The Experts below are selected from a list of 25935 Experts worldwide ranked by ideXlab platform
Alessio Branchini - One of the best experts on this subject based on the ideXlab platform.
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The chaperone‐like sodium phenylbutyrate improves factor IX Intracellular Trafficking and activity impaired by the frequent p.R294Q mutation
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Silvia Pignani, Aldo Todaro, Mattia Ferrarese, Saverio Marchi, Silvia Lombardi, Dario Balestra, Francesco Bernardi, Mirko Pinotti, Paolo Pinton, Alessio BranchiniAbstract:Essentials Missense mutations often impair protein folding, and thus Intracellular Trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved Intracellular Trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. Background Missense mutations often impair protein folding and Intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), Intracellular Trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired Intracellular Trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved Trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy. © 2018 International Society on Thrombosis and Haemostasis.
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the chaperone like sodium phenylbutyrate improves factor ix Intracellular Trafficking and activity impaired by the frequent p r294q mutation
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Silvia Pignani, Aldo Todaro, Mattia Ferrarese, Saverio Marchi, Silvia Lombardi, Dario Balestra, Francesco Bernardi, Mirko Pinotti, Paolo Pinton, Alessio BranchiniAbstract:Essentials Missense mutations often impair protein folding, and thus Intracellular Trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved Intracellular Trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. SUMMARY: Background Missense mutations often impair protein folding and Intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), Intracellular Trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired Intracellular Trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved Trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.
Silvia Pignani - One of the best experts on this subject based on the ideXlab platform.
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The chaperone‐like sodium phenylbutyrate improves factor IX Intracellular Trafficking and activity impaired by the frequent p.R294Q mutation
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Silvia Pignani, Aldo Todaro, Mattia Ferrarese, Saverio Marchi, Silvia Lombardi, Dario Balestra, Francesco Bernardi, Mirko Pinotti, Paolo Pinton, Alessio BranchiniAbstract:Essentials Missense mutations often impair protein folding, and thus Intracellular Trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved Intracellular Trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. Background Missense mutations often impair protein folding and Intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), Intracellular Trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired Intracellular Trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved Trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy. © 2018 International Society on Thrombosis and Haemostasis.
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the chaperone like sodium phenylbutyrate improves factor ix Intracellular Trafficking and activity impaired by the frequent p r294q mutation
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Silvia Pignani, Aldo Todaro, Mattia Ferrarese, Saverio Marchi, Silvia Lombardi, Dario Balestra, Francesco Bernardi, Mirko Pinotti, Paolo Pinton, Alessio BranchiniAbstract:Essentials Missense mutations often impair protein folding, and thus Intracellular Trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved Intracellular Trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. SUMMARY: Background Missense mutations often impair protein folding and Intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), Intracellular Trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired Intracellular Trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved Trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.
Rudolph L Juliano - One of the best experts on this subject based on the ideXlab platform.
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cellular uptake and Intracellular Trafficking of oligonucleotides
Advanced Drug Delivery Reviews, 2015Co-Authors: Rudolph L Juliano, Kyle CarverAbstract:Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and Intracellular Trafficking of both ‘free’ oligonucleotides and oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and Intracellular Trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects.
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cellular uptake and Intracellular Trafficking of oligonucleotides implications for oligonucleotide pharmacology
Nucleic Acid Therapeutics, 2014Co-Authors: Rudolph L Juliano, Kyle Carver, Xin Ming, Brian LaingAbstract:One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and Intracellular Trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and Trafficking and then review recent literature on targeted delivery and subcellular Trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.
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cellular uptake and Intracellular Trafficking of antisense and sirna oligonucleotides
Bioconjugate Chemistry, 2012Co-Authors: Rudolph L Juliano, Xin Ming, Osamu NakagawaAbstract:Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in cellular and animal models and in some clinical studies. Nonetheless, our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and Intracellular Trafficking of oligonucleotides, whether as “free” molecules or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review, we examine the recent literature on oligonucleotide internalization and subcellular Trafficking in the context of current insights into the basic machinery for endocytosis and Intracellular vesicular traffic.
Hideyoshi Harashima - One of the best experts on this subject based on the ideXlab platform.
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Multifunctional envelope-type nano device for controlled Intracellular Trafficking and selective targeting in vivo.
Journal of Controlled Release, 2014Co-Authors: Kazuaki Kajimoto, Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi HarashimaAbstract:Nanomedicine is expected to be a basic technology for using nucleic acids as a drug, in which treating the cause of diseases represent the ultimate therapy. However, a sophisticated delivery system is required for efficient delivery of RNA/DNA, since these compounds need precise control of Intracellular Trafficking as well as biodistribution. Here we report on the use of a multifunctional envelope-type nano device (MEND) which is capable of Intracellular Trafficking such as endosomal escape, delivery to mitochondria, as well as active targeting to selective tissues/cells in vivo. In this review, we focused on the controlled Intracellular Trafficking of antigens for advanced immunotherapy, and then introduced a mitochondrial delivery system as an organelle targeting system for unmet medical needs. We also provide a successful in vivo delivery of siRNA to the liver based on a newly designed pH-responsive cationic lipid. Finally we will discuss an important role of an active targeting system using a peptide ligand to adipose vasculature. These progresses in drug delivery system will break through the barriers exist in our body, tissues and cells and open a window for future Nanomedicine.
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Octaarginine-modified liposomes: enhanced cellular uptake and controlled Intracellular Trafficking.
International journal of pharmaceutics, 2007Co-Authors: Ikramy A Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi HarashimaAbstract:Gene therapy is a promising new approach for treating a variety of genetic and acquired diseases. While viral vectors are highly efficient for gene therapy, their use is associated with high toxicity and immunogenicity. Synthetic or nonviral vectors are attractive alternatives to viral vectors because of their low immunogenicity and low acute toxicity. The main disadvantage of the nonviral vectors is the low transfection efficiency compared to viral vectors. Novel functional devices to enhance the transfection activities of nonviral vectors are needed. In this review, we discuss the modification of liposomal drug carriers with a novel functional device, the octaarginine (R8) peptide, for drug and gene delivery. Decoration of liposomes with R8 enhanced their cellular uptake. In addition, by optimizing the density of the peptide as well as its topology, the liposomes could be internalized via clathrin-independent pathways, which improved the Intracellular Trafficking through avoiding lysosomal degradation. A special emphasis is given to the need for optimizing the conditions of using the peptide to not only enhance the cellular uptake but also to improve the Intracellular Trafficking of its cargos. In addition, the use of R8-modified liposomes and nano-particles in gene delivery is discussed.
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Development of non-viral vector based on the quantitative comparison of Intracellular Trafficking with viral vector
Yakugaku Zasshi-journal of The Pharmaceutical Society of Japan, 2006Co-Authors: Hidetaka Akita, Susumu Hama, Hiroyuki Mizuguchi, Hideyoshi HarashimaAbstract:For the development of efficient gene vector, Intracellular processes such as cellular uptake, endosomal release and nuclear delivery must be overcome. Viruses have also evolved and have developed sophisticated mechanisms for controlling Intracellular Trafficking for the efficient delivery of their genomes to nuclei in host cells for symbiosis. In the light of these mechanisms, various kinds of artificial devices have been developed to overcome the Intracellular barriers. However, in the majority of studies, variation of the transfection activity before and after the modification of devices was evaluated, and Intracellular Trafficking remained unclear. Therefore, it is understand to recognize which of the Intracellular barrier should be intensively improved to enhance the transfection activity. To clarify the rate-limited process in the current non-viral vector, we compared the Intracellular Trafficking between adenovirus and LipofectAMINE PLUS. As a result, we found that difference of the transfection efficiency between adenovirus and LipofectAMINE PLUS was dominantly derived from the differences on transcription activity. Therefore it is essential to consider the regulation of the intranuclear events to improve the transfection activity of artificial vector.
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high density of octaarginine stimulates macropinocytosis leading to efficient Intracellular Trafficking for gene expression
Journal of Biological Chemistry, 2006Co-Authors: Ikramy A Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi HarashimaAbstract:Abstract The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and Intracellular Trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an Intracellular Trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to Intracellular Trafficking, resulting in different levels of gene expression.
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Intracellular Trafficking and transgene expression of viral and non-viral gene vectors.
Advanced Drug Delivery Reviews, 2001Co-Authors: Hiroyuki Kamiya, Hiroyuki Tsuchiya, J Yamazaki, Hideyoshi HarashimaAbstract:The objective of this review is to summarize the critical steps in Intracellular Trafficking and the principal factors involved in transgene expression by a comparison of viral and non-viral gene vectors. Intracellular Trafficking of viral and non-viral gene vectors are reviewed kinetically as well as from the mechanistic point of view. The peptide-dependent specific mechanism of endosomal escape by viral vectors is compared with the non-specific mechanism of non-viral vectors. Regarding the nuclear transport of DNA, a number of recently developed strategies in non-viral vectors such as the application of nuclear localization signals or cell specific transcription factors are summarized in comparison with viral nuclear gene delivery. The molecular mechanisms of transcription and the translation of delivered genes to nucleus are also summarized in view of drug delivery systems. This information is intended to serve as a basis for developing a new gene delivery system for both viral and non-viral gene vectors. Optimizing the gene delivery system by integrating this Intracellular Trafficking as well as transgene expression will be required in order to develop an efficient and an safe gene delivery system.
Mattia Ferrarese - One of the best experts on this subject based on the ideXlab platform.
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The chaperone‐like sodium phenylbutyrate improves factor IX Intracellular Trafficking and activity impaired by the frequent p.R294Q mutation
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Silvia Pignani, Aldo Todaro, Mattia Ferrarese, Saverio Marchi, Silvia Lombardi, Dario Balestra, Francesco Bernardi, Mirko Pinotti, Paolo Pinton, Alessio BranchiniAbstract:Essentials Missense mutations often impair protein folding, and thus Intracellular Trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved Intracellular Trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. Background Missense mutations often impair protein folding and Intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), Intracellular Trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired Intracellular Trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved Trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy. © 2018 International Society on Thrombosis and Haemostasis.
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the chaperone like sodium phenylbutyrate improves factor ix Intracellular Trafficking and activity impaired by the frequent p r294q mutation
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Silvia Pignani, Aldo Todaro, Mattia Ferrarese, Saverio Marchi, Silvia Lombardi, Dario Balestra, Francesco Bernardi, Mirko Pinotti, Paolo Pinton, Alessio BranchiniAbstract:Essentials Missense mutations often impair protein folding, and thus Intracellular Trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved Intracellular Trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. SUMMARY: Background Missense mutations often impair protein folding and Intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), Intracellular Trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired Intracellular Trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved Trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.