8th Biennial Investigators’Meeting
October19,2024
Miami,Florida
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Adel El Boueiz, MD
Mike Wells, MD
Camilla Bell, PhD
Monica Goldklang, MD
Lela Lackey, PhD
Shah Hussain, PhD
Francesco Annunziata, PhD
Bibek Gooptu, MD, PhD
Rick Sifers, PhD
Nunzia Pastore, PhD
Pasquale Piccolo, PhD
Carmine Settembre, PhD
Jeffrey Teckman, MD
James Irving, PhD
Tomás Carroll, PhD
Valerie Gouon-Evans, PhD
Lisa Cabrita, PhD
Purpose of the Conference
The Alpha-1 Foundation’s (A1F) Medical and Scientific Advisory Committee welcomes youtotheA1F8thBiennialInvestigators’Meeting
Promoting research with the vision of finding better treatments and ultimately a cure for Alpha-1 Antitrypsin Deficiency (AATD) is a core mission of A1F. Investigatorinitiated grants have been the driving force of the Foundation’s research program fromitsinceptionin1999,basedonthetime-testedprincipleinbiomedicaldiscovery that innovative ideas typically arise in the research community. In the past 30 years, A1F has invested over $100 million to support AATD research and programs at over 130 institutions in North America, Europe, the Middle East, and Australia A1Fsponsored research has had a significant impact on our understanding of the mechanisms underlying the clinical manifestations of AATD and has identified novel drug targets likely to lead to new therapeutic solutions A1F works collaboratively with biotech and pharmaceutical industries as well as government regulators to promotethefastestpossibledevelopmentandregulatoryapprovalofnewtherapies. This would not be possible without the many academic investigators whose discoverieshavebroughtthefieldtothiscriticaljuncture.
The purpose of the A1F Biennial Investigators’ Meeting is to provide A1F-supported investigators with a forum to exchange research findings and interact with the A1F's Medical and Scientific Advisory Committee (MASAC), Grants Advisory Committee (GAC), Board of Directors (BOD), Alphas, and Donors Investigators who have held A1F-sponsored grants during the two years preceding the meeting were invited to present research data, which is expected to initiate free discussion among all attendees,theconference’sprincipalgoal.
We look forward to a productive meeting that will advance the science of AATD and fostertheexchangeofideasamongAATDinvestigators,theultimateobjectiveofthe A1FBiennialInvestigators’Meeting
A1F acknowledges our sponsors AlphaNet, Beam Therapeutics, CSL Behring, Grifols andTakedafortheirongoingcommitmenttotheadvancementofresearch.
Scott Santarella President & Chief Executive Officer Alpha-1 Foundation
Andrew Wilson, MD Scientific Director Alpha-1 Foundation
A1F8thBiennialInvestigators’MeetingAgenda
October19,2024
8:00am—4:00pmET
AlhambraBallroom
Introduction and Welcome
08:00 am – 08:20 am
Scott Santarella and Andrew Wilson, MD
Session 1: Omics Studies in Alpha-1 Antitrypsin Deficiency (AATD)
(Andrew Wilson, MD, Chair, 15 minute presentation, 5 minute Q&A)
08:20 am – 08:40 am
08:40 am – 09:00 am
Imaging and Multi-omics Analyses of Emphysema Patterns in MM and MZ Smokers
Adel El Boueiz, MD
Brigham and Women's Hospital, Inc., USA
Molecular Profiling AATD Respiratory Specimens: A Pilot
Mike Wells, MD, Camilla Bell, PhD
The University of Alabama at Birmingham, USA
Session 2: Lung disease mechanisms in AATD
(Andrew Wilson, MD, Chair, 15 minute presentation, 5 minute Q&A)
09:00 am – 09:20 am
09:20 am – 09:40 am
Alterations in Inflammation and Proteases during Acute Exacerbations of COPD in AATD
Monica Goldklang, MD
Columbia University, USA
Misregulation of Polyadenylation during Stress Contributes to AATD Phenotypes
Lela Lackey, PhD
Clemson University, USA
09:40 am – 10:00 am
Severity and Progression of Airway Dropout in AAT Knockout Transgenic Ferret Model of COPD
Shah Hussain, PhD
The University of Alabama at Birmingham, USA
10:00 am – 10:30 am
Coffee Break
Session 3: Liver Disease Mechanisms in AATD
(Virginia Clark, MD, Chair, 15 minute presentation, 5 minute Q&A)
10:30 am – 10:50 am
10:50 am – 11:10 am
Identification and Modulation of Local and Systemic Environmental Factors in AATD
Francesco Annunziata, PhD
Telethon Institute of Genetics and Medicine, Italy
Structural Studies of Misfolded and Polymeric Alpha-1
Antitrypsin in ERAD and ERLAD
Bibek Gooptu, MD, PhD
University of Leicester, UK
11:10 am – 11:30 am
Identification of ESLD Prognostic Indicators
Rick Sifers, PhD
Baylor College of Medicine, USA
11:30 am – 11:50 am
11:50 am – 12:10 pm
Revealing the Role of Lysosomes in the Pathology of the AATD-Related Liver Disease
Nunzia Pastore, PhD
Fondazione Telethon ETS, Italy
Mitochondrial Dysfunction in AATD-Associated Liver Disease
Pasquale Piccolo, PhD
Telethon Institute of Genetics and Medicine, Italy
12:10 pm – 12:30 pm
Exploring the Role of FAM134B-MEDIATED ER-PHAGY in AATD
Carmine Settembre, PhD
Fondazione Telethon ETS, Italy
Lunch 12:30 pm – 01:45 pm
01:45 pm – 02:05 pm
Adult Alpha-1 Liver Clinical and Genetic Linkage
Jeffrey Teckman, MD
Saint Louis University, USA
Session 4: Novel Diagnostic and Treatment Approaches in AATD (Andrew Wilson, MD, Chair, 15 minute presentation, 5 minute Q&A)
02:05 pm – 02:25 pm
02:25 pm – 02:45 pm
Rational Design of Better Diagnostic Reagents
James Irving, PhD
University College London, UK
A Family Affair - Clarifying the Risk of Lung Disease in ZZ AATD
Tomás Carroll, PhD
Royal College of Surgeons in Ireland, Ireland
02:45 pm – 03:15 pm
03:15 pm – 03:35 pm
03:35 pm – 03:55 pm
Coffee Break
Engineered and Edited Patient-Derived iPSC for AATDAssociated Liver Disease Cell Therapy
Valerie Gouon-Evans, PhD
Boston Medical Center, USA
Modulating the Co-Translational Misfolding and Polymerisation of Antitrypsin
Lisa Cabrita, PhD, BSc
University College London, UK
Concluding Remarks
03:55pm
Scott Santarella
Session 1:
Omics Studies in Alpha-1
Antitrypsin Deficiency (AATD)
Adel El Boueiz, MD
Mike Wells, MD
Camilla Bell, PhD
Speaker Bios
Adel El Boueiz, MD
Brigham and Women’s Hospital, USA
Project title: Imaging and Multi-omics Analyses of Emphysema Patterns in MM and MZ Smokers
Dr El Boueiz is a pulmonary and critical care physician-scientist at Brigham and Women’s Hospital and an Assistant Professor of Medicine at Harvard Medical School.Hespecializesintreatingpatientswithawidevarietyofconditions,witha focus on unexplained dyspnea, COPD, pulmonary hypertension, post-COVID-19 complications, and tracheobronchomalacia. His research, funded by the Alpha-1 Foundation and the National Institute of Health, spans three related areas: identifying novel genes associated with COPD and pulmonary hypertension, applying machine learning to better characterize disease heterogeneity, and developing clinical prediction tools using multi-omics data. He has published in leading journals and frequently presents at national and international conferences.Alongsidehisclinicalandresearchwork,Dr.ElBoueiziscommitted to teaching and mentoring, earning several awards for his contributions. He is also active in outreach efforts, providing education and medications to underservedcommunities.
Objectives: Alpha-1 Antitrypsin Deficiency is associated with an elevated risk of emphysema,particularlyinsmokers Despitehavingnear-normallevelsofalpha1 antitrypsin (AAT) protein, MZ smokers often experience more severe emphysema than MM individuals with COPD, yet they frequently remain untreated. Identifying emphysema biomarkers in MZ smokers may help target those most likely to benefit from therapy. Although advanced texture-based CT quantificationmethodsareavailable,globalthreshold-basedmetricscontinueto be differentiated between centrilobular, panlobular, and paraseptal emphysema patterns Inthisstudy,weanalyzedtheseemphysemapatternsandusedcluster analysis to examine the clinical and imaging differences between MM and MZ smokers.
Methods:Weappliedthelocalhistogram(LH)chestCTquantificationmethodto examine centrilobular, panlobular, and paraseptal emphysema patterns in 10,113 non-Hispanic white and African American smokers with COPD from the COPDGene cohort, spanning various GOLD stages Differences in emphysema patterns between MZ and MM smokers were analyzed, along with the relationshipbetweensmokingexposureandemphysemaseverity
UsingK-medoidsclustering,wegroupedall10,113smokersbasedontheirdistinct emphysema patterns and compared the distribution of MM and MZ individuals across clusters. Additionally, we evaluated emphysema severity, dyspnea scores, lungfunction,comorbidities,andexacerbationfrequencywithineachcluster
Results: MZ smokers had significantly higher proportions of paraseptal, panlobular, and centrilobular emphysema compared to MM smokers and showedgreatersusceptibilitytoemphysemaperpack-yearofsmoking(P-values <0.05).Clusteringanalysisidentifiedfourclusters,eachcharacterizedbydifferent LH emphysema patterns, smoking history, dyspnea severity, exacerbation frequency,andratesoflungfunctiondeclineandemphysemaprogressionover5 years (P-values < 0.05). These clusters also differed in the prevalence of selfreported comorbidities such as coronary heart disease, hypertension, and diabetes (P-values < 0.05). MZ smokers were predominantly in the severe emphysema cluster, which was associated with more pronounced spirometric impairment, worse dyspnea, more frequent exacerbations, and accelerated diseaseprogression(P-values<0.05).Thesefindingsremainedsignificantinboth univariableandmultivariablemodels
Conclusion: Local histogram analysis revealed notable emphysema and clinical differences between MM and MZ smokers. Ongoing multi-omics research and validationinindependentcohortsmayhelpuncoverthemolecularmechanisms of Alpha-1 Antitrypsin Deficiency and advance personalized COPD treatment strategies.
Mike Wells, MD
The University of Alabama at Birmingham, USA
Project title: Molecular Profiling AATD Respiratory Specimens: A Pilot
Investigator(s): Camilla (Margaroli) Bell, PhD; Kristopher Genschmer, PhD; J. Michael Wells, MD, MSPH
Dr J Michael (Mike) Wells is a physician-scientist with expertise in translational and clinical research focusing on Alpha-1 Antitrypsin Deficiency (AATD), chronic obstructivepulmonarydisease(COPD),andpulmonaryvasculardisease.Heisan Associate Professor in the Division of Pulmonary, Allergy, and Critical Care Medicine at the University of Alabama at Birmingham (UAB), the Endowed ProfessorforAirwaysBiologyintheDepartmentofMedicine,MedicalDirectorfor theUABHeersinkSchoolofMedicineLungHealthCenter,andtheSectionChief for Pulmonary and Critical Care Medicine at the Birmingham VA Healthcare System.
InhisroleastheMedicalDirectorfortheLungHealthCenter,Dr.Wellsoversees a clinical research program that is currently facilitating 27 NIH-funded and 61 industry-sponsored clinical or translational research projects and trials. In addition to this role, Dr Wells leads the UAB Lung Health Center biospecimen repository,co-directstheUABTranslationalCardiopulmonaryResearchProgram, and leads a basic-science laboratory as an R01-funded investigator studying mechanisms of pulmonary vascular disease in COPD. In addition to his work in research, Dr. Wells is the Medical Director for the UAB Alpha-1 Clinical Resource CenterwhereheprovidescareforpatientswithAlpha-1AntitrypsinDeficiency
As a physician-scientist, Dr Wells has led numerous clinical trials, translational research, and basic science research projects. His work has been supported continuouslysincejoiningthepulmonaryfacultyatUABin2012throughNIHand Veterans Affairs grants, competitive university awards, foundation grants, and industry-sponsored clinical trials He is an investigator on several NIH-sponsored multi-site studies including the Alpha-1 Biomarker Consortium, SPIROMICS/SOURCE, COPDGene, Lung Health Cohort, and is an investigator through the Alpha-1 Foundation and the American Lung Association Airways Clinical Research Center (ALA-ACRC). Over the past 12 years, he has trained 13 post-doctoralfellows,7students,andmentored7juniorfaculty
Camilla Bell, PhD
The University of Alabama at Birmingham, USA
Project title: Molecular Profiling AATD Respiratory Specimens: A Pilot
Investigator(s): Camilla (Margaroli) Bell, PhD; Kristopher Genschmer, PhD; J Michael Wells, MD, MSPH
Dr.CamillaBellreceivedhermaster’sdegreeinmedicalbiologyattheUniversity of Lausanne (Switzerland) in 2015, and her PhD in Immunology and Molecular Pathogenesis in 2019 from the Graduate Division of Biological and Biomedical SciencesatEmoryUniversity(Atlanta,GA).DuringherthesisinDr.Tirouvanziam’s group,Dr Bellstudiedthemechanismsleadingtoneutrophilplasticityinchronic diseases, with a primary focus on lung inflammation in infants and adults affected by cystic fibrosis She continued working on mechanisms regulating airway inflammation upon infection (including SARS-CoV-2) during her postdoctoral training in Dr Amit Gaggar’s group at the University of Alabama at Birmingham(UAB).Currently,Dr.BellisanassistantprofessorintheDepartment ofPathologyatUABandherresearchfocusesonpathologicalimmuneresponse inthecardiopulmonarysystem
Objectives:Todemonstratethefeasibilityofrespiratoryspecimencollectionfrom Pi*ZZ participants enrolled in the Alpha-1 Biomarkers Consortium (A1BC) To measurecell-typespecificgeneexpressionpatternsusingdigitalspatialprofiling on alveolar tissue To investigate the NE+ extracellular vesicles (EVs) burden and the PGP pathway in invasive and non-invasive respiratory specimens in Alpha-1 AntitrypsinDeficiency(AATD)
Methods: We prospectively recruited participants to undergo respiratory specimensampling.Participantswereeligibleiftheywereenrolledintheparent A1BC study and had no contraindications to bronchoscopy or biopsy including known coagulopathy, bleeding diathesis, allergies or intolerance to sedation medications,severerestinghypoxemia,recentCOPDexacerbation,orlimitedlife expectancy The study was split into two visits, a pre-bronchoscopy screening visit and a bronchoscopy visit scheduled within 30 days. Participants had eligibility review and collection of safety labs, exhaled breath condensate (EBC), and spirometry performed at screening. Bronchoalveolar lavage (BAL), endobronchial brushes, and trans-bronchial biopsies were collected during the bronchoscopy visit Multiplex assays and mass spectrometry were used for proteinmeasurement;exosomeisolationandcountingviaNanosightNS300,NE activitybyFRETassay,andspatialtranscriptomicsbyNanostringGeoMx
Results: We enrolled 5 participants with a mean age of 59±14 years, 100% female sex, and 100% White race; 40% had COPD, 40% use weekly augmentation therapy,meanFEV1was89±11%predicted,averagelungdensitywas-908±27HU, and %LAA-950 was 3.1±2.9%. All participants had successful EBC, BAL, brush, and biopsycollectionwithoutcomplications.NEactivityonEVswasdetectableinall5 BAL specimens and in 3 out of 5 EBC specimens Cytokine profiling was successful primarily in the BAL samples and in 3 out of 5 EBC specimens. Transbronchialbiopsiesyieldedenoughtissuetoperformspatialtranscriptomics inall5patients.Geneexpressionanalysesareongoingforbrushesandbiopsies.
Conclusion: Respiratory sampling provides novel insights into disease mechanismsofAlpha-1AntitrypsinDeficiency(AATD).Thisprocessisfeasibleand invasive sample collection is well tolerated in the target patient population with AATD.
Session 2: Lung
Disease Mechanisms in Alpha-1 Antitrypsin Deficiency (AATD)
Monica Goldklang, MD
Lela Lackey, PhD
Shah Hussain, PhD
Speaker Bios
Monica Goldklang, MD
Columbia University, USA
Project title: Alterations in Inflammation and Proteases during Acute Exacerbations of COPD in AATD
Dr Monica Goldklang is a pulmonary and critical care physician-scientist at Columbia University. She grew up in Michigan, completing undergraduate, medical school, and residency training at the University of Michigan. She then moved to Columbia University for a pulmonary and critical care fellowship and post-doctoral research training in the laboratory of Dr. Jeanine D’Armiento. In addition to caring for patients with rare lung diseases including Alpha-1 AntitrypsinDeficiencyandLymphan-gioleiomyomatosis,andworkingintheICU, Dr Goldklang has an active research portfolio She has several clinical and translational studies in Alpha-1 Antitrypsin Deficiency, including an investigation intonovelimagingbiomarkersoflungdiseaseaswellasstudiesregardinglongterm disease trajectory and serum biomarkers following acute exacerbations of COPD. Outside of work, she enjoys running and can be found most weekends with her husband, Mike, loudly cheering for sons Ben and Josh at hockey and baseballgames.
Objectives:Alpha-1AntitrypsinDeficiency(AATD)wasthefirstidentifiedheritable cause of emphysema, with emphysema largely due to imbalances between neutrophil elastase and its endogenous inhibitor, alpha-1 antitrypsin (AAT). In evaluating the initiation and progression of COPD, chronic imbalances between proteases and antiproteases are most referenced, with increased interest in acute exacerbations of chronic obstructive pulmonary disease (AECOPD) as potential disease modifying events Therefore, our group sought to evaluate the burden of AECOPD in the community of patients with AATD (PiZZ genotype) participating in the Alpha-1 Biomarkers Consortium (A1BC), with specific interest ininflammatoryandproteasechangesasaresultofexacerbations.
Methods: The A1BC is a multi-center longitudinal cohort of patients across the disease spectrum of AATD. The 275 enrolled patients answered specific queries regarding exacerbation history preceding enrollment Every month questionnaires regarding exacerbation incidence were generated, and every six monthsclinicalcoordinatorsadjudicatedexacerbationevents
Drieddriedbloodspot(DBS)cardswereobtainedatenrollment,andparticipants were instructed at each AECOPD to obtain a DBS card at day 1, 3, 7 for later analysis. Cards were shipped to the data coordinating center for further biomarker evaluation DBS were eluted and IL-6 assayed as an inflammatory marker.
Results: Of the 275 enrolled patients, only 46 (16.7%) reported being a frequent exacerbator (FE), someone experiencing two or more exacerbations in the year prior to enrollment. The FE had an average FEV1 of 67.2% (SD 28.2%) at baseline. There were 116 exacerbations in 61 patients over the course of the study to date. Of the FE, 13 (283%) experienced an AECOPD after enrollment 717% of historic FE have not experienced an exacerbation. Of the patients reporting an exacerbation, 241% returned DBS cards to the data coordinating center Of the returned cards, 46.4% of patients returned all three cards, 28.6% returned one card, and 250% returned two cards 607% who reported AECOPD have had follow-up spirometry performed. The average change in FEV1 in patients with exacerbations was -6%. IL-6 as detected by ELISA was below the lower limit of detectableacrossseveralsampleexacerbationcards
Conclusion: There were suboptimal DBS return rates from patients following exacerbations, and methodology to assay for biomarkers from DBS cards will require optimization for low abundance proteins While there is a high exacerbationratereportedintheAATDpatientpopulation,theA1BCcohorthasa lower exacerbation rate than historically reported. The reduction may be due to enrollment of patients across all degrees of airway disease. However, even amongst frequent exacerbators, there is a lower exacerbation rate than historically reported in this patient population Powering studies in AATD based on exacerbation frequency will require contemporaneous data regarding incidenceinthiscommunity
Lela Lackey, PhD
Clemson University, USA
Project title: Misregulation of Polyadenylation during Stress Contributes to AAT Deficiency Phenotypes
Dr. Lela Lackey is an Assistant Professor in the Department of Genetics and Biochemistry at Clemson University. She started her laboratory at the Center for HumanGeneticsin2020.ShecamefromtheUniversityofNorthCarolina,Chapel Hill, where she did her post-doctoral research with Dr. Alain Laederach. Dr. Lackey started researching Alpha-1 Antitrypsin Deficiency (AATD) as a postdoctoralfellowwiththeAlpha-1Foundation.
She was involved in characterizing SERPINA1 mRNA alternative 5’UTR splicing and alternative 3’UTR polyadenylation These RNA processing steps influence AAT protein expression. She has continued to work on AATD and SERPINA1 mRNA regulation in her independent position at Clemson University Dr Lackey received her PhD in Molecular Biology at the University of Minnesota with Dr. Reuben Harris Her thesis work was on the APOBEC family of cytosine deaminases and their role in genomic mutation. Before graduate school, Dr. Lackey served as a Peace Corps Volunteer in South Africa in rural elementary education
Objectives: Alpha-1 antitrypsin (AAT) protein is key for repression of the inflammatory immune response. Disruption of AAT secretion can lead to lung and liver disease Such diseases frequently have a genetic component, such as the SERPINA1 Pi*Z allele, and environmental components, such as cigarette smokeandalcoholuse AATistranslatedfromSERPINA1mRNA SERPINA1mRNA have extensive regulatory pathways that remain poorly understood. The goal of our research is to understand the dynamic mechanisms underlying SERPINA1 RNA 3’ end processing and their impact on AAT expression We propose experiments to understand how environmental conditions alter RNA processing and identify factors responsible for alternative polyadenylation at proximal and distalpositionsatthe3’endofSERPINA1.
Methods: We measured SERPINA1 RNA processing in a liver cell line (HepG2) following treatment with the inflammatory cytokine interleukin 6 (IL-6), ethanol orperoxide.Toanalyzealternativepolyadenylation,weused3’endspecificlibrary preparation and next-generation sequencing followed by differential gene and polyadenylation analysis We developed a CRISPR cell line with a mutant proximal polyadenylation site (APA-mut). We quantified AAT expression and secretioninWTandAPA-mutcelllineswithIL-6treatment
Results: We validated IL6 treatment of HepG2 cells using qRT-PCR We found clear upregulation of both FGB and IL1R1 mRNA after IL6 treatment, which was confirmed in our 3’ end specific sequencing and gene expression analysis. In addition to transcriptional upregulation after IL-6 treatment, SERPINA1 had altered polyadenylation, resulting in an increase in long 3’UTR isoforms 24 hours post-IL-6 exposure In the CRISPR APA-mut cell line, we find that the long 3’UTR repressesendogenousAATproteinexpressionevenwithhighlevelsofSERPINA1 mRNA SERPINA1expressionand3’endprocessingarenotaffectedbyethanolor peroxide. IL-6-induced changes in transcriptome-wide transcriptional regulation suggest changes to the endoplasmic reticulum and in secretory protein processing.
Conclusion: Our data shows that inflammation influences polyA site choice for SERPINA1 transcripts, resulting in reduced AAT protein expression We also find thatinflammationaffectsgenesassociatedwiththeendoplasmicreticulumand protein localization Remodeling of these secretory pathways during inflammation may contribute to disease and provide therapeutic targets. Our experiments build knowledge on the processing and regulation of SERPINA1 mRNAs and related transcripts, with a goal toward understanding and treating diseaseinindividualswithAlpha-1AntitrypsinDeficiency(AATD).
Shah Hussain, PhD
The University of Alabama at Birmingham, USA
Project title: Severity and Progression of Airway Dropout in AAT Knockout Transgenic Ferret Model of COPD
Dr Shah S Hussain is a faculty member in the UAB Department of Medicine (Pulmonary, Allergy, and Critical Care Medicine). He received his MS degree in Bioscience from Jamia Millia Islamia University in 2009 and his MPhil degree from the University of Delhi in 2011. Dr. Hussain completed his PhD at the University of Delhi in 2017, focusing on the protein chemistry of active recombinant glycoengineered hormones. He also received molecular biology trainingattheIndianInstituteofSciences,Bangalore(IISc),India.Duringhisfirst postdoctoral training at the Department of Immunology, Florida International University in Miami in 2017, Dr. Hussain conducted research on the role of Long Noncoding RNA in the Allergic Asthma Associated Hyperreactive Response of Airway Epithelial Cells. His interest in Respiratory Biology led him to join the Department of Medicine and CFRC Center at the University of Alabama at Birmingham as a Postdoctoral Fellow. Throughout his postdoctoral work, Dr.. Hussain published his research in prestigious journals such as Nature, JCI, ERJ, andMucosalImmunology In2022,Dr HussainwasappointedasanInstructorin the UAB Department of Medicine. His research interests revolve around small airway disease and the impact of abnormal mucus on lung diseases, including chronic obstructive pulmonary disease, Alpha-1 Antitrypsin Deficiency (AATD), idiopathic pulmonary fibrosis, and cystic fibrosis He is particularly dedicated to studying respiratory infectious diseases like SARS-COV-2 and HIV. Dr. Hussain's research encompasses cutting-edge discoveries in airway disease biology, ciliary dynamics, and translational research in IPF, CF, Alpha-1 Antitrypsin COPD, and other airway diseases. During his tenure at UAB, Dr. Hussain has developed a groundbreaking technique for micro-CT imaging of the microstructure of the smallestairways(lessthan2mm)inferretlungs.
This innovation opens new avenues for understanding progressive airway diseasesinvolvingsmallairways,includingCOPD,CF,andIPF Hiscurrentfocusis on unraveling the complex pathological changes in small airway disease progression and gaining mechanistic insights into novel aspects of disease mechanisms that have previously lacked a comprehensive understanding, despitebeingwell-documentedinhumanspecimens
Objectives: Alpha-1 Antitrypsin Deficiency (AATD) is a major genetic risk factor associated with COPD, but our understanding of how it contributes to chronic obstructive pulmonary disease is still evolving. Airway obstruction in COPD due tocigarettesmokingislargelyduetosmallairwaydiseasethatisassociatedwith abnormal mucus clearance and is highly accelerated in individuals with AATD. Recent evidence suggests that the progression of COPD airway obstruction is a result of airway remodeling characterized by tapering and loss of the terminal bronchioles(ie,airwaydropout),andmaybeassociatedwithmucusobstruction
The mechanistic basis of airway loss in COPD and how this may be exacerbated in AATD is currently unknown, in part because an animal model that features human-like loss of terminal bronchioles has not been available We hypothesize that cigarette smoke exposure causes mucus obstruction, leading to airway remodelingandairwaydropout,whileAATDfurtheracceleratesthisprocess
Methods: To address this issue, we a) developed physiologic, radiographic, and pathological methods to quantify airway loss in a novel ferret model of cigarette smoke-induced COPD that exhibits is uniquely associated with mucus obstructionascomparedtootheranimalmodels;b)establishedlongitudinalloss of pulmonary function due loss of terminal bronchioles with smoking; and c) in preliminary data, demonstrate that these abnormalities are more severe and accelerated in a novel AAT-knockout ferret. We created a novel ferret model of COPD with an AATD to study the progression of the disease This unique model allows us to closely examine the loss of airways, and the obstruction caused by mucus,whicharekeyfactorscontributingtoCOPD.Ourresearchhasthreemain objectives: to characterize airway loss in COPD ferrets, to determine whether mucus obstruction directly causes this loss, and to identify specific RNA signaturesthatprecedeairwayremodelingandloss
Results: By achieving these objectives, we aim to uncover the underlying mechanismsthatdriveCOPDprogressioninindividualswithAATD.Ourfindings will provide valuable insights into how AATD influences COPD and how mucus obstruction contributes to airway damage. This knowledge is crucial for developing targeted therapies to protect lung health and improve the quality of lifeforindividualswithCOPD,particularlythosewithAATD.
Conclusion: The insights gained from our research, funded by the Alpha-1 Foundation, will guide future therapeutic development and help mitigate the impactofthisdebilitatingdisease
Session 3:
Liver Disease Mechanisms in Alpha-1 Antitrypsin Deficiency (AATD)
Francesco Annunziata, PhD
Bibek Gooptu, MD, PhD
Rick Sifers, PhD
Nunzia Pastore, PhD
Pasquale Piccolo, PhD
Carmine Settembre, PhD
Jeffrey Teckman, MD
Speaker Bios
Francesco Annunziata, PhD
Telethon Institute of Genetics and Medicine, Italy
Project title: Alterations in Inflammation and Proteases during Acute Exacerbations of COPD in AATD
Dr Francesco Annunziata graduated in Molecular Biology in 2016 from the University of Naples Federico II and then completed a PhD at the Leibniz Institute on Aging (Fritz Lipmann Institute). While completing his PhD at Fritz Lipmann Institute, he focused on on age-related diseases, particularly studying intestinaldiseases.SinceSeptember2021,heworksasapostdoctoralfellowinDr. Nunzia Pastore's laboratory, where they study the molecular pathways responsibleforAATDliverdiseasethatcanbeconsideredtherapeutictargetsfor the treatment of AATD patients In May 2022, he was awarded a 2-year postdoctoral fellowship from the Alpha-1 Foundation to elucidate the role of the gut-liver axis as a modifier of AATD-associated liver disease and to study the hepatic stellate cells involvement in the onset and progression of AATDassociatedliverdisease
Objectives: Alpha-1 Antitrypsin (AAT) is a serine protease inhibitor, which is primarily synthetized in the hepatocyte and then secreted in the blood, preventing extracellular matrix degradation in the lungs. Mutations in the gene encoding for AAT mostly result in reduced levels of serum AAT causing Alpha-1 Antitrypsin Deficiency (AATD). Clinical features of AATD are pulmonary emphysemain the lung, hepatic cirrhosis and elevated risk of developing hepatocarcinoma in the liver. However, AATD has been associated with other conditions, including panniculitis and rheumatoid arthritis. Moreover, AATD has also been described as co-morbidity with intestinal bowel diseases (IBDs), despite no experimental evidence have been reported. In this study, using PiZ mice,weinvestigatedthecorrelationbetweenAATDandIBDs
Methods: We performed histological and electron microscopy analyses to evaluate macroscopic and structural changes of intestinal cells. By transcriptomic analyses we identified the molecular pathways underlying the observed differences between WT and PiZ mice In addition, we used metagenomic analyses to evaluate fecal microbial composition. Finally, we applied a well-known protocol based on administration of 3% dextran sodium sulfate(DSS)indrinkingwatertoinducecolitisinmice.
Results: We found that PiZ mice were more susceptible to DSS treatment compared to WT mice, showing significant body weight reduction and increased intestinal damage and inflammation. Interestingly, this was accompanied by a worsening of the hepatic phenotype, with an increased number of ATZ polymers, liver inflammation and fibrosis suggesting a key role for the gut-liver axis in the AATD liver pathology. We observed that polymeric ATZ accumulates in different intestinal epithelial cells impairing their functionality. We focused on Paneth cells (PCs) that are mostly involved in IBD. Electron microscopy analysis revealed an increase in apoptosis, abnormal lysozyme granules, ER fragmentation and degenerating mitochondria in PCs. In agreement with PCs abnormalities, transcriptomic analysis confirmed an increase in ER stress and inflammation. Of note, we found abnormal production and secretion of lysozyme in PiZ mice associated with reduced alpha- and beta-diversity of the intestinal microbiota.
Conclusion: Overall, these data indicate AATD as an etiological factor in IBDs and suggest a crucial role for the gut-liver axis in the liver phenotype associated with AATD
Bibek Gooptu, MD, PhD
University of Leicester, UK
Project title: Structural Studies of Misfolded and Polymeric Alpha-1 Antitrypsin in ERAD and ERLAD
Dr.BibekGooptuisaclinicianscientistwhostudiesmechanismsofinflammation and fibrosis in Alpha-1 Antitrypsin Deficiency (AATD), serpinopathies, and interstitial lung disease (ILD). His research group uses structural and cell biology and ex vivo tissue studies to address questions of clinical relevance to identify and validate new therapeutic strategies. He trained as an MD/PhD at the Universities of Manchester and Cambridge, UK His doctoral studies with David Lomas (1996-99) characterized how conformational change and polymerisation in disease-associated mutants of α1-antichymotrypsin and neuroserpin caused deficiency and a novel dementia. After establishing his hospital medical career, he returned to research with a Wellcome Trust Intermediate Clinical Fellowship at Birkbeck and University College London In this Fellowship (2006-2011) he applied X-ray crystallography, ion-mobility mass spectrometry, NMR spectroscopy, and single particle (cryo-)electron microscopy to wild-type and mutantAAT.(Pulmonology),withsub-specialtyinterestsinAATD,COPDandILD. After working as a Clinical Senior Lecturer at King’s College London (2012-2016) and helping establish the London AATD Service, he moved to take up a role as Professor of Respiratory Biology at the University of Leicester and Consultant in RespiratoryMedicineatGlenfieldHospital
Objectives: To characterize in definitive detail, critical molecular interactions of endoplasmic reticulum (ER) proteins with the Z variant of (AAT), that mediate circulatingdeficiencyandclearanceofassociatedpolymersinAlpha-1Antitrypsin Deficiency(AATD)
Methods: Expression and purification of PDI:EDEM and UGGT1 in the apo-states and in complex with misfolding variants of AAT. Cryo-EM studies and singleparticle analysis to solve the structures of these complexes Validation of the significance of observed interactions by site-directed mutagenesis and biophysicalstudies.
Results: We have solved the cryo-EM structures of PDI:EDEM and UGGT1 apostates to 27 Å resolution We have successfully purified ternary complexes of PDI:EDEM with misfolded Z and NHK AAT species from the ER of human cells, and obtained initial cryo-EM datasets Our current 3D reconstruction supports interactionsviathemobileprotease-associateddomain(PAD)ofEDEMmoietyof the ERAD checkpoint complex. Mutagenesis studies and mass spectrometry of the ternary complex also define linkages between misfolded AAT and redox motifs within the PDI moiety.These are the first structural data on ERAD checkpoint:substrate interactions, to our knowledge We are now pursuing approaches to improve the resolution of the ternary complex and extend from the thermostabilized (C thermophilum) PDI:EDEM to the human paralogue complexes to establish relevance to disease. We have made recombinant, glycosylated human AAT and UGGT1. We are therefore now optimizing conditions to prepare samples of UGGT1 interacting with misfolded monomeric AATintherefoldingpathway,andwithpolymericAATintheERLADpathway.
Conclusion: Our initial findings provide insights into the range of interactions between misfolded AAT and different regions of the ERAD checkpoint complex.They indicate the of dynamic disulphide bonding and large-scale flexibilitywithinthecomplex
Rick Sifers, PhD
Baylor College of Medicine, USA
Project title: Identification of ESLD Prognostic Indicators
Dr. Richard N. Sifers is a Professor at Baylor College of Medicine in the Texas Medical Center in Houston, TX. He began working on Alpha-1 Antitrypsin Deficiency(AATD)asapostdocatthatsameinstitutionwhereheparticipatedin the generation and initial analysis of the PiZ transgenic mouse. Dr. Sifers helped pioneer the identification of the Proteostasis Network in which Man1b1 plays a keyrole.
Also, he found that the Z protein could be degraded by proteasomes, but predominantlybylysosomes Morerecently,hereportedthatMan1b1participates in directing the sorting of the Z protein into a disposal pathway that involves extracellular vesicles (EVs) The distinct factors and regulatory systems responsible for the sequential use of the EV pathway and activation of the HepatocyteProliferativeStressResponsearethefocusofhiscurrentresearch
Objectives: The central objective is to identify the underlying causality factors associated with the progression of some ZZ patients to end-stage liver disease (ESLD), necessitating orthotopic liver transplantation. The hypothesis to be tested is that the operative failure of a "protective" stress response system underliesprogressionofhepatocyteinjurytoESLD.
Methods: A multi-omics approach including biochemistry, cell biology, epigenetics, and the identification of SNPs was developed to ask whether the analysis of extracted livers from a cadre of unrelated Z/Z ESLD individuals vs blood-derived gDNA from non-ESLD would provide informative mechanistic information.
Results: A published SNP located in the MAN1B1 gene initiated our findings. Man1b1playsacentralroleintheoperationofSIEVE(Stress-InducedExtracellular Vesicle Expulsion) whereas additional components (to be presented) are responsible for regulating/activating the actual Hepatocyte Proliferative Stress Response (HPSR)ERLAD and SIEVE are proposed to function as the first and second Preemptive Sentinel Components, but the recently reported SNP pair in ERLAD was not detected in our samples, implying that they function as a rare defect.
Conclusion: A decentralized 3-stage Hepatocyte Proliferative Stress Response (HPSR) consisting of both preemptive and multi-component stress response elements protects the progression of hepatocyte liver injury to ESLD Additional studies will utilize patient-derived iHeps, in combination with RNA-seq and spatial biology (transcriptomics and proteomics), before and after specific CRISPR-modified alterations, to establish mechanistic insights into disease causality.
Nunzia Pastore, PhD
Fondazione Telethon ETS, Italy
Project title: Revealing the Role of Lysosomes in the Pathology of the AATD-Related Liver Disease
Dr NunziaPastoreisanAssistantProfessorofMedicalGeneticsattheUniversity of Naples Federico II and a Project Leader at Telethon Institute of Genetics and Medicine (TIGEM), Italy. Dr. Pastore graduated in Molecular Biology from the UniversityofNaplesFedericoIIin2008.Soonafter,shestartedaPhDprogramin Life Science with Open University (UK) at TIGEM working on gene therapy approachesformetabolicinheriteddisordersinthelaboratoryofProfessorNicola Brunetti-Pierri. Next, she moved to the United States to the Neurological Research Institute- Baylor College of Medicine in Houston (TX, USA) as a postdoctoralfellowandthenasAssistantProfessorinthelabofProfessorAndrea Ballabioworkingontheidentificationofthephysiologicalandpathologicalroles of the Transcription Factor EB (TFEB) in immunity, whole-body homeostasis, circadian entrainment and liver regeneration. After 6 years, she returned to Naples where she is currently interested in investigating novel molecular and therapeuticapproachesforAlpha-1AntitrypsinDeficiency(AATD)atTIGEM.
Objectives: Lysosomes are acidic membrane-bound organelles, containing hydrolytic enzymes required for the degradation of a wide variety of substrates includingproteins,lipids,carbohydrates,andnucleicacids.Lysosomeshavebeen consideredforlongtimejustas“trashbins”ofthecell.However,latelythevision oftheroleoflysosomeincellhomeostasischangedandtheyarenowconsidered as “signaling hub” orchestrating metabolic responses to nutrient signals. As consequence, lysosomal dysfunction has been described in many human conditions, including lysosomal storage disorders (LSDs) and neurodegenerative diseases All these diseases share as a common feature the progressive accumulation of undigested macromolecules within the cell that are either intermediates of the cellular catabolism, or proteins that tend to form pathogenicaggregates.
Methods: We analyzed the functionality of lysosomes and the activation of the autophagy pathway in AATD models using gold-standard techniques. Moreover, we evaluated the effect of the genetic and pharmacological inhibition of the calpain system on lysosomal functionality as therapeutic option for AATD liver disease
Results: Our data demonstrated that chronic accumulation of ATZ in the ER of hepatocytes impairs lysosomal function In particular, lysosomes resulted engulfedwithundigestedmaterial,aspecifichallmarkofseveralLSDs.Wefound that calpain-2, a known inhibitor of lysosomal function inducing membrane permeabilization and impaired autophagosome formation, is upregulated in cells, organoids, mice and human samples of AATD Moreover, genetic and pharmacological inhibition of Calpain-2 in mice reduced the accumulation of ATZ and the secondary hallmark associated with the liver disease by inducing autophagy
Conclusion: Our study demonstrated a pivotal role for lysosomes in the pathobiology of the AATD liver disease and may pave the way toward the identificationofnovelbiomarkersandtherapeutictargetsforpatients
Pasquale Piccolo, PhD
Telethon Institute of Genetics and Medicine, Italy
Project title: Mitochondrial Dysfunction in A1-Antitrypsin
Deficiency-Associated Liver Disease
Dr PasqualePiccolograduatedinMedicalBiotechnologyin2005fromUniversity ofNaples“FedericoII”andthencompletedaPhDinHumanGenetics,alsoatthe UniversityofNaplesin2009 Hethencarriedoutpost-doctoralresearchinPhilip Ng’slabatBaylorCollegeofMedicineinHoustonTX,herehebecameinterested inliver-directedgenetherapywithhelper-dependentadenoviralvectors.Hethen returnedtoItaly,movingtoTIGEMtoworkinNicolaBrunetti-Pierri’slab Herehe investigatedtheroleoftranscriptionfactorsandmicroRNAsinthepathogenesis of liver disease related to Alpha-1 Antitrypsin Deficiency (AATD) Given the importance of this work, he was awarded with the AAT Laurell's Training Award and the Gordon L Snider Scholar Award His research has also explored the potentialofsmallmoleculesforthetreatmentofinheriteddiseases;heidentified losartanasaneffectivetreatmentfortheextracellularmatrixdepositiondefectof Myhre syndrome, a rare connective tissue disease Losartan has been shown to ameliorate ECM deposition in Myhre patients in a recent clinical trial. Since his appointmentasanindependentassistantinvestigatoratTIGEM,hisresearchhas been focused on the development of novel liver-directed gene therapy approachesforgeneticdisorderswithliverfibrosis
Objectives: Mitochondrial injury and autophagy (mitophagy) have been previously described as part of mutant Z α1-Antitrypsin (ATZ)-triggered injury cascade.Removalofdamagedmitochondriabymitophagyavoidsexcessivefree radicalproductionandoxidativestressandpreventsthereleaseofmitochondriaderived damage-associated molecular patterns (mito-DAMPs). Mito-DAMPS, like mitochondrialDNA(mtDNA),formylatedproteinsandATP,arepotenttriggersof inflammation and their release from injured hepatocytes can drive innate immune response and fibrosis, aggravating liver injury. We hypothesized that mitochondrial dysfunction represents not only a consequence of hepatic ATZ accumulationbutplaysaroleindevelopmentandprogressionofliverdiseasein AATD There are three specific aims for this proposal: i) to investigate mitochondrial biogenesis in liver disease associated with AATD; ii) to investigate mitochondrial dynamics and mitophagy flux in response to hepatic ATZ accumulation; iii) to investigate the role of circulating mito-DAMPs in liver diseaseassociatedwithAATD.
Methods: We investigated mitochondrial potential, dynamics, and mitophagy flux in juvenile PiZ, transgenic for human ATZ, and wild-type mice Liver fibrosis and circulating levels of mtDNA were assessed in both PiZ and control mice at differentages.
Results: PiZ hepatocytes displayed mitochondrial depolarization and higher levels of mitochondrial superoxide compared to controls, confirming mitochondrial malfunctioning. Additionally, we found increased fission and decreased fusion markers, leading to mitochondrial fragmentation and abnormal fission in PiZ mice. Livers from PiZ mice also exhibited increased mtDNAcontentcomparedtocontrols,supportingtheaccumulationofdamaged mitochondria. Consistently, upon induction of mitochondrial damage and autophagy, PiZ hepatocytes showed reduced degradation of mitochondrial protein, suggesting impaired mitophagy leading to impaired mitochondrial degradation. To investigate the link between mitochondrial damage and liver diseaseprogression,weassessedcirculatingmtDNAandliverfibrosisinPiZmice atdifferentages.CirculatingmtDNAlevelspeakedat16weeksofage,whilemice developed significant liver fibrosis at 20 weeks of age A marked reduction of hepatic ATZ burden between 12 and 16 weeks of age suggests that increased mtDNAreleaseintothecirculationislinkedtoliverregeneration.
Conclusion: These findings suggest that altered mitochondrial dynamic and degradationcontributetomitochondrialdisfunctioninPiZmice.Thesedataalso support mito-DAMPs as drivers of fibrosis in PiZ mice, proposing a new pathogeneticmechanismcontributingtoliverdiseaseinAATD
Carmine Settembre, PhD
Fondazione Telethon ETS, Italy
Project title: Exploring the Role of FAM134B-MEDIATED ERPHAGY in Alpha-1 Antitrypsin Deficiency
Dr SettembreisagroupleaderofthecellbiologyanddiseaseprogramatTIGEM instituteandprofessorofhistologyatFedericoIIuniversityofNaples.Hereceived his PhD in Molecular Medicine from the Federico II University, Naples, Italy, and did postdoctoral work at both Columbia University and Baylor College of Medicine. The main research interest of his laboratory is to understand the regulation and role of the lysosomal-autophagy pathway in both physiological and disease processes and to develop novel therapeutic approaches based on autophagymodulation
Objectives: Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate removal of faulty products. ER-associated protein degradation and ER-phagy pathways target misfolded proteins for proteasomal and lysosomal degradation, respectively. The mechanisms initiating ER-phagy in response to misfolded proteinsarenotwellunderstood
Methods: To investigate ER-phagy in response to accumulation of misfolded cargoeswithintheER,westudyvariouscellularmodelsofdiseasescharacterized by the build-up of misfolded proteins within the ER lumen, leading to its enlargement. We also used primary hepatocytes obtained from a mouse model ofAlpha-1AntitrypsinDeficiency(AATD)(PiZmouse)expressingaE342Kmutant versionofhumanSERPINA1(ATZ)thataccumulatesintheERlumen
Results: We show that the accumulation of misfolded proteins within the ER in cellularmodelsofERstoragedisorders,suchasAA,triggersaresponseinvolving SESTRIN2, a nutrient sensor controlling mTORC1 signaling SESTRIN2 induction by XBP1 inhibits mTORC1's phosphorylation of TFEB/TFE3, allowing these transcriptionfactorstoenterthenucleusandupregulatetheER-phagyreceptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via the FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ER storage diseasemodels.
Conclusion:OurstudyidentifiestheinterplaybetweennutrientsignalingandER quality control and suggests therapeutic strategies for disorders related to proteinfoldingintheER.
Jeffrey Teckman, MD
Saint Louis University School of Medicine, USA
Project title: Adult Alpha-1 Liver Clinical and Genetic Linkage
Dr Jeffrey Teckman is the Drs James and Patricia Monteleone Endowed Chair, Vice Chair of the Department of Pediatrics, Professor of Pediatrics and Biochemistry at the Saint Louis University School of Medicine and Cardinal GlennonChildren’sHospital.Hehasbeeninvolvedinresearchonliverdiseasefor more than 30 years in both basic science and clinical studies. Although his studies have ranged from viral hepatitis to liver failure in short bowel syndrome, hisprimarybasicsciencefocushasbeentheliverdiseaseassociatedwithAlpha-1 Antitrypsin Deficiency (Alpha-1) His laboratory was the first to recognize the link betweenAlpha-1mutantZproteinintheliverandautophagy,hewasthefirstto recognizethatmitochondrialinjuryplayedaroleinliverinjury,andhepublished the first unified explanation of the mechanism of liver injury in Alpha-1. Dr. TeckmanhasledseveralseminalstudiesofbothpediatricandadultAlpha-1liver disease and has consulted with pharma industry partners and the FDA Dr Teckman has also served the patient community as an advisor, educator, and advocate
Objectives: Understand the course of adults with ZZ Alpha-1 Antitrypsin Deficiency (AATD) liver disease, which is unpredictable. The utility of markers, includingliverbiopsy,isundefined.
Methods: A prospective cohort, including protocol liver biopsies, was enrolled to addressthesequestions
Results: We enrolled 96 homozygous ZZ AATD adults prospectively at 3 US sites with standardized clinical evaluations, and protocol liver biopsies. Fibrosis was scored using Ishak (stage 0 – 6) 51% of the 96 subjects had Ishak score >1fibrosis(49% Ishak 0-1, 36% Ishak 2-3, and 15% >4). Elevated AST but not ALT, high BMI, obesity, APRI, and elevated serum Z AAT polymer levels were associated with increased fibrosis. Steatosis did not correlate to fibrosis. Increased fibrosis was associated with increased mutant Z polymer globular inclusions (p=0002) and increased diffuse cytoplasmic Z polymer on biopsy (p=0.0029) in a direct relationship. Increased globule Z polymer was associated with increased serum AST (p=0007) and increased periportal inflammation on histopathology (p=0.004), but there was no relationship of Z polymer hepatocellular accumulation with ALT, GGT, inflammation in other parts of the lobule, necrosis or steatosis. Serum Z polymer levels were directly correlated to hepatic Z protein polymer content. Lung function, smoking and alcohol consumptionpatternswerenotassociatedwithfibrosis
Conclusion: In AATD high BMI, obesityand elevated AST are associatedwith increased fibrosis. Liver biopsy features are correlated to some serum tests. Serum Z AAT polymer levels could be a future biomarker to detect fibrosis early andisdirectlycorrelatedtoliverZcontent
Session 4:
Novel Molecular Mechanisms in Alpha-1 Antitrypsin Deficiency (AATD)
James Irving, PhD
Tomás Carroll, PhD
Valerie Gouon-Evans, PhD
Lisa Cabrita, PhD, BSc
Speaker Bios
James Irving, PhD
University College London, UK
Project title: Rational Design of Better Diagnostic Reagents
Dr James Irving is a Principal Research Fellow in the Centre for Respiratory Biology and the Institute of Structural and Molecular Biology, University College LondonandHonoraryResearchFellowatBirkbeckCollege,UniversityofLondon. He undertook his doctorate in biochemistry, structural and computational biology at Monash University in Melbourne, Australia. He has previously worked intheDivisionofStructuralBiologyatOxfordUniversityonanNHMRC(Australia) CJ Martin Fellowship and at the University of Cambridge. Much of his research concerns the molecular processes that lead to protein misfolding and aggregation, how they can inform diagnostics development and how these processes can be prevented. He has accumulated two decades’ experience studying members of the serpin superfamily of proteins, primarily alpha-1 antitrypsin (AAT), and his research has spanned a range of techniques including NMR, X-ray crystallography, single-particle cryo-electron microscopy, biochemicalandbiophysicaltechniques,andcomputationalbiology.
Objectives:Thelifecycleofanalpha-1antitrypsin(AAT)moleculeischaracterized by different structural arrangements (or ‘conformations’) This protein is produced by hepatocytes, where it passes through a (i) partially unfolded intermediate structure, to the (ii) fully folded native form. In the case of the Z variant, here it can also remain in a (iii) misfolded state and ultimately be targeted for destruction; or form long chains of molecules called (iv) polymers, which accumulate in soluble and insoluble fractions in the cell or are secreted The native protein is released into the circulation; upon encountering a target protease it undergoes another structural rearrangement to the (v) cleaved form Each of these conformations provide distinct ‘fingerprints’ on the surface of the molecule. We have developed a repertoire of monoclonal antibodies that are able to recognize different conformations of AAT. We aim to understand the epitopefingerprintsboundbytheantibodieswhichwillallowustointerpret,ina structural sense, the changes in the molecule that they report Using protein engineeringtechniques,thisreactivityisbeingincorporatedintosmallantibodyderived engineered ‘sensor’ molecules that can provide additional useful functionality.
Methods:Usingantibodies,antibodyfragments,recombinantwild-typeAATand point mutants, antibody-A complexes have been reconstructed by integrating data from X-ray crystallography, small angle X-ray scattering, electron microscopy, conjugation of thiol reactive probes and molecular modelling Combiningproteinengineeringapproacheswithcomputationalanalysis,weare developing a single-chain intrinsically fluorescent antibody platform We have synthesized variants of a prototypical chimeric protein with alternative ordering of the antibody-derived segments and introducing solubilizing point mutations predictedbyinsilicotools Optimumexpressionandpurificationconditionshave beenexplored.
Results: The use of an integrative structural biology approach has allowed identification of regions that are altered during polymerization of AAT This provides a structural interpretation of the differential immunorecognition of polymer and native forms of AAT in assays The fine details of the interface reported by crystal structures give further opportunities for optimization by mutagenesisofengineeredantibodies.
In parallel, we have identified a configuration of antibody and fluorescent domains as well as expression and purification conditions to obtain soluble single-chainfluorescentantibodychimeras
Conclusion:Thestructuralfingerprintsrecognizedbyantibodiesthatdistinguish different forms of AAT provide clues as to the pathogenic changes that occur in the molecule. By incorporating this functionality into small, functionalized reporters of conformation, our aim is to generate tools that can be used in mammalian cell models of disease, in biochemical assays and to characterize complexbiologicalmixtures
Royal College of Surgeons in Ireland, Ireland
Project title: A Family Affair - Clarifying the Risk of Lung Disease in ZZ AATD
Dr.TomásCarrollisaseniorlecturerinRCSIinDublin,Ireland,andchiefscientist with Alpha-1 Foundation Ireland After studying at University College Galway (B.Sc.Biotechnology,M.Sc.BiomedicalScience)hebeganaPhDincysticfibrosis at RCSI working with Professor Gerry McElvaney. Following this, Tomás started working on Alpha-1 Antitrypsin Deficiency (AATD) in a position with Alpha-1 Foundation Ireland in 2004. The Foundation is dedicated to raising awareness, increasingdiagnosis,promotingresearch,andimprovingthetreatmentofAATD inIreland(seewww.alpha1.ie).
Tomás Carroll, PhD
Tomás has carried out research leading to over 50 publications spanning basic, clinical, and translational research In addition, he coordinates the national targeted detection program for AATD which has tested over 24,000 Irish people sinceitbeganin2004 TheprogramprovidesfreetestingforAATDandhassofar diagnosedover6,000IrishpeoplewitheithersevereormoderateAATD.
Objectives: Individuals homozygous for the Z mutation (p.Glu342Lys) in the SERPINA1 gene, encoding the alpha-1 antitrypsin (AAT) protein, are known to be at high risk of lung disease due to severe Alpha-1 Antitrypsin Deficiency (AATD). Despite this knowledge, a large majority of ZZ individuals remain undetected or escape detection until severe lung disease is first diagnosed Furthermore, clinical studies in AATD have been predominantly cohort or registry-based. As a result,theysufferfrompronouncedascertainmentbiasandoftenlackadequate or appropriate “normal risk” controls. Thus, after decades of research, there are significant gaps in our understanding of the risk and nature of lung disease associated with severe AATD. Specifically, studies to date have failed to accurately quantify the magnitude of increased COPD risk in the ZZ genotype relative to the normal risk MM genotype, or precisely evaluate the differences in lungdiseaseriskbetweenZZnever-smokersandZZever-smokers.Furthermore, we do not know if ZZ ever-smokers post-smoking cessation continue to demonstrateacceleratedlungfunctiondeclinecomparedtoZZnever-smokers.
Methods:Wewillcarryoutauniquefamilystudytomitigateconfoundingfactors presentinpreviousstudies.ThisstudywillcompareZZindividualstonormalrisk MM controls within the same families so that family members are matched for ageandhomeenvironment.Wewillemployoscillometryinasub-groupanalysis of subjects with preserved spirometry to investigate subtle, early airway disease Inaddition,allstudysubjectsinthestudywillundergoalow-radiationCTscanof the chest to measure lung density Finally, we will assemble a “real world” ZZ cohort from 3 national AATD registries containing longitudinal pulmonary function data. This multinational population (over 600 ZZ) will enable us to evaluatetherateofdeclineinZZindividualsfollowingsmokingcessation.
Results: The risk and natural history of lung disease for ZZ genotypes remain unclear.
Conclusion: We believe that if successful our study will have a tremendous impact on how we view lung risk in the ZZ genotype, with immediate consequences for the appropriate management of ZZ individuals and longertermconsequencesfordiagnosisandtreatment,aswellasdetectionefforts
Valerie Gouon-Evans, PhD
Boston Medical Center, USA
Project title: Engineered and Edited Patient-Derived iPSC for AATD-Associated Liver Disease Cell Therapy
Dr Valerie Gouon-Evans is an Associate Professor of Medicine in the Section of GastroenterologyattheChobanianandAvedisianSchoolofMedicine,Directorof the Program of Boston University Liver Biologists (BULB), and Associate Director of the Molecular & Translational Medicine (MTM) PhD Program of the Department of Medicine. After completing her graduate studies in Paris, Dr. Gouon-Evans joined Dr Pollard's Lab as a postdoctoral fellow where she studied mammary gland development and breast cancer. She then began her career as anInstructorofGeneandCellMedicineattheIcahnSchoolofMedicineatMount SinaiSchoolofMedicineinNewYorkinthelaboratoryofDr.GordonKeller,where she pioneered protocols to efficiently generate hepatocyte-like cells from pluripotent stem cells. She quickly rose up the ranks and was promoted to Assistant Professor before arriving at Boston University. As a PharmD PhD lab leader for 16 years, Dr Gouon-Evans has overseen the creation of a research program to advance understanding of liver development and establishing therapeutic strategies to alleviate liver disease using an induced pluripotent stem cell platform and mouse models. Recently, Dr. Gouon-Evans lab also pioneeredaninnovativetechnologytodeliverregenerativefactorstotheliverto treat various liver diseases, by using mRNA complexed to lipid nanoparticles, which is a validated platform with the widely used mRNA-based COVID-19 vaccines
Objectives:Alpha-1AntitrypsinDeficiency(AATD)isageneticdiseasecausedbya single base pair mutation of the SERPINA1 gene and increases risk of liver and lung disease The most common disease-causing mutation, called the Z mutationorPiZallele,causesAATproteintomisfoldleadingtopolymerizationof Z-AAT in hepatocytes, cell death, fibrosis, and cirrhosis. Liver transplantation is the only treatment for AATD patients that develop end stage liver disease, though donor organs are quite scarce. Alternatively, transplantation of healthy liver cells – either primary human hepatocytes (PHH) or patient-specific induced pluripotent stem cell (iPSC) derived hepatocyte-like cells (HLC) – is a promising approach to restore liver function PHH transplantation has been validated as safe in humans, yet major challenges remain such as low efficiency and lack or sustained benefit. Hepatocyte transplantation remains preclinical, and factors that limit engraftment in liver disease mouse models include poor cell survival, proliferation,andmaturation.
We hypothesize that stimulating key regenerative pathways in transplanted hepatocytes using hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in an environment mimicking hepatocyte senescence by preconditioning the host liver with P21 expression will improve survival, proliferation, and engraftmentofPHHsandHLCsinmice.
Methods: We established a safe way to transiently express HGF and EGF specifically in the liver using nonintegrative nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP). We use AAV8-Tbg-P21 to precondition the mouse liver with long lasting P21 expression in host hepatocytes to prevent their proliferation NSG-PiZ mice serve as our injury model,recapitulatingAATDassociatedliverdisease.
Results:WefindthatbothpreconditioningthehostmouseliverwithAAV8-TbgP21 and the HGF and EGF mRNA-LNP treatments significantly augment transplanted PHH survival and proliferation in vivo in NSG-PiZ mice, evidenced by histological quantification of transplanted cells and human albumin levels in mouse sera in comparison to control with a 37-fold increase. Combined preconditioning with P21 and treatment with HGF+EGF leads to robust repopulationofthemouseliverreaching30%oftheliverrepopulationbyhuman cells and amelioration of AATD liver disease. We specifically showed significant decrease in the misfolded polymer load in the liver, higher level of secretion of normal AAT protein, decreased levels of serum Z-AAT, and a reduction in serum ALT, a hallmark of liver damage. Furthermore, HGF and EGF mRNA-LNP transientlyimprovestransplantediPSC-derivedHLCsurvivalinNSG-PiZmice.
Conclusion: In this study, we established a refined AATD mouse model that recapitulates the p21-induced senescence in hepatocytes observed in AATD patients In this refined PiZ-NSG/AAV8-p21 mouse model, we showed that HGF and EGF mRNA-LNP promoted ~15-fold engraftment of primary human hepatocytes;andthatthislevelofengraftmentsignificantlyalleviatedtheAATDassociated liver disease. Altogether, we have established a novel strategy that robustly improves PHH engraftment and alleviates the AATD-associated liver disease burden that could potentially also improve patient-specific iPSC-derived HLCtherapy.
Lisa Cabrita, PhD, BSc
University College London, UK
Project title: Modulating the Co-Translational Misfolding and Polymerisation of Antitrypsin
Dr LisaCabritacompletedherPhDinBiochemistryandMolecularBiologyinthe Department of Biochemistry at Monash University in Melbourne, Australia studying serine proteinase inhibitors. She then took up a C.J. Martin fellowship with Chris Dobson at the University of Cambridge where she worked on developing a recombinant system for studying protein folding on the ribosome. She then took up a postdoctoral position with John Christodoulou at University College London continuing these studies. In 2017, she took up an independent positionatUCLandisnowanAssociateProfessor Herresearchgroupfocuseson understanding the molecular links between protein biosynthesis and dysfunctioninhumandisease.
Objectives: My research studies the alpha-1 antrypsin’s (AAT) folding and misfolding at a molecular level and its connection to the onset of Alpha-1 AntitrypsinDeficiency(AATD).Ourfocusisunderstandingtheseprocesseswhere they first begin, during translation on the ribosome, the cell’s dedicated machinery for protein biogenesis. Our aim is to provide molecular-level descriptionsofAAT’sco-translationalfoldingandmisfoldingpathways
Methods: We have been developing an in vitro, reticulocyte-based cell-free system together with an inducible cell-based model in HEK293, to generate “biosynthetic snapshots” of AAT, which use ribosome-nascent chain complexes (RNC) to capture discrete stages of protein biosynthesis on the ribosomeUsing quantitative biochemical approaches including PAGE and cysteine-mapping (PEGylation),andcellbiologytools,wearedevelopingkineticmodelstoevaluate the fate of the nascent polypeptide chain, co-translationally. These biochemical observations are guiding structural studies using cryo-electron microscopy and NMR spectroscopy to understand the conformational properties of the RNCs at highresolution.
Results: We provide new mechanistic details for Z AAT’s early biosynthesis and folding on the ribosomes Using molecular dynamics simulations and cryoelectronmicroscopy,wehavegeneratedamolecularmodelwhichillustratesthe range of conformations that are sampled by a full-length Z polypeptide chain while it is bound to the ribosome.The average conformation resembles a partially-foldedintermediateensemble,typicallyreferredtoasZ’s“kineticfolding trap”.
WefindusingimmunofluorescenceandbiochemicalstudiesofHEK293cellsthat ribosomes translating Z-AAT can pause and recruit released Z polypeptides to initiate polymerization. We propose that this mode of polymerization requires the availability of Z’s compacted N-terminal region (D1-K191) and involves a (more) substantial swap of the C-terminal region (ca. 75 amino acids) than what is currently understood for the mature protein We have initiated proof-ofconcept studies to show how small molecules can modulate this process of misfoldingontheribosome.
Conclusion: Our structural and mechanistic studies propose a novel mode of misfoldingforZAATwithinintheendoplasmicreticulum Thisinvolvesexistence of parallel pathways for polymerization that can begin during translation on ribosomes(co-translationally),aswellasforthereleased,matureZprotein(posttranslationally). These results contribute to a contemporary model for the pathogenesisofAlpha-1AntitrypsinDeficiency(AATD)andwillprovideabasisfor the development of early intervention therapeutics that target Z during its biosynthesis.
A1F 8th Biennial Investigators’ Meeting
October 19, 2024
A1F 8th Biennial Investigators’ Meeting
October 19, 2024
A1F 8th Biennial Investigators’ Meeting
October 19, 2024
A1F 8th Biennial Investigators’ Meeting
October 19, 2024
The Alpha-1 Foundation (A1F) acknowledges the following sponsors for their ongoing commitment to the advancement of research:
AlphaNet
Beam Therapeutics
CSL Behring Grifols
Takeda
The Alpha-1 Foundation (A1F) is committed to finding a cure for Alpha-1 Antitrypsin Deficiency (Alpha-1) and to improving the lives of people affected by Alpha-1 worldwide.
A1F has invested over $100 million to support Alpha-1 research and programs in 130 institutions in North America, Europe, the Middle East and Australia.
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