Farshid N. Rouhani

Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy

Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 × 1012, 2.2 × 1013, and 6.0 × 1013 vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated serious adverse event (bacterial epididymitis). There were no changes in hematology or clinical chemistry parameters. M-specific AAT was expressed above background in all subjects in cohorts 2 and 3 and was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort, despite development of neutralizing antibody and IFN-γ enzyme-linked immunospot responses to AAV1 capsid at day 14 in all subjects. These findings suggest that immune responses to AAV capsid that develop after i.m. injection of a serotype 1 rAAV vector expressing AAT do not completely eliminate transduced cells in this context.

Phase 2 Clinical Trial of a Recombinant Adeno-Associated Viral Vector Expressing α1-Antitrypsin: Interim Results

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20u2009μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

Shedding of the Type II IL-1 Decoy Receptor Requires a Multifunctional Aminopeptidase, Aminopeptidase Regulator of TNF Receptor Type 1 Shedding

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1β. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.

Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

Single nucleotide polymorphism–mediated translational suppression of endoplasmic reticulum mannosidase I modifies the onset of end‐stage liver disease in alpha1‐antitrypsin deficiency

Inappropriate accumulation of the misfolded Z variant of alpha1‐antitrypsin in the hepatocyte endoplasmic reticulum (ER) is a risk factor for the development of end‐stage liver disease. However, the genetic and environmental factors that contribute to its etiology are poorly understood. ER mannosidase I (ERManI) is a quality control factor that plays a critical role in the sorting and targeting of misfolded glycoproteins for proteasome‐mediated degradation. In this study, we tested whether genetic variations in the human ERManI gene influence the age at onset of end‐stage liver disease in patients homozygous for the Z allele (ZZ). We sequenced all 13 exons in a group of unrelated Caucasian ZZ transplant recipients with different age at onset of the end‐stage liver disease. Homozygosity for the minor A allele at 2484G/A (refSNP ID number rs4567) in the 3′‐untranslated region was prevalent in the infant ZZ patients. Functional studies indicated that rs4567(A), but not rs4567(G), suppresses ERManI translation under ER stress conditions. Conclusion: These findings suggest that the identified single‐nucleotide polymorphism can accelerate the onset of the end‐stage liver disease associated with alpha1‐antitrypsin deficiency and underscore the contribution of biosynthetic quality control as a modifier of genetic disease. (HEPATOLOGY 2009.)

Extracellular TNFR1 release requires the calcium-dependent formation of a nucleobindin 2-ARTS-1 complex

Extracellular tumor necrosis factor (TNF) receptors function as TNF-binding proteins that modulate TNF activity. In human vascular endothelial cells (HUVEC), extracellular TNFR1 (type I TNF receptor, TNFRSF1A) is generated by two mechanisms, proteolytic cleavage of soluble TNFR1 ectodomains and the release of full-length 55-kDa TNFR1 in the membranes of exosome-like vesicles. TNFR1 release from HUVEC is known to involve the association between ARTS-1 (aminopeptidase regulator of TNFR1 shedding), an integral membrane aminopeptidase, and TNFR1. The goal of this study was to identify ARTS-1 binding partners that modulate TNFR1 release to the extracellular space. A yeast two-hybrid screen of a human placenta cDNA library showed that NUCB2 (nucleobindin 2), via its helix-loop-helix domains, binds the ARTS-1 extracellular domain. The association between endogenous ARTS-1 and NUCB2 in HUVEC was demonstrated by co-immunoprecipitation experiments, which showed the formation of a calcium-dependent NUCB2·ARTS-1 complex that associated with a subset of total cellular TNFR1. Confocal microscopy experiments demonstrated that this association involved a distinct population of NUCB2-containing intracytoplasmic vesicles. RNA interference was utilized to specifically knock down NUCB2 and ARTS-1 expression, which demonstrated that both are required for the constitutive release of a full-length 55-kDa TNFR1 within exosome-like vesicles as well as the inducible proteolytic cleavage of soluble TNFR1 ectodomains. We propose that calcium-dependent NUCB2·ARTS-1 complexes, which associate with TNFR1 prior to its commitment to pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains, play an important role in mediating TNFR1 release to the extracellular compartment.

Genome-wide study of percent emphysema on computed tomography in the general population. The Multi-Ethnic Study of Atherosclerosis Lung/SNP Health Association Resource Study

RATIONALEnPulmonary emphysema overlaps partially with spirometrically defined chronic obstructive pulmonary disease and is heritable, with moderately high familial clustering.nnnOBJECTIVESnTo complete a genome-wide association study (GWAS) for the percentage of emphysema-like lung on computed tomography in the Multi-Ethnic Study of Atherosclerosis (MESA) Lung/SNP Health Association Resource (SHARe) Study, a large, population-based cohort in the United States.nnnMETHODSnWe determined percent emphysema and upper-lower lobe ratio in emphysema defined by lung regions less than -950 HU on cardiac scans. Genetic analyses were reported combined across four race/ethnic groups: non-Hispanic white (n = 2,587), African American (n = 2,510), Hispanic (n = 2,113), and Chinese (n = 704) and stratified by race and ethnicity.nnnMEASUREMENTS AND MAIN RESULTSnAmong 7,914 participants, we identified regions at genome-wide significance for percent emphysema in or near SNRPF (rs7957346; P = 2.2 × 10(-8)) and PPT2 (rs10947233; P = 3.2 × 10(-8)), both of which replicated in an additional 6,023 individuals of European ancestry. Both single-nucleotide polymorphisms were previously implicated as genes influencing lung function, and analyses including lung function revealed independent associations for percent emphysema. Among Hispanics, we identified a genetic locus for upper-lower lobe ratio near the α-mannosidase-related gene MAN2B1 (rs10411619; P = 1.1 × 10(-9); minor allele frequency [MAF], 4.4%). Among Chinese, we identified single-nucleotide polymorphisms associated with upper-lower lobe ratio near DHX15 (rs7698250; P = 1.8 × 10(-10); MAF, 2.7%) and MGAT5B (rs7221059; P = 2.7 × 10(-8); MAF, 2.6%), which acts on α-linked mannose. Among African Americans, a locus near a third α-mannosidase-related gene, MAN1C1 (rs12130495; P = 9.9 × 10(-6); MAF, 13.3%) was associated with percent emphysema.nnnCONCLUSIONSnOur results suggest that some genes previously identified as influencing lung function are independently associated with emphysema rather than lung function, and that genes related to α-mannosidase may influence risk of emphysema.

Alpha1-Antitrypsin Deficiency–Related Alleles Z and S and the Risk of Wegener’s Granulomatosis

OBJECTIVEnDeficiency of α(1) -antitrypsin (α(1) AT) may be a determinant of susceptibility to Wegeners granulomatosis (WG). Several previous, mainly small, case-control studies have shown that 5-27% of patients with WG carried the α(1) AT deficiency Z allele. It is not clear whether the S allele, the other major α(1) AT deficiency variant, is associated with WG. This study investigated the relationship of the α(1) AT deficiency Z and S alleles with the risk of developing WG in a large cohort.nnnMETHODSnWe studied the distribution of the α(1) AT deficiency alleles Z and S in 433 unrelated Caucasian patients with WG and 421 ethnically matched controls. Genotyping was performed using an allele discrimination assay. Results were compared between cases and controls using exact statistical methods.nnnRESULTSnAmong the patients with WG, the allele carriage frequencies of Z and S were 7.4% and 11.5%, respectively. The frequencies of the 6 possible genotypes differed in a statistically significant manner between cases and controls (P = 0.01). The general genetic 2-parameter codominant model provided the best fit to the data. Compared with the normal MM genotype, the odds ratio (OR) for MZ or MS genotypes was 1.47 (95% confidence interval [95% CI] 0.98-2.22), and the OR for ZZ, SS, or SZ genotypes was 14.58 (95% CI 2.33-∞). ORs of similar direction and magnitude were observed within the restricted cohorts that excluded cases and controls carrying ≥1 Z or ≥1 S allele.nnnCONCLUSIONnBoth Z and S alleles display associations with risk of WG in a codominant genetic pattern. These findings strengthen the evidence of a causal link between α(1) AT deficiency and susceptibility to WG.

Alveolar macrophage dysregulation in Hermansky-Pudlak syndrome type 1.

RATIONALEnIndividuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown.nnnOBJECTIVESnTo investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied.nnnMETHODSnForty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed.nnnMEASUREMENTS AND MAIN RESULTSnConcentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P < 0.001). In vitro, HPS-1 pulmonary fibrosis alveolar macrophages, which did not express HPS1 mRNA, secreted significantly higher concentrations of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and regulated upon activation, normal T cell expressed and secreted (RANTES) protein compared with normal cells (P = 0.001, P = 0.014, and P = 0.011, respectively). Pirfenidone suppressed HPS-1 alveolar macrophage cytokine and chemokine secretion in vitro in a dose-dependent manner.nnnCONCLUSIONSnIn HPS-1, alveolar inflammation predominantly involves macrophages and is associated with high lung concentrations of cytokines and chemokines. HPS-1 alveolar macrophages provide a model system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.

Alpha-1-antitrypsin augmentation therapy in deficient individuals enrolled in the Alpha-1 Foundation DnA and Tissue Bank

Introduction: Intravenous augmentation therapy with purified intravenous alpha-1 antitrypsin replaces the deficient protein and is the only currently approved treatment for alpha-1 antitrypsin deficiency (AATD) related lung disease. While augmentation therapy has been available for more than 20 years, there are a limited number of studies evaluating the effect of augmentation on lung function. Material and methods: We examined the decline in forced expiratory volume in one second (FEV1) in patients enrolled in the Alpha-1 Foundation DNA and Tissue Bank in relation to the use or not of alpha-1 antitrypsin augmentation therapy. For the purpose of our analysis we included 164 patients with AATD and PI ZZ genotype. Results: Mean age of the patients was 60 years, 52% were females, 94% were white and 78% ex-smokers. The mean FEV1 at baseline was 1.7 L and the mean FEV1 % of predicted was 51.3%. The mean follow-up time was 41.7 months. A total of 124 (76%) patients received augmentation therapy (augmented group) while 40 patients (24%) did not received it (non-augmented group). When adjusted by age at baseline, sex, smoking status, baseline FEV1 % of predicted, the mean overall change in FEV1 was 47.6 mL/year, favoring the augmented group (ΔFEV1 10.6 ± 21.4 mL/year) in comparison with the non-augmented group (ΔFEV1 −36.96 ± 12.1 mL/year) (P = 0.05). Beneficial ΔFEV1 were observed in ex-smokers and the group with initial FEV1 % of predicted of <50%. No differences were observed in mortality. Conclusions: In conclusion, augmentation therapy improves lung function in subjects with AATD when adjusted by age, gender, smoking status and baseline FEV1 % of predicted. The beneficial effects were noted in ex-smoker subjects with FEV1 below 50% of predicted.