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

Sosulski, Meredith L.; Stiles, Katie M.; Frenk, E.; Hart, Fiona M.; Matsumura, Yuki; De, Bishnu P.; Kaminsky, Stephen M.; Crystal, Ronald G.

doi:10.1172/jci.insight.135951

Cited by 13 publications

(9 citation statements)

References 92 publications

(222 reference statements)

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“…Oxidative inactivation of A1AT is likely contributing to lung damage caused by elastase in individuals exposed to oxidizing agents (Sosulski et al, 2020). It is well known that oxidation of Met 351 and Met 358 residues located at the reactive site of A1AT results in the loss of its anti-elastase activity (Johnson & Travis, 1979).…”

Section: Discussionmentioning

confidence: 99%

High‐level production of wild‐type and oxidation‐resistant recombinant alpha‐1‐antitrypsin in glycoengineered CHO cells

Koyuturk

Kedia

Robotham

et al. 2022

Biotech & Bioengineering

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Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α−1,6-fucosylation (Fut8) and α−2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α−2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5-to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media

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Section: Discussionmentioning

confidence: 99%

High‐level production of wild‐type and oxidation‐resistant recombinant alpha‐1‐antitrypsin in glycoengineered CHO cells

Koyuturk

Kedia

Robotham

et al. 2022

Biotech & Bioengineering

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“…Our findings are consistent with recent studies showing that mice expressing high levels of the M351V/M358Lsubstituted AAT variant, were protected against an excess of oxidants in vivo. (42) A schematic illustrating key findings of protective AAT effects in the AAV context is shown in Figure 8. Our data support the notion that quantitative AAT aspects do not sufficiently reflect the complex AAV disease situation and that additional qualitative AAT aspects and the balance with PR3 are important.…”

Section: Discussionmentioning

confidence: 99%

“…The AVL-AAT plasmid was a kind gift from Dr. Ron Crystal (Ithaca, NY, USA) and was previously described. (42) We amplified a c-terminal AVL-AAT part containing the oxidation-resistant methionine substitutions in the reactive center loop (M351V/M358L) using the forward primer-…”

Section: Pr3 and Aat Mrna Expression In Isolated Neutrophilsmentioning

confidence: 99%

Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation

Ebert¹,

Jerke²,

Eulenberg-Gustavus³

et al. 2022

Journal of Clinical Investigation

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Background. Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCA directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surfacepresented antigen, activate neutrophils and cause vasculitis. An imbalance between PR3 and its major inhibitor 1-antitrypsin (AAT) was proposed to underlie PR3-but not MPO-AAV. We measured AAT and PR3 in healthies and AAV patients and studied protective AAT effects pertaining to PR3-and MPO-ANCA. Methods.Plasma and blood neutrophils were assessed for PR3 and AAT. Wild-type, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, FRET assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring.Results. We found significantly increased PR3 and AAT pools in both PR3-and MPO-AAV patients, however, only in PR3-AAV did the PR3 pool correlate with ANCA titer, inflammatory response and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active PR3-AAV patients showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when two critical methionines were substituted by valine and leucine. Conclusion.Pathogenic differences between PR3-and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.

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“…Janosz and colleagues reported in vitro generation of AAT MΦ, enabling to engraft into the pulmonary microenvironment and convert into alveolar macrophages [ 110 ]. Recently, serotype 8 adeno-associated virus (AAV 8/AVL) has been presented as a second-generation gene therapy for AATD, encouraged by superior antiprotease protection even in an oxidative stress situation [ 111 ]. Another group evaluated and described the cytosine and adenine base editing for potential treatment of AATD [ 112 ].…”

Section: Alpha-1-anti Trypsin Deficiencymentioning

confidence: 99%

Novel Gene-Correction-Based Therapeutic Modalities for Monogenic Liver Disorders

et al. 2022

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The majority of monogenic liver diseases are autosomal recessive disorders, with few being sex-related or co-dominant. Although orthotopic liver transplantation (LT) is currently the sole therapeutic option for end-stage patients, such an invasive surgical approach is severely restricted by the lack of donors and post-transplant complications, mainly associated with life-long immunosuppressive regimens. Therefore, the last decade has witnessed efforts for innovative cellular or gene-based therapeutic strategies. Gene therapy is a promising approach for treatment of many hereditary disorders, such as monogenic inborn errors. The liver is an organ characterized by unique features, making it an attractive target for in vivo and ex vivo gene transfer. The current genetic approaches for hereditary liver diseases are mediated by viral or non-viral vectors, with promising results generated by gene-editing tools, such as CRISPR-Cas9 technology. Despite massive progress in experimental gene-correction technologies, limitations in validated approaches for monogenic liver disorders have encouraged researchers to refine promising gene therapy protocols. Herein, we highlighted the most common monogenetic liver disorders, followed by proposed genetic engineering approaches, offered as promising therapeutic modalities.

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

Cited by 13 publications

References 92 publications

High‐level production of wild‐type and oxidation‐resistant recombinant alpha‐1‐antitrypsin in glycoengineered CHO cells

High‐level production of wild‐type and oxidation‐resistant recombinant alpha‐1‐antitrypsin in glycoengineered CHO cells

Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation

Novel Gene-Correction-Based Therapeutic Modalities for Monogenic Liver Disorders

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