Smoking accelerates aging of the small airway epithelium.

BACKGROUND: Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking. METHODS: Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function. RESULTS: Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers. CONCLUSION: These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.

Assessment of Residual Full-Length SV40 Large T Antigen in Clinical-Grade Adeno-Associated Virus Vectors Produced in 293T Cells.

Efficient production of adeno-associated virus (AAV) vectors is a significant challenge. Human embryonic kidney HEK293T cells are widely used in good manufacturing practice facilities, producing higher yield of AAV vectors for clinical applications than HEK293 through the addition of a constitutive expression of SV40 large T antigen (SV40T), which stimulates Rep expression. However, the theoretical potential for tumorigenic consequences of a clinical AAV product containing residual DNA encoding SV40T, which may inhibit p53 growth suppressive functions is a safety concern. Although the risk is theoretical, to assure a low risk/high confidence of safety for clinical drug development, we have established a sensitive assay for assessment of functional full-length transcription competent SV40T DNA in HEK293T cell-produced AAV vectors. Using HEK293T generated 8, 9, and rh.10 serotype AAV vectors, the presence of SV40T in purified vector was assessed in vitro using quantitative polymerase chain reaction (qPCR) targeting a 129 bp amplicon combined with nested PCR targeting full-length SV40T DNA. Although low levels of the smaller amplicon were present in each AAV serotype, the full-length SV40T was undetectable. No transcription competent full-length SV40T DNA was observed by reverse transcription-quantitative polymerase chain reaction using an in vivo amplification of signal in mouse liver administered (2-10 × 1010 gc) 129 bp amplicon-positive AAV vectors. As a control for gene transfer, high levels of expressed transgene mRNAs were observed from each serotype AAV vector, yet, SV40T mRNA was undetectable. In vivo assessment of these three liver-tropic AAV serotypes, each with amplicon-positive qPCR SV40T DNA, demonstrated high transgene mRNA expression but no SV40T mRNA, that is, detection of small segments of SV40T DNA in 293T cell produced AAV inappropriately leads to the conclusion of residuals with the potential to express SV40T. This sensitive assay can be used to assess the level, if any, of SV40T antigen contaminating AAV vectors generated by HEK293T cells. ClinicalTrials.gov identifier: NCT03634007; NCT05302271; NCT01414985; NCT01161576.

Safety of Intravenous Administration of an AAV8 Vector Coding for an Oxidation-Resistant Human α1-Antitrypsin for the Treatment of α1-Antitrypsin Deficiency.

α1-antitrypsin (AAT) deficiency is a common autosomal recessive hereditary disorder, with a high risk for the development of early-onset panacinar emphysema. AAT, produced primarily in the liver, functions to protect the lung from neutrophil protease; with AAT deficiency, unimpeded neutrophil proteases destroy the lung parenchyma. AAT is susceptible to oxidative damage resulting in an inability to inhibit its target proteases, neutrophil elastase, and cathepsin G. The major sites of oxidative modification on the AAT molecule are methionine residues 351 and 358. We have previously demonstrated that an engineered variant of AAT that resists oxidation by modifying both protein surface methionines (M351V and M358L) provides antiprotease protection, despite oxidative stress. In mice, intravenous delivery of the modified AAT(AVL) variant by AAV serotype 8, AAV8hAAT(AVL), primarily to the liver resulted in long-term expression of an AAT that resists oxidative inactivation. In this study, we evaluated the safety of intravenous administration of AAV8hAAT(AVL) in a dose-escalating, blinded, placebo-controlled toxicology study in wild-type mice. The study assessed organ histology and clinical pathology findings of mice, intravenously administered AAV8hAAT(AVL) at three doses (5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 genome copies [gc]/kg), compared to control mice injected intravenously with phosphate-buffered saline. As previously demonstrated, administration of AAV8hAAT(AVL) resulted in dose-dependent expression of high, potentially therapeutic, levels of serum human AAT protein that persist for at least 6 months. Antibodies against the AAV8 capsid were elicited as expected, but there was no antibody detected against the AAT(AVL) protein generated by the AAV8hAAT(AVL) vector. There was no morbidity or mortality observed in the study. The data demonstrate that intravenous administration of AAV8hAAT(AVL) is safe with no significant adverse effect attributed to AAV8hAAT(AVL) vector at any dose. This study demonstrates that AAV8hAAT(AVL) has a safety profile consistent with the requirements for proceeding to a clinical study.

Positron Emission Tomography Quantitative Assessment of Off-Target Whole-Body Biodistribution of I-124-Labeled Adeno-Associated Virus Capsids Administered to Cerebral Spinal Fluid.

Based on studies in experimental animals demonstrating that administration of adeno-associated virus (AAV) vectors to the cerebrospinal fluid (CSF) is an effective route to transfer genes to the nervous system, there are increasing number of clinical trials using the CSF route to treat nervous system disorders. With the knowledge that the CSF turns over four to five times daily, and evidence in experimental animals that at least some of CSF administered AAV vectors are distributed to systemic organs, we asked: with AAV administration to the CSF, what fraction of the total dose remains in the nervous system and what fraction goes off target and is delivered systemically? To quantify the biodistribution of AAV capsids immediately after administration, we covalently labeled AAV capsids with iodine 124 (I-124), a cyclotron generated positron emitter, enabling quantitative positron emission tomography scanning of capsid distribution for up to 96 h after AAV vector administration. We assessed the biodistribution to nonhuman primates of I-124-labeled capsids from different AAV clades, including 9 (clade F), rh.10 (E), PHP.eB (F), hu68 (F), and rh91(A). The analysis demonstrated that 60-90% of AAV vectors administered to the CSF through either the intracisternal or intrathecal (lumbar) routes distributed systemically to major organs. These observations have potentially significant clinical implications regarding accuracy of AAV vector dosing to the nervous system, evoking systemic immunity at levels similar to that with systemic administration, and potential toxicity of genes designed to treat nervous system disorders being expressed in non-nervous system organs. Based on these data, individuals in clinical trials using AAV vectors administered to the CSF should be monitored for systemic as well as nervous system adverse events and CNS dosing considerations should account for a significant AAV systemic distribution.

Assessment of Safety and Biodistribution of AAVrh.10hCLN2 Following Intracisternal Administration in Nonhuman Primates for the Treatment of CLN2 Batten Disease.

CLN2 disease is a fatal, childhood autosomal recessive disorder caused by mutations in ceroid lipofuscinosis type 2 (CLN2) gene, encoding tripeptidyl peptidase 1 (TPP-1). Loss of TPP-1 activity leads to accumulation of storage material in lysosomes and resultant neuronal cell death with neurodegeneration. Genotype/phenotype comparisons suggest that the phenotype should be ameliorated with increase of TPP-1 levels to 5-10% of normal with wide central nervous system (CNS) distribution. Our previous clinical study showed that intraparenchymal (IPC) administration of AAVrh.10hCLN2, an adeno-associated vector serotype rh.10 encoding human CLN2, slowed, but did not stop disease progression, suggesting that this may be insufficient to distribute the therapy throughout the CNS (Sondhi 2020). In this study, we assessed whether the less invasive intracisternal delivery route would be safe and provide a wider distribution of TPP-1. A study was conducted in nonhuman primates (NHPs) with intracisternal delivery to cerebrospinal fluid (CSF) of AAVrh.10hCLN2 (5 × 1013 genome copies) or phosphate buffered saline (PBS). No abnormal behavior was noted. CNS magnetic resonance imaging and clinical chemistry data were all unremarkable. Histopathology of major organs had no abnormal finding attributable to the intervention or the vector, except that in one out of two animals treated with AAVrh.10hCLN2, dorsal root ganglia showed mild-to-moderate mononuclear cell infiltrates and neuronal degeneration. In contrast to our previous NHP study (Sondhi 2012) with IPC administration where TPP-1 activity was >2 × above controls in 30% of treated brains, in the two intracisternal treated NHPs, the TPP-1 activity was >2 × above controls in 50% and 41% of treated brains, and 52% and 84% of brain had >1,000 vector genomes/µg DNA, compared to 0% in the two PBS NHP. CSF TPP1 levels in treated animals were 43-62% of normal human levels. Collectively, these data indicate that AAVrh.10hCLN2 delivered by intracisternal route is safe and widely distributes TPP-1 in brain and CSF at levels that are potentially therapeutic. Clinical Trial Registration: NCT02893826, NCT04669535, NCT04273269, NCT03580083, NCT04408625, NCT04127578, and NCT04792944.

Identification of Safe and Effective Intravenous Dose of AAVrh.10hFXN to Treat the Cardiac Manifestations of Friedreich's Ataxia.

Friedreich's ataxia (FA) is a life-threatening autosomal recessive disorder characterized by neurological and cardiac dysfunction. Arrhythmias and heart failure are the main cause of premature death. From prior studies in murine models of FA, adeno-associated virus encoding the normal human frataxin gene (AAVrh.10hFXN) effectively treated the cardiac manifestations of the disease. However, the therapeutic dose window is limited by high level of human frataxin (hFXN) gene expression associated with toxicity. As a therapeutic goal, since FA heterozygotes have no clinical manifestations of FA, we estimated the level of frataxin (FXN) necessary to convert the heart of a homozygote to that of a heterozygote. In noncardiac cells, FA heterozygotes have 30-80% of normal FXN levels (17.7-47.2 ng/mg, average 32.5 ng/mg) and FA homozygotes 2-30% normal levels (1.2-17.7 ng/mg, average 9.4 ng/mg). Therefore, an AAV vector would need to augment endogenous in an FA homozygote by >8.3 ng/mg. To determine the required dose of AAVrh.10hFXN, we administered 1.8 × 1011, 5.7 × 1011, or 1.8 × 1012 gc/kg of AAVrh.10hFXN intravenously (IV) to muscle creatine kinase (mck)-Cre conditional knockout Fxn mice, a cardiac and skeletal FXN knockout model. The minimally effective dose was 5.7 × 1011 gc/kg, resulting in cardiac hFXN levels of 6.1 ± 4.2 ng/mg and a mild (p < 0.01 compared with phosphate-buffered saline controls) improvement in mortality. A dose of 1.8 × 1012 gc/kg resulted in cardiac hFXN levels of 33.7 ± 6.4 ng/mg, a significant improvement in ejection fraction and fractional shortening (p < 0.05, both comparisons) and a 21.5% improvement in mortality (p < 0.001). To determine if the significantly effective dose of 1.8 × 1012 gc/kg could achieve human FA heterozygote levels in a large animal, this dose was administered IV to nonhuman primates. After 12 weeks, the vector-expressed FXN in the heart was 17.8 ± 4.9 ng/mg, comparable to the target human levels. These data identify both minimally and significantly effective therapeutic doses that are clinically relevant for the treatment of the cardiac manifestations of FA.

Cocaine analog coupled to disrupted adenovirus: a vaccine strategy to evoke high-titer immunity against addictive drugs.

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.

Safety of Direct Intraparenchymal AAVrh.10-Mediated Central Nervous System Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy, a fatal pediatric neurodegenerative lysosomal storage disease caused by mutations in the arylsulfatase A (ARSA) gene, is characterized by intracellular accumulation of sulfatides in the lysosomes of cells of the central nervous system (CNS). In previous studies, we have demonstrated efficacy of AAVrh.10hARSA, an adeno-associated virus (AAV) serotype rh.10 vector coding for the human ARSA gene to the CNS of a mouse model of the disease, and that catheter-based intraparenchymal administration of AAVrh.10hARSA to the CNS of nonhuman primates (NHPs) white matter results in widespread expression of ARSA. As a formal dose-escalating safety/toxicology study, we assessed the safety of intraparenchymal delivery of AAVrh.10hARSA vector to 12 sites in the white matter of the CNS of NHPs at 2.85 × 1010 (total low dose, 2.4 × 109 genome copies [gc]/site) and 1.5 × 1012 (total high dose, 1.3 × 1011 gc/site) gc, compared to AAVrh.10Null (1.5 × 1012 gc total, 1.3 × 1011 gc/site) as a vector control, and phosphate buffered saline for a sham surgical control. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA. However, animals treated with the high dose AAVrh.10ARSA and the high dose Null vector had highly localized CNS abnormalities on magnetic resonance imaging scans at the sites of catheter infusions, and histopathology demonstrated that these sites were associated with infiltrates of T cells, B cells, microglial cells, and/or macrophages. Although these findings had no clinical consequences, these safety data contribute to understanding the dose limits for CNS white matter direct intraparenchymal administration of AAVrh.10 vectors for treatment of CNS disorders.

Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers.

Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.

Cocaine vaccine dAd5GNE protects against moderate daily and high-dose "binge" cocaine use.

The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose "binge" use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against "binge" cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose "binge" scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose "binge" model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced "highs" even in the context of moderate daily and high-dose "binge" use that might otherwise cause a drug-induced overdose.

Gene therapy for alpha 1-antitrypsin deficiency with an oxidant-resistant human alpha 1-antitrypsin.

Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell-mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease-inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.

Stress-Induced Mouse Model of the Cardiac Manifestations of Friedreich's Ataxia Corrected by AAV-mediated Gene Therapy.

Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction (p < 2 × 10-4) and 71% reduction in stress-related fractional shortening (p < 10-5). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls (p < 0.02). A one-time intravenous administration of 1011 genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN, corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls (p > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.

Quantitative Whole-Body Imaging of I-124-Labeled Adeno-Associated Viral Vector Biodistribution in Nonhuman Primates.

A method is presented for quantitative analysis of the biodistribution of adeno-associated virus (AAV) gene transfer vectors following in vivo administration. We used iodine-124 (I-124) radiolabeling of the AAV capsid and positron emission tomography combined with compartmental modeling to quantify whole-body and organ-specific biodistribution of AAV capsids from 1 to 72 h following administration. Using intravenous (IV) and intracisternal (IC) routes of administration of AAVrh.10 and AAV9 vectors to nonhuman primates in the absence or presence of anticapsid immunity, we have identified novel insights into initial capsid biodistribution and organ-specific capsid half-life. Neither I-124-labeled AAVrh.10 nor AAV9 administered intravenously was detected at significant levels in the brain relative to the administered vector dose. Approximately 50% of the intravenously administered labeled capsids were dispersed throughout the body, independent of the liver, heart, and spleen. When administered by the IC route, the labeled capsid had a half-life of ∼10 h in the cerebral spinal fluid (CSF), suggesting that by this route, the CSF serves as a source with slow diffusion into the brain. For both IV and IC administration, there was significant influence of pre-existing anticapsid immunity on I-124-capsid biodistribution. The methodology facilitates quantitative in vivo viral vector dosimetry, which can serve as a technique for evaluation of both on- and off-target organ biodistribution, and potentially accelerate gene therapy development through rapid prototyping of novel vector designs.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

Dysfunctional glycogen storage in a mouse model of alpha1-antitrypsin deficiency.

Autophagy is an intracellular pathway that contributes to the degradation and recycling of unfolded proteins. Based on the knowledge that autophagy affects glycogen metabolism and that alpha(1)-antitrypsin (AAT) deficiency is associated with an autophagic response in the liver, we hypothesized that the conformational abnormalities of the Z-AAT protein interfere with hepatocyte glycogen storage and/or metabolism. Compared with wild-type mice (WT), the Z-AAT mice had lower liver glycogen stores (P < 0.001) and abnormal activities of glycogen-related enzymes, including acid alpha-glucosidase (P < 0.05) and the total glycogen synthase (P < 0.05). As metabolic consequences, PiZ mice demonstrated lower blood glucose levels (P < 0.05), lower body weights (P < 0.001), and lower fat pad weights (P < 0.001) compared with WT. After the stress of fasting or partial hepatectomy, PiZ mice had further reduced liver glycogen and lower blood glucose levels (both P < 0.05 compared WT). Finally, PiZ mice exhibited decreased survival after partial hepatectomy (P < 0.01 compared with WT), but this was normalized with postoperative dextrose supplementation. In conclusion, these observations are consistent with the general concept that abnormal protein conformation and degradation affects other cellular functions, suggesting that diseases in the liver might benefit from metabolic compensation if glycogen metabolism is affected.

Coordinate control of expression of Nrf2-modulated genes in the human small airway epithelium is highly responsive to cigarette smoking.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oxidant-responsive transcription factor known to induce detoxifying and antioxidant genes. Cigarette smoke, with its large oxidant content, is a major stress on the cells of small airway epithelium, which are vulnerable to oxidant damage. We assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample the small airway epithelium in healthy-nonsmoker and healthy-smoker, and gene expression was assessed using microarrays. Relative to nonsmokers, Nrf2 protein in the small airway epithelium of smokers was activated and localized in the nucleus. The human homologs of 201 known murine Nrf2-modulated genes were identified, and 13 highly smoking-responsive Nrf2-modulated genes were identified. Construction of an Nrf2 index to assess the expression levels of these 13 genes in the airway epithelium of smokers showed coordinate control, an observation confirmed by quantitative PCR. This coordinate level of expression of the 13 Nrf2-modulated genes was independent of smoking history or demographic parameters. The Nrf2 index was used to identify two novel Nrf2-modulated, smoking-responsive genes, pirin (PIR) and UDP glucuronosyltransferase 1-family polypeptide A4 (UGT1A4). Both genes were demonstrated to contain functional antioxidant response elements in the promoter region. These observations suggest that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cells, and that there is variability within the human population in the Nrf2 responsiveness to oxidant burden.

Rapid/sustained anti-anthrax passive immunity mediated by co-administration of Ad/AAV.

Achieving both immediate and sustained protection against diseases caused by bacterial toxins and extracellular pathogens is a challenge in developing biodefense therapeutics. We hypothesized that a single co-administration of an adenovirus (Ad) vector and an adeno-associated virus (AAV) vector, both expressing a pathogen-specific monoclonal antibody, would provide rapid, persistent passive immunotherapy against the pathogen. In order to test this strategy, we used the lethal toxin of Bacillus anthracis as a target of a monoclonal antibody directed against the protective antigen (PA) component of the toxin, using co-administration of an Ad vector encoding an anti-PA monoclonal antibody (AdalphaPA) and an AAV vector encoding an anti-PA monoclonal antibody (AAVrh.10alphaPA). As early as 1 day after co-administration of AdalphaPA and AAVrh.10alphaPA to mice, serum anti-PA antibody levels were detectable, and were sustained through 6 months. Importantly, animals that received both vectors were protected against toxin challenge as early as 1 day after administration and throughout the 6 month duration of the experiment. These data provide a new paradigm of genetic passive immunotherapy by co-administration of Ad and AAV vectors, each encoding a pathogen-specific monoclonal antibody, as an effective approach for both rapid and sustained protection against a bio-terror attack.

Intrapleural Gene Therapy for Alpha-1 Antitrypsin Deficiency-Related Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) manifests primarily as early-onset emphysema caused by the destruction of the lung by neutrophil elastase due to low amounts of the serine protease inhibitor alpha-1 antitrypsin (AAT). The current therapy involves weekly intravenous infusions of AAT-derived from pooled human plasma that is efficacious, yet costly. Gene therapy applications designed to provide constant levels of the AAT protein are currently under development. The challenge is for gene therapy to provide sufficient amounts of AAT to normalize the inhibitor level and anti-neutrophil elastase capacity in the lung. One strategy involves administration of an adeno-associated virus (AAV) gene therapy vector to the pleural space providing both local and systemic production of AAT to reach consistent therapeutic levels. This review focuses on the strategy, advantages, challenges, and updates for intrapleural administration of gene therapy vectors for the treatment of AATD.

Biology of the Adrenal Gland Cortex Obviates Effective Use of Adeno-Associated Virus Vectors to Treat Hereditary Adrenal Disorders.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder occurring in 1:10,000 to 1:20,000 live births. In >95% of the cases, CAH results from mutations in the CYP21A2 gene, encoding the adrenal steroid enzyme 21-hydroxylase (21OH). Cardinal phenotypic features of CAH include genital ambiguity and sexual precocity, and in severe cases, neonatal salt loss and death. Current standard of care consists of lifelong oral steroid replacement to reverse the cortisol deficiency. Although significant advances in the treatment of CAH have been made, the burden of a lifelong therapeutic intervention is not ideal for quality of life. Gene therapy for CAH by adeno-associated virus (AAV) vectors has been shown to efficiently transduce the adrenal cortex, restoring normal steroidogenesis in the short term. However, adrenocortical cells are continuously renewed by stem cells located at the adrenal capsule, which differentiate as they centripetally migrate towards the adrenal medulla where they undergo apoptosis. In this context, we hypothesized that AAV-mediated genetic correction of the adrenal cortex will work short term but will eventually lead to a loss of correction. To test this hypothesis, we administered intravenously an AAV serotype rh.10 gene transfer vector (AAVrh.10-21OH-HA) to 21-hydroxylase deficient mice (21OH-/-). The data demonstrates that a single intravenous administration efficiently transduces adrenocortical cells leading to 21OH-HA expression and restoration of normal steroidogenesis. However, the duration of therapeutic efficacy lasted for only 8 weeks, accompanied by loss of 21OH-HA expression in the adrenal gland. Analysis in immunodeficient mice confirmed that the disappearance of transgene expression was not due to an antiviral/transgene immune response. Taken together, these results demonstrate that a single treatment with an adeno-associated viral vector expressing a functional copy of the mutated gene can only transiently treat adrenocortical hereditary disorders and that strategies to genetically modify the adrenocortical stem cells population will likely be required.

Attenuation of the Niemann-Pick type C2 disease phenotype by intracisternal administration of an AAVrh.10 vector expressing Npc2.

Niemann-Pick type C2 (NPC2) disease is a rare, neurodegenerative disorder caused by mutations in the NPC2 gene, leading to lysosomal accumulation of unesterified cholesterol and other lipids. It is characterized by hepatosplenomegaly, liver dysfunction and severe neurological manifestations, resulting in early death. There is no effective therapy for NPC2 disease. Here, we evaluated the effectiveness of an adeno-associated virus (AAV), serotype rh.10 gene transfer vector expressing the mouse Npc2 gene (AAVrh.10-mNpc2-HA, HA tagged to facilitate analysis) to treat the disease in an Npc2-/- mouse model. A single intracisternal administration of the AAVrh.10-mNpc2-HA to 6 week old Npc2-/- mice mediated vector DNA, transgene mRNA and protein expression in brain and other organs. Compared to untreated Npc2-/- mice, AAV-treated Npc2-/- mice demonstrated amelioration of disease pathology in the brain, reduced lysosomal storage, reduced Purkinje cell death, decreased gliosis, and improved performance in behavioral tasks. Treatment-related reduction in serum disease markers was detected early and this effect persisted. Liver and spleen pathology were improved with significant reduction of liver cholesterol and sphingomyelin levels in treated Npc2-/- mice. Finally, administration of AAVrh.10-mNpc2-HA significantly extended life-span. Taken together, these data demonstrate the benefit of a one-time intracisternal administration of AAVrh.10-mNpc2-HA as a life-long treatment for NPC2 disease.