Genetically engineered cell lines for α1-antitrypsin expression
To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination.
α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines. Cell clones obtained had the AAT gene integrated at the AAVS1 locus and secreted approx. 0.04 g/l recombinant AAT into the medium. Moreover, the secreted AAT showed an inhibitory activity that is comparable to plasma AAT.
CRISPR/Cas9-mediated engineering of human cells is a promising alternative for generating isogenic cell lines with consistent AAT production. This work sheds new light on the generation of therapeutic liver stem cells for AATD.
KeywordsAAVS1 locus α1-Antitrypsin deficiency CRISPR/Cas9 Homologous recombination
This work was supported by China Postdoctoral Science Foundation (Grant No. 2015M582465).
Supplementary Table 1—Primer sequences.
Supplementary Figure 1—SDS-PAGE of AAT in puromycin-resistant HEK293T cells after transfection.
Compliance with ethical standards
Conflicts of interest
The authors have no conflict of interest to declare, and no competing financial interests exist.
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