Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein

Abstract

Citrullinemia type I (CTLN-I) results from the absence or deficiency of argininosuccinate synthetase (AS), a 46 kDa enzyme that acts in the cytosol of hepatocytes to convert aspartic acid and citrulline into argininosuccinic acid. AS is an essential component of the urea cycle, and its absence or deficiency results in the harmful accumulation of ammonia in blood and cerebrospinal fluid. No disease-modifying treatment of CTLN-I exists. Here we report that the cell-permeant miniature protein (CPMP) ZF5.3 (ZF) can deliver AS to the cytosol of cells in culture and the livers of healthy mice. The fusion protein ZF-AS is catalytically active in vitro, stabilized in plasma, and traffics successfully to the cytosol of cultured Saos-2 and SK-HEP-1 cells, achieving cytosolic concentrations greater than 100 nM. This value is 3−10-fold higher than the concentration of endogenous AS (11 ± 1 to 44 ± 5 nM). When injected into healthy C57BL/6 mice, ZF-AS reaches the mouse liver to establish concentrations almost 200 nM above baseline. These studies demonstrate that ZF5.3 can deliver a complex enzyme to the cytosol at therapeutically relevant concentrations and support its application as an improved delivery vehicle for therapeutic proteins that function in the cytosol, including enzyme replacement therapies.

Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2

“…These results largely mirrored the results obtained in Saos-2 cells, although overall the protein concentration in the nucleus was 1.8-3.8 fold lower than observed in Saos-2 cells. Similar cell type-to-cell type variations in delivery efficiency have been observed before among HeLa cells, SK-HEP1 cells, and Saos-2 cells 9,11 . Nevertheless, these results provide confidence that ZF5.3 and Tat improve by 2.0-2.5 fold the nuclear delivery of t MeCP2-Rho in two model cell lines.…”

“…The cellular fractionation experiment was adapted from previous reports 9–11 with modifications to ensure pure nuclei isolation. Saos-2 cells were grown to ∼2.5 × 10 6 cells in a 100 mm dish in McCoy’s 5A medium (+15% FBS, -phenol red).…”

“…Previous efforts to deliver MeCP2 protein suffered from low delivery efficiency and significant cargo degradation 25–29 . We showed previously that the mini-protein ZF5.3 7–11 is taken up by the endosomal pathway and released efficiently into the cytosol and nuclei of live cells 8 , alone and when fused to certain protein cargos. Proteins successfully delivered using ZF5.3 include the model protein SNAP-tag 9 , the metabolic enzyme argininosuccinate synthetase 11 , and the proximity labeling tool APEX2 9 .…”

“…Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice 3–5 , suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity. We report that ZF- t MeCP2, a conjugate of MeCP2(Δaa13-71, 313-484) 6 and the cell-permeant mini-protein ZF5.3 7–11 , both binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF- t MeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters.…”

Parallel Automated Flow Synthesis of Covalent Protein Complexes That Can Inhibit MYC-Driven Transcription

“…The DNA binding regions of several transcription factors often are polycationic and α-helical, two traits of cell-penetrating peptides . Indeed, several transcription factor proteins were independently shown to be intrinsically cell-penetrating, − including Soucek’s discovery regarding Omomyc . Our group recently reported the synergistic effect of multiple cell-penetrating moieties combined in one sequence .…”