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Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder α-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy.

More than a half of plasma proteins are N-glycosylated. Most of them are synthesized, glycosylated, and secreted to the bloodstream by liver and lymphoid tissues. While associations with N-glycosylation are implicated in the rising number of liver, cardiometabolic, and immune diseases, little is known about the genetic regulation of this process. Here, we performed the largest genome-wide association study of N-glycosylation of the blood plasma proteome in 10,000 individuals. We doubled the number of genetic loci known to be associated with blood N-glycosylation by identifying 16 novel loci and prioritizing 13 novel genes contributing to N-glycosylation. Among these were the GCKR , TRIB1 , HP, SERPINA1 and CFH genes. These genes are predominantly expressed in the liver and show a previously unknown genetic link between plasma protein N-glycosylation, metabolic and liver diseases, and inflammatory response. By integrating glycomics, proteomics, transcriptomics, and genomics, we provide a resource that facilitates deeper exploration of disease pathogenesis and supports the discovery of glycan-based biomarkers. Proteins are often modified by complex carbohydrates (N-glycans). Here, authors identified gene regulators of this process and uncovered links between plasma protein Nglycosylation, metabolic and liver diseases, and anti-inflammatory proteins.

Site-directed RNA editing might provide a safer or more effective alternative to genome editing in certain clinical scenarios. Until now, RNA editing has relied on overexpression of exogenous RNA editing enzymes or of endogenous human ADAR (adenosine deaminase acting on RNA) enzymes. Here we describe the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). We observed almost no off-target editing, and natural editing homeostasis was not perturbed. We successfully applied RESTORE to a panel of standard human cell lines and human primary cells and demonstrated repair of the clinically relevant PiZZ mutation, which causes α1-antitrypsin deficiency, and editing of phosphotyrosine 701 in STAT1, the activity switch of the signaling factor. RESTORE requires only the administration of an oligonucleotide, circumvents ectopic expression of proteins, and represents an attractive approach for drug development. Endogenous RNAs are edited using antisense oligos that recruit endogenous RNA-editing enzymes.

Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson’s disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.

During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z’s misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease. Alpha-1-antitrypsin (AAT) deficiency results from misfolding-prone AAT variants. Here the authors show that AAT forms co-translational folding intermediates on the ribosome that persist upon release and determine its folding fate. They show too that the ribosome can also modulate misfolding-prone AAT intermediates during their synthesis.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology. Borel et al. describe two studies based on misfolded human α-1 antitrypsin (A1AT). First, when A1AT is expressed in livers of NSG, mice it allows for reproducible engraftment of human hepatocytes. Second, gene editing of hepatocytes to decrease misfolded protein results in expansion of corrected cells and amelioration of liver disease.

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%–3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%–2.5% of the target level from years 1–5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated. Alpha-1 antitrypsin deficient patients injected intramuscularly with a rAAV1 vector demonstrated 5 years of stable transgene expression after a single dose, coincident with an anti-capsid Treg response and exhaustion of CD8 cells. Mueller et al. also observed partial correction of disease biomarkers despite serum levels below the target level for protein replacement.

Background Mutations in the SERPINA1 gene can result in alpha-1 antitrypsin deficiency (AATD), which may be associated with lung or liver injury. Although the S and Z alleles account for over 95% of cases of AATD, a wide variety of rare variants have been linked to deficiency and dysfunction, while other variants are associated with normal alpha-1 antitrypsin (AAT) levels and activity. Here, we present the identification and characterization of thirteen rare SERPINA1 variants discovered during the genetic diagnosis of AATD by the Progenika diagnostic network. Methods The new variants were identified by sequencing the exons of SERPINA1 gene in cases with discrepancies between AAT serum levels and initial genotyping. In order to determine their pathogenic impact, the variants were expressed in a cellular model and evaluated for AAT secretion, intracellular accumulation and elastase inhibitory activity. In addition, protein structural mapping of the variants and analysis of positioning and residue/atomic contacts were performed. Results The in silico and functional in vitro analysis allowed us to classify these AAT variants as six deficient (p.Val234Glu, p.Val242_Pro243insLeu, p.Leu291Phe, p.Ala308Ser, p.Pro393Thr and p.Pro393Arg), one dysfunctional (p.Thr96Ile), three normal (p.Ser71Arg, p.Ala349Pro and p.Asp365Glu) and three null alleles (p.Gln33*, p.Gln285* and p.Leu310Phefs*14). Conclusions Functional assays and protein structural information are useful tools in the characterization of novel variants of the SERPINA1 gene. The newly characterized mutations expand the number of SERPINA1 variants with proven pathogenic effects, facilitating the diagnoses of future cases of AATD.