PB1316 - Impact of Synonymous Gene Recoding on ADAMTS13

Tuesday, June 27, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Brian Lin, PhD

Research Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States

Co-Author(s)

RA

Randilu Amarasinghe

Research Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States
Upendra K. Katneni, PhD

Staff Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States
KJ

Katarzyna I. Jankowska

Research Biologist
US Food and Drug Administration
Silver Spring, Maryland, United States
NH

Nobuko Hamasaki-Katagiri

Research Biologist
US Food and Drug Administration
Silver Spring, Maryland, United States
DH

David D. Holcomb

Research Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States
NP

Nigam Padhiar

Research Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States
SF

Sarah E. Fumagalli

Research Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States
JP

Je-Nie Phue

Research Biologist
US Food and Drug Administration
Silver Spring, Maryland, United States
WW

Wells W. Wu

Research Chemist
US Food and Drug Administration
Silver Spring, Maryland, United States
AG

Arnab Ghosh

Research Fellow
Cleveland State University
Cleveland, Ohio, United States
DF

Darón I. Freedberg

Research Chemist
US Food and Drug Administration
Silver Spring, Maryland, United States
OK

Ofer Kimchi

Research Fellow
Princeton University
Princeton, New Jersey, United States
RS

Rong-Fong Shen

Head Director, Facility for Biotechnology
US Food and Drug Administration
Silver Spring, Maryland, United States
AK

Anton A. Komar

Professor
Cleveland State University
Cleveland, Ohio, United States
ZS

Zuben E. Sauna

Biologist
U.S. Food and Drug Administration
Silver Spring, Maryland, United States
CK

Chava Kimchi-Sarfaty

Research Chemist
US Food and Drug Administration
Silver Spring, Maryland, United States

Background: Synonymous gene recoding is used by the biotechnology industry to enhance yields of protein therapeutics. While synonymous variants do not alter the amino acid sequence, the substitutions can have deleterious effects on recombinant therapeutics by altering protein structure, function, and immunogenicity. ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13) is a plasma protease that cleaves ultra-large Von Willebrand Factor (VWF) multimers to maintain hemostasis. ADAMTS13 deficiency is common in thrombotic thrombocytopenic purpura (TTP) and sickle cell disease, and recombinant ADAMTS13 is currently under development as a potential treatment.

Aims: To investigate the effects of two synonymous gene recoding strategies, codon and codon-pair optimization, on the biological and physicochemical properties of ADAMTS13.

Methods: We expressed recoded ADAMTS13 genes in HEK293 cells through three transduction methods: transient, single copy Flp-In integration, and stable lentiviral systems. We investigated the impact of recoding and these delivery systems on ADAMTS13 activity, stability, conformation, and immunogenicity. We evaluated the cellular effects of expressing recoded ADAMTS13 by assessing cellular bioenergetics using Seahorse assays, and endoplasmic reticulum (ER) stress and chaperone activity by immunoblotting for the ER chaperone, binding immunoglobulin protein (BiP), and protein-protein interaction assays.

Results: We find that gene recoding and use of different cellular delivery systems can alter expression of recoded ADAMTS13 and results in (i) a more than 3-fold decrease in specific-activity in transient and stable lentiviral integration systems, (ii) more stable ADAMTS13 when recoded through codon optimization, as measured by a cycloheximide-chase assay, (iii) altered ADAMTS13 conformations as determined by circular dichroism spectral analysis and (iv) potentially less immunogenic ADAMTS13. Cells expressing recoded ADAMTS13 adopt more active bioenergetic states, produce more ATP, exhibit ER stress, and necessitate increased chaperone surveillance.

Conclusion(s): Our results demonstrate that gene recoding of therapeutic proteins could affect their safety and efficacy.