Non-clinical, quality and environmental impact assessments of cell and gene therapy products: Report on the 5th Asia Partnership Conference of Regenerative Medicine - April 7, 2022.

The 5th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 7, 2022 to promote regulatory harmonization of regenerative medicine products throughout Asia. The recognition of domestic regulatory guidelines within each country and region and the underpinning rationales are important initial steps toward the harmonization of regulations. The 5th APACRM featured open dialog regarding non-clinical, quality and environmental impact assessment settings for cell and gene therapy products through presentations from the industry and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region were also introduced. This paper summarizes the proceedings of the 5th APACRM for public dissemination to foster future discussion.

Albumin and apolipoprotein H mRNAs in human plasma as potential clinical biomarkers of liver injury: analyses of plasma liver-specific mRNAs in patients with liver injury.

CONTEXT: Plasma liver-specific mRNAs are useful biomarkers of hepatotoxicity in rats. OBJECTIVE: To investigate the potential application of liver-specific mRNAs as biomarkers for liver injury in humans. METHODS: We determined the plasma levels of liver-specific mRNAs by real-time qRT-PCR in healthy donors and patients with liver injury. RESULTS: Plasma levels of albumin (ALB) and apolipoprotein H (APOH) mRNAs increased in patients with elevated serum alanine aminotransferase. These mRNAs also increased in plasma after transcatheter arterial chemoembolization, which induces specific injury to liver. CONCLUSIONS: We demonstrated the potential application of plasma ALB and APOH mRNAs as clinical biomarkers for liver injury.

Identification of novel liver-specific mRNAs in plasma for biomarkers of drug-induced liver injury and quantitative evaluation in rats treated with various hepatotoxic compounds.

Circulating liver-specific mRNAs such as albumin (Alb) and α-1-microglobulin/bikunin precursor (Ambp) have been reported to be potential biomarkers for drug-induced liver injury (DILI). We identified novel circulating liver-specific mRNAs and quantified them, together with the two previously reported mRNAs, in plasma from rats treated with various hepatotoxicants to validate circulating liver-specific mRNAs as biomarkers for DILI. Among six genes selected from the database, high liver specificity of apolipoprotein h (Apoh) and group-specific component (Gc) mRNAs were confirmed by reverse transcription (RT)-PCR and the copy numbers of these mRNAs elevated in plasma from rats treated with thioacetamide. Liver-specific mRNAs (Alb, Ambp, Apoh, and Gc) were quantified by real-time RT-PCR in plasma from rats with single dosing of seven hepatotoxicants. There were noticeable interindividual and intercompound variabilities in the severity of liver injury. The levels of four mRNAs increased almost in parallel and correlated with changes in the alanine aminotransferase (ALT) values and the hepatocellular necrosis scores at 24h after dosing. It was noteworthy that the magnitude of the increases in mRNA levels was greater than that in the ALT value. Time course analysis within 24h after dosing revealed that the timing of the increase was different among mRNA species, and the plasma levels of Alb and Gc mRNAs increased substantially earlier than the ALT values, suggesting that patterns of changes in circulating liver-specific mRNAs indicate the progression of liver injury. These results strongly support the reliability and usefulness of the four circulating liver-specific mRNAs as biomarkers for DILI.

Real-time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock.

Resetting the peripheral clock and understanding the integration between the circadian rhythm and metabolic pathways are fundamental questions. To test whether insulin acts as a synchronizer for the hepatic clock by cell-autonomous mechanisms, the phase-resetting capabilities of insulin were investigated in cultured hepatic cells. We provide evidence that three-dimensional (3D) cell culture conditions that preserve the differentiated state of primary hepatocytes sustained the robustness of the molecular clock, while this robustness rapidly dampened under classical monolayer cell culture conditions. Herein, we established a 3D cell culture system coupled with a real-time luciferase reporter, and demonstrated that insulin directly regulates the phase entrainment of hepatocyte circadian oscillators. We found that insulin-deficient diabetic rats had a pronounced phase advance in their hepatic clock. Subsequently, a single administration of insulin induced phase-dependent bi-directional phase shifts in diabetic rat livers. Our results clearly demonstrate that insulin is a liver clock synchronizer.

Electronic structures of single-walled carbon nanotubes encapsulating ellipsoidal C70.

The molecular orientation of ellipsoidal C(70) in single-walled carbon nanotubes (SWCNTs) depends on the tube diameter (d(t)). Photoluminescence (PL) studies reveal that the fullerene encapsulation effects on the optical transition energy of SWCNTs are significantly different for C(70) and C(60) at d(t) = 1.405-1.431 nm. This indicates that the transition from the "lying" alignment to the "standing" alignment occurs at d(t) ≈ 1.41 nm and the electronic states of SWCNTs are very sensitive to the interspacing between the encapsulated molecules and the SWCNTs. The present findings suggest that the electronic structure of SWCNTs is tunable not only by alternating the encapsulated molecules but also by controlling their molecular orientations, thus paving the way for development of novel SWCNT-based devices.

Regular feeding plays an important role in cholesterol homeostasis through the liver circadian clock.

RATIONALE: Peripheral clock control and the relevance of the circadian rhythm to physiology and disease are major questions in mammalian circadian biology. OBJECTIVE: We examined the physiological functions of the liver clock. METHODS AND RESULTS: We established a suppressed feeding schedule regimen constituting a high-cholesterol diet delivered every 6 hours without changes in energy and cholesterol intake. We found that rats exposed to this regimen developed hypercholesteremia. In the liver, the rhythmicity of expression of several clock genes was disrupted. Furthermore, the nocturnal expression of the CYP7A1 gene, which encodes the rate-limiting enzyme for the conversion of cholesterol to bile acids, was shifted to a diurnal pattern. Indeed, suppression of a regular feeding rhythm increased the secretion rate of very-low-density lipoprotein cholesterol from the liver and decreased the excretion of fecal bile acids. CONCLUSIONS: Our results demonstrated that not only the amount and quality of food but also the timing of meals has crucial health implications.

Optical band gap modification of single-walled carbon nanotubes by encapsulated fullerenes.

We report optical band gap modifications of single-walled carbon nanotubes upon C60 insertions by using photoluminescence and the corresponding excitation spectroscopy. The shifts in optical transition energies strongly depend on the tube diameter (dt) and the "2n + m" family type, which can be explained by the local strain and the hybridization between the nanotube states and the C60 molecular orbitals. The present results provide possible design rules for nanotube-based heterostructures having a specific type of electronic functionality.

Enhanced 1520 nm photoluminescence from Er3+ ions in di-erbium-carbide metallofullerenes (Er2C2)@C82 (isomers I, II, and III).

Di-erbium and di-erbium-carbide endohedral metallofullerenes with a C(82) cage such as Er(2)@C(82) (isomers I, II, and III) and (Er(2)C(2))@C(82) (isomers I, II, and III) have been synthesized and chromatographically isolated (99%). The structures of Er(2)@C(82) (I, II, III) and (Er(2)C(2))@C(82) (I, II, III) metallofullerenes are characterized by comparison with the UV-vis-NIR absorption spectra of (Y(2)C(2))@C(82) (I, II, III), where molecular symmetries of the structures are determined to be C(s), C(2v) and C(3v), respectively. Furthermore, enhanced near-infrared photoluminescence (PL) at 1520 nm from Er(3+) ions in Er(2)@C(82) (I, III) and (Er(2)C(2))@C(82) (I, III) have been observed at room temperature. The PL intensities have been shown to depend on the symmetry of the C(82) cage. In particular, the PL intensity of (Er(2)C(2))@C(82) (III) has been the strongest among the isomers of Er(2)@C(82) and (Er(2)C(2))@C(82). Optical measurements indicate that the PL properties of Er(2)@C(82) (I, II, III) and (Er(2)C(2))@C(82) (I, II, III) correlate strongly with the absorbance at 1520 nm and the HOMO-LUMO energy gap of the C(82) cage.

Isolation and characterization of a carbene derivative of La@C82.

The photochemical reaction of La@C82 with 2-adamantane-2,3-[3H]-diazirine affords adduct 2, La@C82(Ad), in a quantitative and highly selective manner. The structure of compound 2 is confirmed by ESR, MS, and UV-vis-NIR spectroscopies, and the first X-ray crystallographic characterization of an endohedral monometallofullerene derivative is reported.