Human Low Density Lipoprotein Receptor Research Articles

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host–pathogen interactions

BackgroundSporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host–pathogen protein–protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host–pathogen protein–protein interactions involved are poorly understood.MethodsTo gain a better understanding of the protein–protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites.ResultsA total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified.Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes.ConclusionPf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

Chrysin 7-O-β-d-glucopyranoside increases hepatic low-density lipoprotein receptor expression through AMP-activated protein kinase activation.

Elevated plasma low-density lipoprotein (LDL) cholesterol level is a risk factor for developing atherosclerosis. Increased LDL receptor (LDLR) expression is expected to reduce the risk of atherosclerotic disease since hepatic LDLR is essential for clearing plasma LDL cholesterol. Here, we screened human LDLR promoter effectors and observed that extracts from peduncles of sweet cherry (Prunus avium) 'Sato-Nishiki' induce LDLR gene promoter activity. We used several analytical and chemical methods to show that chrysin 7-O-β-d-glucopyranoside (chrysin-7G) is one of the compounds that stimulate LDLR gene promoter activity in cherry peduncle extracts. Furthermore, synthetic chrysin-7G increased the expression and activity of LDLR. The chrysin-7G-mediated increase in LDLR expression and activity was completely abolished by treatment with an AMP-activated protein kinase (AMPK) inhibitor, compound C. These results indicate that chrysin-7G increases LDLR expression through AMPK activation and may be a useful compound that can be recycled from waste parts of agricultural products.

Unregulated LDL cholesterol uptake is detrimental to breast cancer cells.

Tumor uptake of exogenous cholesterol has been associated with the proliferation of various cancers. Previously, we and others have shown that hypercholesterolemia promotes tumor growth and silencing of the LDL receptor (LDLR) in high LDLR-expressing tumors reduces growth. To advance understanding of how LDL uptake promotes tumor growth, LDLR expression was amplified in breast cancer cell lines with endogenously low LDLR expression. Murine (Mvt1) and human (MDA-MB-468) breast cancer cell lines were transduced to overexpress human LDLR (LDLROE). Successful transduction was confirmed by RNA and protein analysis. Fluorescence-labeled LDL uptake was increased in both Mvt1 and MDA-MD-468 LDLROE cells. The expression of the cholesterol-metabolizing genes, ABCA1 and ABCG1, was increased, while HMGCR was decreased in the MDA-MB-468 LDLROE cells. In contrast, Mvt1 LDLROE cells showed no differences in Abca1 and Abcg1 expression and increased Hmgcr expression. Using a Seahorse analyzer, Mvt1 LDLROE cells showed increased respiration (ATP-linked and maximal) relative to controls, while no statistically significant changes in respiration in MDA-MB-468 LDLROE cells were observed. Growth of LDLROE cells was reduced in culture and in hypercholesterolemic mice by two-fold. However, the expression of proliferation-associated markers (Ki67, PCNA and BrdU-label incorporation) was not decreased in the Mvt1 LDLROE tumors and cells. Caspase-3 cleavage, which is associated with apoptosis, was increased in both the Mvt1 and MDA-MB-468 LDLROE cells relative to controls, with the Mvt1 LDLROE cells also showing decreased phosphorylation of p44/42MAPK. Taken together, our work suggests that while additional LDL can promote tumor growth, unregulated and prolonged LDL uptake is detrimental.

Very Low‐Density Lipoprotein (VLDL) Signals Through Sphingosine‐1‐Phosphate Receptor 1, Upon Binding to VLDL Receptor, to Induce Aldosterone Synthesis and Secretion in Human Adrenocortical Cells

Hypertension is prevalent in 46% of the US adult population with 40% of cases being attributable to obesity. Obese individuals have high serum levels of very low‐density lipoprotein (VLDL) and sphingosine‐1‐phosphate (S1P). VLDL and S1P have been shown to stimulate aldosterone production in multiple zona glomerulosa cell models, with aldosterone thought to be the link between hypertension and obesity. S1P is transported in blood bound to lipoproteins such as VLDL, low‐density lipoprotein (LDL), and high‐density lipoprotein (HDL); the VLDL particle contains the highest S1P levels. S1P in HDL has been shown to promote interactions between the scavenger receptor class B, type I (SR‐BI) and S1P receptor 1 (S1PR1). We hypothesized that like HDL, VLDL will signal through S1PRs, upon binding to SR‐BI; therefore, VLDL‐induced aldosterone secretion will be inhibited by S1PR and SR‐BI antagonists. Human adrenocortical (HAC15) cells were treated with VLDL and/or an S1PR1 antagonist (Ex26) or an anti‐SR‐BI blocking antibody for 24 h. Steroidogenic gene expression and aldosterone secretion were monitored by qRT‐PCR and radioimmunoassay, respectively. Ex26 significantly inhibited VLDL‐induced increases in CYP11B2 (22‐fold) and StAR (1.5‐fold) expression and aldosterone secretion (5‐fold) by 43%, 10%, and 36%, respectively. Unexpectedly, the anti‐SR‐BI antibody enhanced VLDL‐induced increases in CYP11B2 (29‐fold) and NR4A1 (2.2‐fold) expression by 17% and 33%, respectively; no effect was observed on VLDL‐induced increases in StAR and NR4A2 expression. However, when the VLDL receptor was blocked with recombinant human LDL receptor related protein associated protein 1, VLDL‐induced increases in CYP11B2 (2.8‐fold) and NR4A2 (5.3‐fold) expression were significantly reduced by 42% and 67%, respectively. Our results, therefore, indicate that VLDL signals through S1PR1, upon binding to the VLDL receptor and not SR‐BI, to induce aldosterone synthesis and secretion. Our study warrants further investigation into VLDL‐induced steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets like S1PR1 to potentially treat obesity‐associated hypertension.

Meet the First Authors.

Meet the First Authors.

Abstract P099: Radionuclide imaging of low-density-lipoprotein receptor (LDLR)-overexpressing glioblastoma: A preclinical study of Gallium-68 RMX-VH

Abstract Introduction: One of the factors that limit the efficacy of the drugs, especially in primary brain tumors, is the permeability of the blood-brain barrier (BBB). The low-density lipoprotein receptor (LDL-R) expressed at the BBB mediates the transport of endogenous ligands through the BBB. We developed a new radiolabeled peptide targeting both the human and murine LDLR and able i) to cross the BBB and ii) to target tumors such as glioblastoma that express high levels of the LDLR. The objective of this study was to determine the LDLR targeting properties, the pharmacokinetics of 68Ga radiolabeled RMX-VH in a glioblastoma model that expresses the human LDLR (hLDLR). Methods: The nonclinical studies of 68Ga-RMX-VH were completed in U87MG, A172, U373 glioblastoma cancer cell lines and xenografts mice models. The studies determined in vivo time-dependent accumulation of this agent, in vitro dose-depended cellular uptake, and cellular competition studies. We compared the tumor-specific accumulation of 68Ga-RMX-VH and normal organ distribution in female and male athymic nude mice. Radiotracer, RMX-VH (10-30ug) was labeled with isotope-Ga68 (10-25mCi, ITM GmBH). U87MG and A172- derived xenografts were generated in athymic nude mice (10 weeks) and PET/CT images were acquired using G4 PET/Xray camera (Sofie Biosciences) at 1h, 2h, 3h, and 4h post-injection. The followed up biodistribution studies were done at 30 min, 1h, 2h, and 3 hrs. post-injection. The organs and tumor were collected, weighed, and the tissue radioactivity was measured with Wizard2 Gamma Counter (Perkin-Elmer, Waltham, MA). The percentage of injected dose per gram of tissue (%ID/g) was calculated and decay-corrected. Results: Our studies confirmed the in vitro and in vivo selectivity and specificity of 68Ga-RMX-VH toward LDLR-positive tumors. 68Ga-RMX-VH is a small peptide (MW: 1432.7 g/mol) and renal excretion was expected as a route of agent elimination. The tumor-specific uptake of radiotracer in U87MG xenografts was 1.8%ID/g at 1h and remained unchanged at 3h post-injection. The kidney retention of the agent reached 12.2%ID/g and decreased to 10.2%ID/g. The accumulation of radiotracer in the liver, ovaries, and intestine correlated well with the known normal expression of LDLR. The tumor-to-muscle ratio was 5.97 at 1h; increased to 26.1 and 22.39 at 2h and 3h. The elimination t1/2 of radiotracer was only 18.6 min, with a clearance CL of 502 ml/min. Adsorption from the site of administration is rapid as the Cmax was 5 min. The tumor-specific uptake and normal organ distribution of 68Ga-RMX-VH were lower in male mice than in females. This correlated with differences in lipid and lipoproteins metabolism in males and females. Conclusions: RMX-VH showed favorable hLDLR targeting properties in vitro and in vivo in glioblastoma mice models. Our results suggest that hLDLR may serve as a target for imaging for glioblastoma. The first-in-human exploratory IND study of Ga68-RMX-VH will be initiated in Q2 of 2021. Citation Format: Izabela Tworowska, Leo Garcia Flores II, Xuewei Qu, Cédric Malicet, Nilesh Wagh, David Ranganathan, Jonathan Nowak, Pascaline Lecorche, Jamal Temsamani, Ebrahim S. Delpassand. Radionuclide imaging of low-density-lipoprotein receptor (LDLR)-overexpressing glioblastoma: A preclinical study of Gallium-68 RMX-VH [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P099.

Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome

The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

Abstract 11197: Nano-Triciribine Reduces SARS-CoV-2 Infection by Sequestering ACE2 and the Novel Host Factor LDLR

Introduction: People with previous CVD hospitalized for COVID-19 have elevated death rate. We reported that patients with diabetes and HF higher protein levels of the low density lipoprotein receptor (LDLR). We hypothesized that LDLR is a novel host factor for the SARS-CoV-2-Spike (S2S) protein that may be regulated by the Akt inhibitor Triciribine (TCN), a drug being tested in Phase III studies for breast cancer. We also hypothesized that nano-formulation of Triciribine (NanoTriciribine; NTCN) would enhance its efficacy and allow for intranasal delivery. Methods: Interactions between the recombinant proteins Spike-RBD (receptor binding domain), ACE2, LDLR and its ectodomains (EGFA-EFFB, C2-C5 and C2) were analyzed by binding assays and co-IP in HepG2, HK2, and 293T cells. Viral entry assays were performed with 2 S2S pseudoviruses using 293T cells + hACE2 and TMPRSS2 or Furin protease. The effect of NTCN or the LXR agonist GW-3965 on viral uptake (pseudotyped VSVΔG-GFP*S2S or chimera VSV-S2S-eGFP virus) was assessed. Akt, pAkt, ACE2, and LDLR levels were determined in 293T+hACE2 by flow cytometry. Assays were done in triplicates and 1-way-ANOVA with Tukey’s correction was used for statistics. Results: RBD protein binds modestly to the human LDLR (EC 50 :10μM) and its C2-C5 ectodomain (EC 50 :13.8μM). Co-IP revealed a novel and strong LDLR-ACE2 interaction in several human cell lines. LDLR overexpression in human cells increased the uptake of VSVΔG-GFP*S2S (FC=2.32;p<0.001) and chimera virus (FC=.33; p<.0001). NTCN and TCN each reduced pAkt/Akt ratio. 1μM TCN or NTCN reduced LDLR (7.2%;p<.01 & 15.6%;p<0.0001) and ACE2 (32%;p<0.05 & 44.7%;p<.01) cell surface expression, respectively. 1μM NTCN or GW-3965 reduced S2S viral entry by 64.2% (p<.0001) and 40.7% (p<.01), respectively, confirming a role for LDLR in S2S infection. In hACE2tg mice, chimera VSV-S2S caused significant lung infection as measured by qPCR, GFP expression in proximal and distal lung airway epithelial cells, and electron microscopy. Intranasal delivery of NTCN was well tolerated. Conclusions: LDLR enhanced S2S viral entry supporting the elevated COVID-19 susceptibility seen in patients with heart disease. NTCN is a promising candidate for prophylactic treatment against COVID-19.

Lipid Lowering Natural Products Targeting hPCSK9 May Prevent the Severity of COVID-19 Infection

Background: The deadly outbreak of COVID-19 disease caused by novel SARS CoV2 has created an unprecedented global health crisis affecting every sectors of human life and enormous damage to world’s economy. With >16.1 million infections and >650,000 deaths worldwide as of July 27, 2020, there is no treatment for this disease neither is there any available vaccine. Serious research efforts are ongoing on all fronts including treatment, prevention and diagnosis to combat the spread of this infection. A number of targets that include both viral and host proteins have been identified and became part of intense investigation. In this respect the viral surface spike (S) glycoprotein caught the attention most. It is cleaved by multiple host proteases to allow recognition by host receptor human Angiotensin Converting Enzyme2 (hACE2) leading to fusion and viral replication. Natural products, small compounds, antioxidants, peptides, proteins, oligonucleotides, antibodies and other compounds are under investigation for development of antiviral agents against COVID- 19. Objective: Recently cholesterol lowering phytocompounds Quercetin, Swertiamarin and Berberine which promote human Low Density Lipoprotein Receptor (hLDLR) via inhibition of human Proprotein Convertase Subtilisin Kexin9 (hPCSK9) have been shown to block viral infections caused by ebola, influenza, Respiratory Syncytial Virus (RSV), Hepatitis C virus (HCV) and other RNA type viruses. Since SARS CoV2 is a RNA virus with similar genetic structure and infection machinery, it is hypothesised that these phytocompounds may also exhibit antiviral property against COVID-19. Methods: Our above concept is based on recently published studies as well as our herein presented in silico modeling and computational data which suggested strong interactions of hPCSK9 with above phytocompounds and most importantly with hACE2 following its complexation with receptor binding domain (RBD) of SARS CoV2 S protein. Results: These results and a proposed schematic model showing association of hPCSK9 with SARS CoV2 infection are presented in this manuscript. It is proposed that hPCSK9 plays the role of a co-receptor in binding with hACE2:RBD complex and thereby facilitates viral fusion. Conclusion: Our studies suggest that PCSK9 inhibitors may provide beneficial effect against COVID-19 infection by retarding viral fusion through displacement of bound hPCSK9 from its complex with ACE2:RBD of SARS CoV2 S protein.

Abstract P420: Nano-Triciribine Reduces SARS2-CoV-2 Infection By Sequestering ACE2 And The Novel Host Factor LDLR

Background: People with previous CVD hospitalized for COVID-19 have elevated death rate. We reported that patients with diabetes and HF higher protein levels of the low density lipoprotein receptor (LDLR). We hypothesized that LDLR is a novel host factor for the SARS-CoV-2-Spike (S2S) protein that may be regulated by the Akt inhibitor Triciribine (TCN), a drug being tested in Phase III studies for breast cancer. We also hypothesized that nano-formulation of Triciribine (NanoTriciribine; NTCN) would enhance its efficacy and allow for intranasal delivery. Methods: Interactions between the recombinant proteins Spike-RBD (receptor binding domain), ACE2, LDLR and its ectodomains (EGFA-EFFB, C2-C5 and C2) were analyzed by binding assays and co-IP in HepG2, HK2, and 293T cells. Viral entry assays were performed with 2 S2S pseudoviruses using 293T cells + hACE2 and TMPRSS2 or Furin protease. The effect of NTCN or the LXR agonist GW-3965 on viral uptake (pseudotyped VSVΔG-GFP*S2S or chimera VSV-S2S-eGFP virus) was assessed. Akt, pAkt, ACE2, and LDLR levels were determined in 293T+hACE2 by flow cytometry. Assays were done in triplicates and 1-way-ANOVA with Tukey’s correction was used for statistics. Results: RBD protein binds modestly to the human LDLR (EC 50 :10μM) and its C2-C5 ectodomain (EC 50 :13.8μM). Co-IP revealed a novel and strong LDLR-ACE2 interaction in several human cell lines. LDLR overexpression in human cells increased the uptake of VSVΔG-GFP*S2S (FC=2.32;p<0.001) and chimera virus (FC=.33; p<.0001). NTCN and TCN each reduced pAkt/Akt ratio. 1μM TCN or NTCN reduced LDLR (7.2%;p<.01 & 15.6%;p<0.0001) and ACE2 (32%;p<0.05 & 44.7%;p<.01) cell surface expression, respectively. 1μM NTCN or GW-3965 reduced S2S viral entry by 64.2% (p<.0001) and 40.7% (p<.01), respectively, confirming a role for LDLR in S2S infection. In hACE2tg mice, chimera VSV-S2S caused significant lung infection as measured by qPCR, GFP expression in proximal and distal lung airway epithelial cells, and electron microscopy. Intranasal delivery of NTCN was well tolerated. Conclusions: LDLR enhanced S2S viral entry supporting the elevated COVID-19 susceptibility seen in patients with heart disease. NTCN is a promising candidate for prophylactic treatment against COVID-19.

Human low-density lipoprotein receptor plays an important role in hepatitis B virus infection.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV vaccine is effective to prevent new HBV infection but does not offer therapeutic benefit to hepatitis B patients. Neither are current antiviral drugs curative of chronic hepatitis B. A more thorough understanding of HBV infection and replication holds a great promise for identification of novel antiviral drugs and design of optimal strategies towards the ultimate elimination of chronic hepatitis B. Recently, we have developed a robust HBV cell culture system and discovered that human apolipoprotein E (apoE) is enriched on the HBV envelope and promotes HBV infection and production. In the present study, we have determined the role of the low-density lipoprotein receptor (LDLR) in HBV infection. A LDLR-blocking monoclonal antibody potently inhibited HBV infection in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) as well as in primary human hepatocytes. More importantly, small interfering RNAs (siRNAs)-mediated knockdown of LDLR expression and the CRISPR/Cas9-induced knockout of the LDLR gene markedly reduced HBV infection. A recombinant LDLR protein could block heparin-mediated apoE pulldown, suggesting that LDLR may act as an HBV cell attachment receptor via binding to the HBV-associated apoE. Collectively, these findings demonstrate that LDLR plays an important role in HBV infection probably by serving as a virus attachment receptor.

High-Throughput Screening Identifies MicroRNAs Regulating Human PCSK9 and Hepatic Low-Density Lipoprotein Receptor Expression.

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3′-UTR) of human microRNA library screen, we identified microRNAs targeting the 3′ untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3′-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

Cohort Generation and Characterization of Patient-Specific Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Homozygous familial hypercholesterolemia (hoFH) is a rare disorder caused primarily by pathological mutations in the low-density lipoprotein receptor (LDLR), which disrupts LDL-cholesterol (LDL-C) metabolism homeostasis. hoFH patients are at extremely high risk for cardiovascular disease and are resistant to standard therapies. LDLR knockout animals and in vitro cell models overexpressing different mutations have proved useful, but may not fully recapitulate human LDLR mutation biology. We and others have generated induced pluripotent stem cells (iPSC) from hoFH patient's fibroblasts and T cells and demonstrated their ability to recapitulate hoFH biology. In this study, we present the generation and characterization of a cohort of seven hoFH-iPSC lines derived from peripheral blood mononuclear cells (PBMC) collected from four homozygous and three compound heterozygous patients. The hoFH-iPSC cohort demonstrated a wide range of LDLR expression and LDL-C internalization in response to rosuvastatin that correlated with the predicted pathogenicity of the mutation. We were able to confirm that hoFH-iPSC cohort were pluripotent by differentiation toward all three germ layers and specifically to hepatocyte-like cells (HLC), the cell with primary LDL-C metabolic regulatory control, by expression of hepatocyte markers. hoFH patient PBMC-derived iPSC recapitulate the LDLR dysfunction of their specific mutation. They were capable of differentiating to HLC and could be useful for early developmental studies, pharmacology/toxicology, and potentially autologous cell therapy.

Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia

Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.

In-silico analysis of non-synonymous SNPs of human LDLR gene and their impact on familial hypercholesterolemia

Single nucleotide polymorphisms (SNPs) are the most regular type of genetic variation among human genome. The low-density lipoprotein receptors (LDLR) gene encoded receptor protein that specifically binds to low density lipoprotein (LDL), to act as prime cholesterol carriers in the blood. Unregulated removal of LDL from the circulation results from inherited mutations in the LDLR gene or, more rarely, in the apo B gene, the ligand for the LDLR causes familial hypercholesterolemia (FH). Several studies have shown that non-synonymous single nucleotide polymorphisms (nsSNPs) induce more than 50% genetic diseases. However, the growing number of SNPs in databases makes it difficult to evaluate the SNPs that come up with disease development. Therefore, we tried to identify the nsSNP within LDLR gene used mainly computational tools like: PROVEAN, Predict-SNP, SIFT, MAAP, SNAP, PhD-SNP, Polyphen v1, Polyphen v2, PANTHER and nsSNPAnalyzer. After analysing a total of 1114 missense SNPs, we found only 39 nsSNPs are most deleterious, whose conservation score ranges from grade ranges 7 to 9 that is assessed through ConSurf web server. Among 39 most significant nsSNPs, 11were predicted for involving in post translational modifications. Additionally, I-Mutant 2.0 and MUpro tool showed a decreased instability for these nsSNPs upon mutation. Protein structural analysis of these amino acid variants was performed by using I-Mutant 2.0. Stability analysis predicted 11 nsSNPs (C313W, F408K, A431P, A431S, A431T, D433H, D433Y, G546D, G546V, G546E and C697W), these are decreased protein stability. The in-silico analysis in current study may be a significant model for future research design studies.

Effects of human LDLR overexpression on apoE-related tau pathology and brain dysfunction

Abstract ApoE4 is the strongest genetic risk factor for late-onset Alzheimer Disease. It has been reported that apoE isoforms directly affect tauopathy and tau-mediated neurodegeneration in P301S tau transgenic mice expressing different human apoE isoforms, with apoE4 resulting in markedly increased tau-mediated neurodegeneration and the absence of apoE being marked protective against neurodegeneration. In the brain, low-density lipoprotein receptor (LDLR) is one of the main apoE receptors that regulates apoE levels, but LDLR has very few identified ligands compared to other apoE receptors. LDLR overexpression in the brain can dramatically lower apoE and Aβ levels, as well as decrease Aβ accumulation and deposition. Therefore, we propose to evaluate whether AAV-mediated overexpression of human LDLR (hLDLR) is an efficient way to reduce apoE levels, tau pathology, and neurodegeneration. To study the effect of LDLR overexpression on apoE4-related tau pathology and neurodegeneration, P301S Tau/ApoE4 (TE4) male mice received bilateral intracerebroventricular injection with AAV expressing hLDLR or GFP-control, starting before the onset of tau pathology development. Brains were isolated after 9 month, divided into hemispheres, and analyzed by histological and biochemical techniques. Widespread expression of hLDLR throughout the brain was observed and apoE levels were significantly decreased. Furthermore, mouse nest-building impairment was rescued after hLDLR overexpression. Strikingly, there is a significant decrease in brain atrophy, phosphorylated tau deposition in TE4 mice overexpressing hLDLR. In summary, these data suggest that LDLR may be a promising target to lower apoE levels and decrease neurodegeneration.

Hsa-miR-140-5p down-regulates LDL receptor and attenuates LDL-C uptake in human hepatocytes

Background and aimsMicroRNAs (miRs) exert important regulatory effects in cholesterol metabolism. Hepatic low density lipoprotein receptor (LDLR) pathway, as the major mechanism for clearing circulating low density lipoprotein cholesterol (LDL-C) in bloodstream, is a pivotal therapeutic target to treat hypercholesterolemia and atherosclerosis. This study aimed to identify novel miRs that regulate LDLR expression. Methods and resultsHsa-miR-140-5p was predicted by bioinformatics analyses to interact with human LDLR mRNA. To evaluate its functional effects in regulating LDLR, hsa-miR-140-5p and anti-miR-140-5p were transfected into human and mouse liver cells, followed by qRT-PCR, western blot, immunofluorescence, flow cytometry, and LDL-C uptake assays. It was observed that hsa-miR-140-5p over-expression dramatically down-regulated LDLR expression and reduced LDL-C uptake, whereas inhibition of hsa-miR-140-5p significantly up-regulated LDLR expression and enhanced LDL-C uptake in human HepG2 and LO2 cells, but not in mouse Hepa1-6 cells. Luciferase reporter assay and site-directed mutagenesis identified that hsa-miR-140-5p interacts with the predicted seed sequence “AAACCACU” in the 3′-UTR of human LDLR mRNA. Hsa-miR-140-5p over-expression attenuated LDL-C uptake and decreased intracellular cholesterol levels in the presence of 50 μg/ml ox-LDL in HepG2 cells. Additionally, palmitic acid and simvastatin suppressed, whereas LDL-C up-regulated the expression of miR-140-5p in HepG2 cells. ConclusionsHsa-miR-140-5p is a negative regulator of LDLR expression in human hepatocytes, but not in mouse hepatocytes. Simvastatin inhibits hsa-miR-140-5p expression in human hepatocytes, which is likely to be a novel mechanism for treating hypercholesterolemia with statins in clinic. Antagonism of hsa-miR-140-5p could be a new therapeutic strategy for the treatment of hypercholesterolemia and atherosclerosis.

Abstract LB-A16: Imaging of glioblastoma using LDLR-based targeted delivery system

Abstract Introduction: Glioblastoma is the most agressive brain tumor with average life expectancy of 12-15 months from diagnosis. One of the factors that limit efficacy of drug, especially in primary brain tumor is permeability of the blood brain barrier (BBB). The low-density lipoprotein receptor (LDLR) expressed at the BBB mediates the transport of endogenous ligands through the BBB, a process referred to as receptor-mediated transcytosis. VECT-HORUS (VH) has identified and chemically optimized a family of peptide-vectors targeting both the human and murine LDLR and able i) to cross the BBB and ii) to target tumors such as glioblastoma that express high levels of the LDLR. The objective of this study was to determine the LDLR targeting properties of 68Ga/177Lu- radiolabeled peptide vectors developed by the VH proprietary platform using a glioblastoma model that expresses the human LDLR (hLDLR) at high levels. Methods: The LDLR targeted DOTA-conjugates (VH-DO31, VH-DO33) and NODAGA conjugate (VH-NO31), (10-30ug, VECT-HORUS SAS, France) were labeled with 68Ga (1.5mCi) eluted from 68Ge/68Ga generator (100mCi, ITG GmBH, Germany) or with 177Lu n.c.a (1mCi, ITG GmBH, Germany). The U87MG cell line has been shown to express high levels of the hLDLR. The LDLR targeting properties of these conjugates were thus determined in vitro in U87MG cellular uptake studies, as well as in vivo in U87MG xenografted mice. The PET/CT images of U87MG xenograft generated in athymic nude mice (10 weeks, n=3) were acquired using G4 PET/Xray camera (Sofie Biosciences; 10min/scan) at 1h, 2h, 3h and 4h post-injection. Results: All 68Ga/177Lu-labeled conjugates were synthesized with radiochemical purity higher than 91 % as determined by radio-HPLC. Radiolytic stability of agents was increased using C18 ethanol purification of the final products. 177Lu-VH-DO33 showed the highest retention of the agent in U87MG cell line at 1h (13.88± 1.6 %ID/mg) and 21h incubation time (8.7± 4 %ID/mg) compared to 177Lu-VH-DO31 (8.28 ±6.2 %ID/mg) The microPET imaging studies showed rapid accumulation and retention of all VH derivatives in tumor as monitored up to 4h post injection. All agents were eliminated through bladder and kidneys. There was no accumulation of agents in the bone marrow. The image based biodistribution studies of 68Ga-VH-DO31, 68Ga-VH-DO33 and 68Ga-VH-NO31 showed that the tumor to muscle ratios (SUV ratio) after 30 min post-injection were 4.12, 5.07 and 3.88, respectively and remained at the same levels up to 3h post-injection. The SUV ratios of tumor to kidneys were as follows: 68Ga-VH-DO31 (0.46), 68Ga-VH-DO33 (0.84) and 68Ga-VH-NO31 (0.46) confirming renal elimination of the agents. Conclusions: VH derivatives showed favorable hLDLR and tumor-targeting properties both in in vitro as well as in vivo in U87MG xenografts. These results can postulate that hLDLR may serve as a target for the development of theranostic probes for the diagnosis and radiotherapy of glioblastoma. Citation Format: Izabela Tworowska, Leo G Flores II, Rafal Zielinski, Jonathan Nowak, Pascaline Lecorche, Cedric Malicet, Michel Khrestchatisky, Jamal Temsamani, Ebrahim Delpassand. Imaging of glioblastoma using LDLR-based targeted delivery system [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A16. doi:10.1158/1535-7163.TARG-19-LB-A16

Development of a Novel DNA Oligonucleotide Targeting Low-Density Lipoprotein Receptor

Low-density lipoprotein receptor (LDL-R) is a cell surface receptor protein expressed in a variety of solid cancers, including lung, colon, breast, brain, and liver, and therefore it opens up opportunities to deliver lysosome-sensitive anti-cancer agents, especially synthetic nucleic acid-based therapeutic molecules. In this study, we focused on developing novel nucleic acid molecules specific to LDL-R. For this purpose, we performed in vitro selection procedure via systematic evolution of ligands by exponential enrichment (SELEX) methodologies using mammalian cell-expressed human recombinant LDL-R protein as a target. After 10 rounds of selections, we identified a novel DNA oligonucleotide aptamer, RNV-L7, that can bind specifically to LDL-R protein with high affinity and specificity (KD = 19.6 nM). Furthermore, flow cytometry and fluorescence imaging assays demonstrated efficient binding to LDL-R overexpressed human cancer cells, including Huh-7 liver cancer cells and MDA-MB-231 breast cancer cells, with a binding affinity of ∼200 nM. Furthermore, we evaluated the functional potential of the developed LDL-R aptamer RNV-L7 by conjugating with a previously reported miR-21 targeting DNAzyme for inhibiting miR-21 expression. The results showed that the miR-21 DNAzyme-RNV-L7 aptamer chimera efficiently reduced the expression of miR-21 in Huh-7 liver cancer cells. As currently there are no reports on LDL-R aptamer development, we think that RNV-L7 could be beneficial toward the development of targeted cancer therapeutics.

Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly171↓Asp172. The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.