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1.
Mol Cell Neurosci ; 130: 103950, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38901655

RESUMEN

Amyloid beta (Aß) peptides, which aggregate to form neocortical plaques in Alzheimer's disease, exist in states that range from soluble monomers and oligomers/protofibrils to insoluble fibrillar amyloid. The present study evaluated the effects of mAb158, a mouse monoclonal antibody version of lecanemab that preferentially binds to soluble Aß protofibrils, in aged transgenic mice (Tg2576) with Aß pathology. Female Tg2576 mice (12 months old) received weekly intraperitoneal mAb158 (35 mg/kg) or vehicle for 4 weeks or for 18 weeks, with or without a subsequent 12-week off-treatment period. Aß protofibril levels were significantly lower in mAb158-treated animals at both 4 and 18 weeks, while longer treatment duration (18 weeks) was required to observe significantly lower Aß42 levels in insoluble brain fractions and lower Aß plaque load. Following the off-treatment period, comparison of the vehicle- and mAb158-treated mice demonstrated that the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load remained significantly lower in mAb158-treated animals, as compared with age-matched controls. However, there was a significant increase of brain accumulation of both the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load after treatment cessation. Thus, repeated mAb158 treatment of aged Tg2576 mice first reduced Aß protofibril levels within 4 weeks of treatment, which then was followed by a reduction of amyloid plaque pathology within 18 weeks of treatment. These effects were maintained 12 weeks after the final dose, indicating that mAb158 had a disease-modifying effect on the Aß pathology in this mouse model. In addition, brain accumulation of both Aß protofibril levels and amyloid pathology progressed after discontinuation of the treatment which supports the importance of continued treatment with mAb158 to maintain the effects on Aß pathology.


Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Anticuerpos Monoclonales , Ratones Transgénicos , Placa Amiloide , Animales , Péptidos beta-Amiloides/metabolismo , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Femenino , Placa Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Envejecimiento
2.
Mol Cell Neurosci ; 130: 103949, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38906341

RESUMEN

Recent advances in immunotherapeutic approaches to the treatment of Alzheimer's disease (AD) have increased the importance of understanding the exact binding preference of each amyloid-beta (Aß) antibody employed, since this determines both efficacy and risk for potentially serious adverse events known as amyloid-related imaging abnormalities. Lecanemab is a humanized IgG1 antibody that was developed to target the soluble Aß protofibril conformation. The present study prepared extracts of post mortem brain samples from AD patients and non-demented elderly controls, characterized the forms of Aß present, and investigated their interactions with lecanemab. Brain tissue samples were homogenized and extracted using tris-buffered saline. Aß levels and aggregation states in soluble and insoluble extracts, and in fractions prepared using size-exclusion chromatography or density gradient ultracentrifugation, were analyzed using combinations of immunoassay, immunoprecipitation (IP), and mass spectrometry. Lecanemab immunohistochemistry was also conducted in temporal cortex. The majority of temporal cortex Aß (98 %) was in the insoluble extract. Aß42 was the most abundant form present, particularly in AD subjects, and most soluble Aß42 was in soluble aggregated protofibrillar structures. Aß protofibril levels were much higher in AD subjects than in controls. Protofibrils captured by lecanemab-IP contained high levels of Aß42 and lecanemab bound to large, medium, and small Aß42 protofibrils in a concentration-dependent manner. Competitive IP showed that neither Aß40 monomers nor Aß40-enriched fibrils isolated from cerebral amyloid angiopathy reduced lecanemab's binding to Aß42 protofibrils. Immunohistochemistry showed that lecanemab bound readily to Aß plaques (diffuse and compact) and to intraneuronal Aß in AD temporal cortex. Taken together, these findings indicate that while lecanemab binds to Aß plaques, it preferentially targets soluble aggregated Aß protofibrils. These are largely composed of Aß42, and lecanemab binds less readily to the Aß40-enriched fibrils found in the cerebral vasculature. This is a promising binding profile because Aß42 protofibrils represent a key therapeutic target in AD, while a lack of binding to monomeric Aß and cerebral amyloid deposits should reduce peripheral antibody sequestration and minimize risk for adverse events.


Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Encéfalo , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Anciano , Masculino , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Anciano de 80 o más Años , Unión Proteica , Anticuerpos Monoclonales Humanizados/uso terapéutico , Fragmentos de Péptidos/metabolismo
4.
J Virol Methods ; 195: 156-63, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24120570

RESUMEN

Although hepatitis C virus (HCV) is a pathogen of global significance, experimental therapies in current clinical development include highly efficacious all-oral combinations of HCV direct-acting antivirals (DAAs). If approved for use, these new treatment regimens will impact dramatically upon our capacity to eradicate HCV in the majority of virus-infected patients. However, recent data from late-stage clinical evaluations demonstrated that individuals infected with HCV genotype (GT) 3 responded less well to all-oral DAA combinations than patients infected with other HCV GTs. In light of these observations, the present study sought to expand the number of molecular tools available to investigate small molecule-mediated inhibition of HCV GT3 NS5A and NS5B proteins in preclinical tissue-culture systems. Accordingly, a novel subgenomic HCV replicon chimera was created by utilizing a GT1b backbone modified to produce NS5A and NS5B proteins from a consensus sequence generated from HCV GT3a genomic sequences deposited online at the European Hepatitis C Virus database. This approach avoided the need to isolate and amplify HCV genomes from sera derived from HCV-infected patients. The replicon chimera, together with a version engineered to express NS5A encoding a Y93H mutation, demonstrated levels of replication in transient assays robust enough to assess accurate antiviral activities of inhibitors representing different HCV DAA classes. Thus, the replicon chimera represents a new simple molecular tool suitable for drug discovery programmes aimed at investigating, understanding, and improving GT3a activities of HCV DAAs targeting NS5A or NS5B.


Asunto(s)
Hepacivirus/fisiología , Proteínas no Estructurales Virales/metabolismo , Virología/métodos , Replicación Viral , Evaluación Preclínica de Medicamentos/métodos , Genotipo , Hepacivirus/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Replicón , Proteínas no Estructurales Virales/genética
5.
Mol Ther ; 5(6): 770-9, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12027562

RESUMEN

Systemic administration of adenoviral vectors leads to a widespread distribution of vector. Therefore, targeting of adenoviral vectors to specific tissues or cell types will require methods to ablate the normal tropism of the vector simultaneously with the introduction of new receptor specificities. To inhibit native receptor binding, we mutated residues in the AB loop of the adenovirus type 5 (Ad5) fiber. We genetically incorporated the S408E-P409A mutation, referred to as KO1, into the adenoviral genome alone or in combination with an RGD-targeting ligand in the HI loop of fiber. Transduction experiments confirmed that the KO1 mutation results in a significant reduction in fiber-dependent gene transfer on A549 and primary fibroblast cells that could be restored via the RGD-targeting ligand. Competition transduction experiments verified the receptor-binding properties of each vector on A549 and hepatocytes in vitro. Unexpectedly, in mice systemic delivery of the vector containing the KO1 mutation resulted in efficient liver transduction that was localized specifically to hepatocytes. We confirmed these results in three different mouse strains, indicating that hepatic adenoviral gene transfer may be independent of the coxsackievirus-adenovirus receptor and that in vivo retargeting will require further viral capsid modifications to generate a fully detargeted adenoviral vector upon which to introduce new tropisms.


Asunto(s)
Adenovirus Humanos/genética , Vectores Genéticos , Receptores Virales/genética , Transducción Genética , Adenovirus Humanos/metabolismo , Animales , Western Blotting , Proteínas de la Cápside/genética , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Femenino , Terapia Genética , Células HeLa , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Oligopéptidos/genética , Receptores Virales/metabolismo , Especificidad de la Especie , Tropismo/genética , Células Tumorales Cultivadas
6.
J Virol ; 76(4): 1892-903, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11799184

RESUMEN

Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.


Asunto(s)
Adenoviridae/genética , Apolipoproteínas E/metabolismo , Marcación de Gen , Vectores Genéticos , Oligopéptidos/metabolismo , Receptores Virales/química , Adenoviridae/metabolismo , Animales , Apolipoproteínas E/genética , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Células HeLa , Humanos , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/genética , Receptores Virales/genética , Receptores Virales/metabolismo , Proteínas Recombinantes de Fusión/genética , Solubilidad , Transducción Genética
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