Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF2-Stp and LAF4-Stp2 structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF2-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF2-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43 % of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.

被引量：- 发表：1970

State-of-the-art and future perspectives in ingestible remotely controlled smart capsules for drug delivery: A GENEGUT review.

被引量：- 发表：1970

Corrigendum to "Epothilone B inactivation of Sirtuin1 promotes mitochondrial reactive oxygen species to induce dysfunction and ferroptosis of Schwann cells" [European Journal of Pharmaeutical Sciences 181 (2023) 106350].

被引量：- 发表：1970

Ultrasound-mediated nanobubbles loaded with STAT6 siRNA inhibit TGF-β1-EMT axis in LUSC cells via overcoming the polarization of M2-TAMs.

M2-like tumor-associated macrophages (M2-TAMs) are closely correlated with metastasis and poor clinical outcomes in lung squamous cell carcinoma (LUSC). Previous studies have demonstrated that STAT6 is an important signaling molecule involved in the polarization of M2-TAMs, EMT is the main way for TAMs to promote tumor progression. However, little attention has been paid to the effect of STAT6 inhibition on LUSC, and it is difficult to achieve an ideal gene silencing effect in immune cells using traditional gene transfection methods. Here, we investigated the optimal concentration of 12-myristic 13-acetate (PMA), lipopolysaccharide (LPS) for the induction of THP-1 into M1-TAMs and M2-TAMs. The expression of pSTAT6 and STAT6 was confirmed in three types of macrophages, and it was demonstrated that pSTAT6 can be used as a specific target of M2-TAMs derived from THP-1. Ultrasound-mediated nanobubble destruction (UMND) is a non-invasive and safe gene delivery technology. We also synthesized PLGA-PEI nanobubbles (NBs) to load and deliver STAT6 small interfering RNA (siRNA) into M2-TAMs via UMND. The results show that the NBs could effectively load with siRNA and had good biocompatibility. We found that UMND enhanced the transfection efficiency of siRNA, as well as the silencing effect of pSTAT6 and the inhibition of M2-TAMs. Simultaneously, when STAT6 siRNA entered M2-TAMs by UMND, proliferation, migration, invasion and EMT in LUSC cells could be inhibited via the transforming growth factor-β1 (TGF-β1) pathway. Therefore, our results confirm that UMND is an ideal siRNA delivery strategy, revealing its potential to inhibit M2-TAMs polarization and ultimately treat LUSC.

被引量：- 发表：1970

Absorption, distribution, metabolism, and excretion of tirzepatide in humans, rats, and monkeys.

Tirzepatide is a once-weekly GIP/GLP-1 receptor agonist used for treatment of type 2 diabetes (T2D) in adults and was recently approved for treatment of obesity. To determine the absorption, distribution, metabolism, and excretion (ADME) of tirzepatide, [14C]-radiolabeled tirzepatide was investigated in both humans and preclinical species. [14C]-Tirzepatide was prepared by incorporating four 14C's in the linker region between the amino acid backbone and the di-acid moiety. Healthy male participants received a single subcutaneous dose of approximately 2.9 mg tirzepatide containing approximately 100 μCi of [14C]-tirzepatide. Preclinical studies were conducted in male Sprague Dawley and Long Evans rats administered a single dose of 3 mg kg-1 (133 µCi/kg) of [14C]-tirzepatide, and male cynomolgus monkeys administered a single dose of 0.5 mg kg-1 (20 µCi/kg) of [14C]-tirzepatide. Following a single SC dose of [14C]-tirzepatide in humans, the majority of the excreted dose was recovered within 480 h. Renal excretion was identified as a principal route of elimination in all species with approximately 66 % of the administered radioactivity recovered in urine, while approximately 33 % was eliminated in feces in humans. Metabolite analysis of tirzepatide revealed the parent drug was the major circulating component in human, rat, and monkey plasma. Metabolites identified in human plasma were similar to circulating metabolites found in rats and monkeys with no circulating metabolites representing >10 % of the total radioactive drug-related exposure. Intact tirzepatide was not observed in urine or feces in any species. Tirzepatide was primarily metabolized via proteolytic cleavage of the amino acid backbone, β-oxidation of the C20 diacid moiety, and amide hydrolysis. ClinicalTrials.gov identifier: NCT 04,311,424.

被引量：- 发表：1970