased muscle function (Choi et al 2016) (Andrich et al 2018). These studies found a negative correlation between intramuscular lipid content and the muscle function (Choi et al 2016) (Andrich et al 2018). In our study, light and transmission electron microscopy examinations clearly demonstrated increased accumulations of enlarged intracellular lipid bodies in the diaphragm and intercostal respiratory muscles in fasted and exercised VLCAD^−/−mice. Greater total lipid droplet area and greater number of droplets reduces power generation in oxidative myofibers type I from obese human adults (Choi et al 2016) and in an obese murine model (Andrich et al 2018). In agreement with published studies, we found increased intramuscular lipid accumulation in respiratory muscles.

We also observed increased lipid accumulation in cardiac muscle and in glycolytic type IIb fibers of the genioglossus (tongue) muscle in fasted and exercised VLCADD mice compared to WT controls. The tongue was included in our study because VLCADD patients may have bulbar weakness (Vengalil et al 2017). This is one of the most important bulbar muscles and plays an important role in breathing. Specifically, contraction of extrinsic tongue muscles, particularly the genioglossus, can dilate and/or stiffen the pharyngeal lumen, thereby minimizing airway narrowing and/or collapse in the face of negative inspiratory pressures (Fuller et al 1998; Fuller et al 1999).

Gene Therapy for FAO Defects

Gene therapy is ideal for for VLCADD because it is a monogenetic disorder. We have previously shown that a single injection of AAV9-VLCAD results in rescue of “cold and fast stressed” mice (Keeler, 2012). In that study, we demonstrated efficient expression of VLCAD proteins to WT levels, facilitated by rAAV9, in skeletal and cardiac muscles of VLCAD deficient mice (Keeler et al 2012). Similarly, in this current study, a single intravenous injection of AAV9-VLCAD gene therapy resulted in VLCAD expression in the respiratory and cardiac muscle. There was a significant increase in breathing frequency, comparable to the frequencies of both WT mice and VLCAD^−/− mice that did not undergo the exercise and fast stress protocol. This outcome was comparable to the effects of a clinical dose of dextrose, which is clinically used in emergencies to compensate for a metabolic crises. In addition, AAV9-VLCAD had a modest effect on inspiratory muscles and resulted in a peak inspiratory flow that was comparable to that of WT challenged animals. This is an indirect measure of inspiratory muscle function. However, AAV9-VLCAD treatment did not improve the tidal volume, minute ventilation, or peak expiratory flow in stressed VLCAD^−/− mice. This is most likely because AAV9 gene therapy resulted in correction of general energy deprivation and did not specifically target the muscles of respiration.

Conclusion

In conclusion, exercise and fast stressed VLCAD^−/− mice have blunted respiratory output and respiratory insufficiency. This deficiency appears to be a result of both energy deprivation and in this study we speculate that the blunted respiratory output is also a result of toxic accumulation of fat in the respiratory muscles. Respiratory insufficiency is an under-recognized cause of significant morbidity and mortality in VLCADD (Tong et al 2006; Giuliani et al 2013). Here, we show that stress conditions can result in persistent respiratory muscle defects. Lipid accumulation was seen in the muscles of respiration and we surmise that, similar to other disorders of excess substance accumulation, the excess fat accumulation impacts muscle contraction (Keeler et al 2018). Therefore, future therapeutic studies using the VLCAD^−/− mouse model should evaluate impacts of the intervention on respiratory insufficiency and restrictive lung disease. Current VLCADD treatments include avoidance of fasting and supplementation with medium chain triglyceride. However, these therapies are suboptimal and 38% of patients receiving them continue to have intermittent muscle weakness and pain (Spiekerkoetter et al 2009). Although a novel therapy with odd-carbon medium-chain triglycerides shows promising results such as decreased rhabdomyolysis, hypoglycemia, and cardiomyopathy (Vockley et al 2018), gene therapy using AAV9-VLCAD has the potential to provide patients with a long-term treatment option. We have previously shown that this improves the metabolic phenotype in VLCAD^−/− mice (Keeler, 2012). Here, we also show that a single injection of AAV9-VLCAD enhanced breathing and resulted in significant rescue of respiratory function during catabolic stress.