Biology subjects

Carrer, M.

Publications and source records attributed to Carrer, M..

7 recordsLinked to original sources

bioRxiv2026

Delivery of small interfering RNA and antisense oligonucleotides across the blood-brain barrier with monovalent transferrin receptor 1 binding VHH-Fc fusion proteins

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

neuroscience↗

bioRxiv2025

Antisense oligonucleotides targeting Valosin-containing protein improve muscle pathology and molecular defects in cell and mouse models of multisystem proteinopathy.

Valosin-containing protein (VCP) related disease, also known as multisystem proteinopathy 1 (MSP1), is an autosomal dominant disease caused by gain-of-function pathogenic variants of the VCP gene. The disease is associated with inclusion body myopathy, early-onset Pagets disease of bone, frontotemporal dementia, and familial amyotrophic lateral sclerosis. There is currently no treatment for this progressive disease associated with early demise resulting from proximal limb girdle and respiratory muscle weakness. We hypothesize that regulating VCP hyperactivity to normal levels can reduce the disease pathology. In this study, we assessed the effect of antisense oligonucleotides (ASOs) specifically targeting the human VCP gene in the patient (R155H) iPSC-derived skeletal muscle progenitor cells (SMPCs). ASOs were well tolerated up to a concentration of 5 {micro}M, and significantly reduced VCP protein expression in the SMPCs by 48% (95% CI [39-56]). We also treated the transgenic mouse model of VCP disease with the overexpressed humanized VCP gene severe A232E pathogenic variant with weekly subcutaneous ASO injections starting from 6 months of age for 3 months. In the skeletal muscle of transgenic mice, ASOs resulted in 30% (95%CI [27-32]) knockdown of VCP protein compared to control ASO. The ASO-mediated reduction of VCP expression in muscle tissue was associated with improvement in autophagy flux and reduction in TDP-43 expression, hallmarks of VCP disease. In addition, ASO-treated VCP A232E mice showed improvements in functional tests of muscle strength, such as rotarod and inverted screen test compared to mice treated with control ASO. These results suggest that targeting VCP could be beneficial in preventing the progression of the VCP myopathy and hold promise for the treatment of patients with VCP MSP1.

molecular biology↗

bioRxiv2025

Non-nucleosomal (CENP-A/H4)2 - DNA complexes as a possible platform for centromere organization

The centromere is a part of the chromosome that is essential for the even segregation of duplicated chromosomes during cell division. It is epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A associates specifically with a group of 16 proteins that form the centromere-associated network of proteins (CCAN). In mitosis, the kinetochore forms on the CCAN to connect the duplicated chromosomes to the microtubules protruding from the cell poles. Previous studies have shown that CENP-A replaces H3 in nucleosomes, and recently the structures of CENP-A-containing nucleosomes in complex with CCANs have been revealed, but they show only a limited interaction between CCANs and CENP-A. Here, we report the cryoEM structure of 2x(CENP-A/H4)2-di-tetramers assembled on DNA in the absence of H2A/H2B histone dimer and speculate how (CENP-A/H4)2-tetramers and -di-tetramers might serve as a platform for CCAN organization. Main pointsO_LICENP-A/H4 in the absence of H2A/H2B dimers associate on DNA to form di-tetrasomes C_LIO_LICENP-A/H4 di-tetrasomes are stable but highly flexible structures resembling nucleosomes C_LIO_LICENP-A/H4 tetrasomes bind CENP-N/L and can serve as platform for centromere organisation C_LI

biochemistry↗

bioRxiv2024

Targeting mechanistic target of rapamycin complex 2 attenuates immunopathology in Systemic Lupus Erythematosus

ObjectiveWe aim to explore the role of mechanistic target of rapamycin complex (mTORC) 2 in systemic lupus erythematosus (SLE) development, the in vivo regulation of mTORC2 by type I interferon (IFN) signaling in autoimmunity, and to use mTORC2 targeting therapy to ameliorate lupus-like symptoms in an in vivo lupus mouse model and an in vitro coculture model using human PBMCs. MethodWe first induced lupus-like disease in T cell specific Rictor, a key component of mTORC2, deficient mice by topical application of imiquimod (IMQ) and monitored disease development. Next, we investigated the changes of mTORC2 signaling and immunological phenotypes in type I IFNAR deficient Lpr mice. We then tested the beneficial effects of anti-Rictor antisense oligonucleotide (Rictor-ASO) in a mouse model of lupus: MRL/lpr mice. Finally, we examined the beneficial effects of RICTOR-ASO on SLE patients PBMCs using an in vitro T-B cell coculture assay. ResultsT cell specific Rictor deficient mice have reduced age-associated B cells, plasma cells and germinal center B cells, and less autoantibody production than WT mice following IMQ treatment. IFNAR1 deficient Lpr mice have reduced mTORC2 activity in CD4+ T cells accompanied by restored CD4+ T cell glucose metabolism, partially recovered T cell trafficking, and reduced systemic inflammation. In vivo Rictor-ASO treatment improves renal function and pathology in MRL/lpr mice, along with improved immunopathology. In human SLE (N = 5) PBMCs derived T-B coculture assay, RICTOR-ASO significantly reduce immunoglobulin and autoantibodies production (P < 0.05). ConclusionTargeting mTORC2 could be a promising therapeutic for SLE.

immunology↗

bioRxiv2024

Skeletal muscle effects of antisense oligonucleotides targeting glycogen synthase 1 in a mouse model of Pompe disease

Pompe disease (PD) is a progressive myopathy caused by the aberrant accumulation of glycogen in skeletal and cardiac muscle resulting from the deficiency of the enzyme acid alpha-glucosidase (GAA). Administration of recombinant human GAA as enzyme replacement therapy (ERT) works well in alleviating the cardiac manifestations of PD but loses sustained benefit in ameliorating the skeletal muscle pathology. The limited efficacy of ERT in skeletal muscle is partially attributable to its inability to curb the accumulation of new glycogen produced by the muscle enzyme glycogen synthase 1 (GYS1). Substrate reduction therapies aimed at knocking down GYS1 expression represent a promising avenue to improve Pompe myopathy. However, finding specific inhibitors for GYS1 is challenging given the presence of the highly homologous GYS2 in the liver. Antisense oligonucleotides (ASOs) are chemically modified oligomers that hybridize to their complementary target RNA to induce their degradation with exquisite specificity. In the present study, we show that ASO-mediated Gys1 knockdown in the Gaa-/- mouse model of PD led to a robust reduction in glycogen accumulation in skeletal and cardiac muscle. In addition, combining Gys1 ASO with ERT further reduced glycogen content in muscle, eliminated autophagic buildup and lysosomal dysfunction, and improved motor function in Gaa-/- mice. Our results provide a strong foundation for further validation of the use of Gys1 ASO, alone or in combination with ERT, as a therapy for PD. We propose that early administration of Gys1 ASO in combination with ERT may be the key to preventative treatment options in PD.

molecular biology↗

bioRxiv2022

The structural basis of the multi-step allosteric activation of Aurora B kinase

Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.

biochemistry↗

bioRxiv2022

What drives chorismate mutase to top performance? Insights from a combined in silico and in vitro study

Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is pre-organized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by MD simulations and confirmed by kinetic investigations of engineered variants.

biochemistry↗