Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70-90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin\'s ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.

UNASSIGNED: Achilles tendon thickening (ATT) can be ameliorated by lowering low-density lipoprotein (LDL) levels in patients with familial hypercholesterolemia (FH). The Japan Atherosclerosis Society (JAS) defines ATT as ≥8.0 mm in males and ≥7.5 mm in females. We aimed to determine the clinical impact of changes in ATT on the development of major adverse cardiovascular events (MACE).
UNASSIGNED: Patients with clinically diagnosed heterozygous FH (HeFH) (N = 1273; 614 males, 659 females) with ATT data from X-ray were assessed. Patients were divided into four groups: patients without ATT from baseline until follow-up (group 1), patients without ATT at baseline but developed ATT at follow-up (group 2), patients with ATT at baseline but regressed at follow-up (group 3), and patients with ATT from baseline until follow-up (group 4). Cox proportional hazard models were used to assess the factors associated with MACE, including cardiovascular death and any coronary events.
UNASSIGNED: On follow-up (median: 10.9 years), 142 MACEs were observed, and the median ATT regressed from 7.8 to 7.6 mm. Changes in ATT were significantly associated with the occurrence of MACE in all groups, when compared to group 1 (hazard ratio [HR]: 2.73; 95 % confidence interval [CI]: 1.33-4.13 [p < 0.001], HR: 2.18, 95 % CI: 1.08-3.28, [p < 0.001], HR: 6.34, 95 % CI: 3.10-9.58, [p < 0.001], in groups 2, 3, and 4, respectively).
UNASSIGNED: Assessing ATT has diagnostic value and allows for risk stratification among patients with HeFH.

Rationale: The gut and its accessory organ, the liver, are crucial determinants of metabolic homeostasis via the regulation of circulating lipids for cardiovascular health. In response to environmental insults, cells undergo diverse adaptation or pathophysiological processes via stress-responsive eukaryotic initiation factor 2 alpha (eIF2α) kinase signaling and subsequent cellular reprogramming. We noted that patients with inflammatory gut distress display enhanced levels of ribosomal stress-responsive eIF2α kinase, which is notably associated with lipid metabolic process genes. Based on an assumption that eukaryotic ribosomes are a promising stress-responsive module for molecular reprogramming, chemical ribosome-inactivating stressors (RIS) were assessed for their involvement in enterohepatic lipid regulation. Methods: Experimental assessment was based on prediction using the clinical transcriptome and single-cell RNA-sequencing analysis of inflammatory bowel diseases and obesity. The prediction was verified using RIS exposure models of mice, gut organoids, and intestinal cells. The lipidomic profiling was performed to address RIS-induced intracellular fat alterations. Biochemical processes of the mechanisms were evaluated using RT-PCR, western blot analysis, luciferase reporter assays, and confocal microscopy of genetically ablated or chemically inhibited mice, organoids, and cells. Results: Chemical RIS including deoxynivalenol promoted enterohepatic lipid sequestration while lowering blood LDL cholesterol in normal and diet-induced obese mice. Although ribosomal stress caused extensive alterations in cellular lipids and metabolic genes, the cholesterol import-associated pathway was notably modulated. In particular, ribosomal stress enhanced gut levels of the low-density lipoprotein receptor (LDLR) via both transcriptional and post-transcriptional regulation. Subsequently, LDLR facilitated enterohepatic cholesterol accumulation, leading to dyslipidemia in response to ribosomal stress. Moreover, genetic features of stress-responsive LDLR modulators were consistently proven in the inflammation- and obesity-associated gut model. Conclusion: The elucidated ribosome-linked gut lipid regulation provides predictive insights into stress-responsive metabolic rewiring in chronic human diseases as an environmental health prediction.

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BACKGROUND: Coronary artery disease (CAD) is the main cause of death in Egypt. Many LDL-R gene locus single nucleotide polymorphisms (SNP) are found to be associated with the risk of CAD. This research aimed to assess the allelic and genotypic frequencies of rs1122608 SNP and their association with the extent of vessel affection and lipid profile in a population of Egyptians.100 CAD patients and 100 healthy controls of Egyptians were included. PCR-RFLP was used to genotype rs1122608 SNPs.
RESULTS: Significantly higher proportion of \'T\' allele among patient (risk allele). This association is of low strength (ϕ lies between 0.1 and 0.3). A participant with \'T\' allele has 1.95 times higher odds to exhibit CAD versus a participant with \'G\' allele. Significantly higher proportion of \'T/T\' genotype among cases versus control (risk genotype). This association is of low strength (Cramer\'s V lies between 0.1 and 0.3). A participant with \'T/T\' genotype has 4.5 times higher odds to exhibit CAD versus a participant with \'G/G\'. Gensini score showed no significant association with rs1122608 genotypes (p = 0.863).
CONCLUSIONS: The mutant GT and TT genotypes and minor T allele of rs1122608 were positively correlated with CAD and considered as independent risk factors for CAD.

UNASSIGNED: This study aimed to evaluate the possible role of urolithin A (UA) and urolithin B (UB) on the mRNA expression levels of LDL receptor (LDLR) and PSCK9 genes, and also of the uptake of LDL particles in HepG2 cells.
UNASSIGNED: The potential role of UA and UB on the induction of LDL uptake and the expression of its regulatory genes was explored using HepG2 cells and curcumin (20 μM), berberine (50 μM), UA (80 μM), and UB (80 μM) as the treatments in the experimental tests.
UNASSIGNED: The LDL uptake and cell-surface LDLR were higher in cells treated with UA in comparison with cells treated with UB, and even in relation to the cells treated with curcumin and berberine as positive controls. In addition, cells treated with UB showed approximately 2 times greater LDLR expression levels compared with curcumin (FC = 2.144, p = 0.013) and berberine (FC = 2.761, p = 0.006). However, UA treatment resulted in significantly lower expression levels of LDLR compared with curcumin (FC = 0.274, p < 0.001) and berberine (FC = 0.352, p = 0.009). UB demonstrated approximately 8 times higher LDLR expression levels when compared with UA (FC = 7.835, p = 0.001). Compared with UB, as well as curcumin and berberine as positive controls, UA was more efficient in reducing PCSK9 expression levels. Although UB did not show any significant differences compared with curcumin and berberine, it showed higher levels of PCSK9 expression when compared with the UA group (FC = 3.694, p < 0.001).
UNASSIGNED: The present results suggest that UA was more effective than UB in promoting LDL uptake as well as cell surface LDLR in HepG2 cells. This effect seems to be mostly mediated through the suppression of PCSK9 expression but not the induction of LDLR expression.

Low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1), also known as receptor associated protein (RAP), is an endoplasmic reticulum (ER) chaperone and inhibitor of LDL receptor related protein 1 (LRP1) and related receptors. These receptors have dozens of physiological ligands and cell functions, but it is not known whether cells release LRPAP1 physiologically at levels that regulate these receptors and cell functions. We used mouse BV-2 and human CHME3 microglial cell lines, and found that microglia released nanomolar levels of LRPAP1 when inflammatory activated by lipopolysaccharide or when ER stressed by tunicamycin. LRPAP1 was found on the surface of live activated and non-activated microglia, and anti-LRPAP1 antibodies induced internalization. Addition of 10 nM LRPAP1 inhibited microglial phagocytosis of isolated synapses and cells, and the uptake of Aβ. LRPAP1 also inhibited Aβ aggregation in vitro. Thus, activated and stressed microglia release LRPAP1 levels that can inhibit phagocytosis, Aβ uptake and Aβ aggregation. We conclude that LRPAP1 release may regulate microglial functions and Aβ pathology, and more generally that extracellular LRPAP1 may be a physiological and pathological regulator of a wide range of cell functions.

OBJECTIVE: In 2022, the Japan Atherosclerosis Society (JAS) has revised its clinical diagnostic criteria of familial hypercholesterolemia (FH) and adopted the use of definite, probable, possible, and unlikely FH according to the Dutch Lipid Clinic Network (DLCN) FH criteria. However, these strata have not been validated and their impact on coronary artery disease (CAD) is yet to be elucidated.
METHODS: In this study, we retrospectively examined the patients with FH aged ≥ 15 years (N=857, male=431) who were admitted to Kanazawa University Hospital between 2010 and 2022. We assessed the prevalence of patients with a pathogenic variant as FH and odds ratio (OR) of CAD among each group determined by the JAS criteria 2022 for adults.
RESULTS: In total, 414, 128, 142, and 173 patients were found to have definite, probable, possible, and unlikely FH, respectively, in this population. The prevalences of patients with a pathogenic variant as FH were 77.1%, 28.7%, 13.0%, and 1.2 %, respectively, among the definite, probable, possible, and unlikely FH patients (P-trend ＜0.001). Compared with the reference group of unlikely FH, patients with definite, probable, and possible FH were noted to have significantly higher adjusted odds of developing CAD (OR, 9.1; 95% confidence interval [CI], 3.2-12.6; P＜0.001 and OR, 4.2; 95% CI, 1.7-6.4; P＜0.001, and OR, 2.8; 95% CI, 1.2-4.4; P=0.002, respectively).
CONCLUSIONS: The new JAS diagnostic criteria for FH have been noted to work well in terms of diagnosing definitive, probable, or possible FH patients. Thus, it is seen to be of great help in terms of risk discrimination.

OBJECTIVE: Refractory hypercholesterolemia (RH), caused primarily by the loss-of-function mutation of LDL receptor (LDLR) gene seen in HoFH and HeFH patients, remains a major risk factor for atherosclerotic cardiovascular disease (ASCVD). Statin and ezetimibe combination therapy lower circulating LDL by 30% in HoFH patients. PCSK9 mAB, being an LDLR-dependent therapy, is not effective in HoFH, but lowers LDL by 25% in HeFH patients. A maximum reduction of 50% was noted in HoFH patients treated with ANGPTL3 mAB, which was not enough to achieve therapeutic goal of LDL. Therefore, new approaches are warranted to offer hopes to individuals intolerant to higher dose statins and not able to achieve recommended LDL level.
RESULTS: New approaches to lower LDL include gene therapy and gene editing. AAV-based gene therapy has shown encouraging results in animal models. Using CRISPR/Cas9-mediated genome/base editing, gain of function and loss of function have been successfully done in animal models. Recent progress in the refinement of genome/base editing has overcome the issues of off-target mutagenesis with ∼1% mutagenesis in case of PCSK9 and almost no off-target mutagenesis in inactivating ANGPTL3 in animal models showing 50% reduction in cholesterol. Current approaches using CRISPR-Cas9 genome/base editing targeting LDLR-dependent and LDLR-independent pathways are underway.
CONCLUSIONS: The new information on gain of LDLR function and inactivation of ANGPTL3 together with developments in genome/base editing technology to overcome off-target insertion and deletion mutagenesis offer hope to refractory hypercholesterolemic individuals who are at a higher risk of developing ASCVD.