Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

Abstract

Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of “repaired” arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.

Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice

“…We designed a TALEN pair targeting intron 6 of the Arg1 locus at a region adjacent to the position targeted previously by CRISPR/Cas9. 24 Each monomer contains an array of RVDs to bind the target DNA sequences ( Figure 1 A). The TALEN expression constructs showed good transfection efficiency when introduced into mouse iPSCs by electroporation ( Figure 1 B).…”

“…Surveyor nuclease cleavage to detect NHEJ events of PCR products from the target region produced two bands in the 162- to 186-bp range, with insertion or deletion (indel) frequencies up to 18% in mouse iPSCs ( Figure 1 C), similar to that with CRISPR/Cas9. 24 The TALEN pair target sites were selected to utilize a BspHI restriction site located within the spacer region to determine editing efficiency. The loss of the BspHI recognition sequence was demonstrated by the presence of uncleaved PCR products compared to the control ( Figure 1 D).…”

“… 21 , 22 , 23 Recently, we generated induced pluripotent stem cells (iPSCs) from our inducible arginase-1-deficient mouse model carrying a deletion of Arg1 exons 7 and 8 ( Arg1 Δ ), which results in defective function of arginase-1. 24 We showed that the deletion was repaired using CRISPR/Cas9, in combination with an excisable piggyBac transposon system to target corrective sequences to the endogenous Arg1 locus. In the current study, we report on TALEN-mediated correction of the Arg1 Δ alleles in iPSCs and their successful differentiation to hepatocyte-like cells (HLCs) coupled with piggyBac transposon selection technology for seamless genetic manipulation, followed by secondary expansion to propagate the repaired iPSC-derived HLCs (named iHLCs hereafter).…”

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice

“…This system introduces a plasmid that encodes a desired gene fragment into T cells and then inserts into the cell genome with the transiently expressed transposase enzyme to recognize inverted repeat sequences. A previous genome-wide study indicated that the piggyBac transposon led to stable integration of the transgene and is suitable for clinical application because of the non-preferential integration into proto-oncogenes and reduction of production cost compared with viral vectors 15 .…”

Cell and Gene Therapy for Carbamoyl Phosphate Synthetase 1 Deficiency

“…The iPSC-derived macrophages also expressed substantial amounts of arginase. They believe that their studies provide proof-of-concept for gene editing in UCDs [25].…”