The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

Introduction: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs.

The CRISPR/Cas9 Minipig-A Transgenic Minipig to Produce Specific Mutations in Designated Tissues.

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells.

FRMD6 has tumor suppressor functions in prostate cancer.

Available tools for prostate cancer (PC) prognosis are suboptimal but may be improved by better knowledge about genes driving tumor aggressiveness. Here, we identified FRMD6 (FERM domain-containing protein 6) as an aberrantly hypermethylated and significantly downregulated gene in PC. Low FRMD6 expression was associated with postoperative biochemical recurrence in two large PC patient cohorts.

FcRn overexpression in human cancer drives albumin recycling and cell growth; a mechanistic basis for exploitation in targeted albumin-drug designs.

Albumin accumulation in tumours could reflect a role of albumin in transport of endogenous nutrient cargos required for cellular growth and not just a suggested source of amino acids; a role driven by albumin engagement with its cognate cellular recycling neonatal Fc receptor. We investigate the hypothesis that albumin cellular recruitment is increased by higher human FcRn (hFcRn) expression in human cancer tissue that provides the mechanistic basis for exploitation in albumin-based drug designs engineered to optimise this process. Eight out of ten different human cancer tissue types screened for hFcRn expression by immunohistochemistry (310 samples) exhibited significantly higher hFcRn expression compared to healthy tissues.

Molecular, Macromolecular, and Supramolecular Glucuronide Prodrugs: Lead Identified for Anticancer Prodrug Monotherapy.

In this work, a tumor growth intervention by localized drug synthesis within the tumor volume, using the enzymatic repertoire of the tumor itself, is presented. Towards the overall success, molecular, macromolecular, and supramolecular glucuronide prodrugs were designed for a highly potent toxin, monomethyl auristatin E (MMAE). The lead candidate exhibited a fold difference in toxicity between the prodrug and the drug of 175, had an engineered mechanism to enhance the deliverable payload to tumours, and contained a highly potent toxin such that bioconversion of only a few prodrug molecules created a concentration of MMAE sufficient enough for efficient suppression of tumor growth.

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling.

Imaging Rheumatoid Arthritis in Mice Using Combined Near Infrared and F Magnetic Resonance Modalities.

Rheumatoid arthritis (RA) is an autoimmune disease that causes pain and tissue destruction in people worldwide. An accurate diagnosis is paramount in order to develop an effective treatment plan. This study demonstrates that combining near infrared (NIR) imaging and F MRI with the injection of labelled nanoparticles provides high diagnostic specificity for RA.

Lipidoid-siRNA Nanoparticle-Mediated IL-1β Gene Silencing for Systemic Arthritis Therapy in a Mouse Model.

Interleukin-1 beta (IL-1β) plays a central role in the induction of rheumatoid arthritis (RA). In the present study, we demonstrated that lipidoid-polymer hybrid nanoparticle (FS14-NP) can efficiently deliver siRNA against IL-1β (siIL-1β) to macrophages and effectively suppress the pathogenesis of experimental arthritis induced by collagen antibody (CAIA mice). FS14-NP/siIL-1β achieved approximately 70% and 90% gene-silencing efficiency in the RAW 264.

Targeted lipid nanoparticle delivery of calcitriol to human monocyte-derived macrophages in vitro and in vivo: investigation of the anti-inflammatory effects of calcitriol.

Background: Vitamin D possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers.

Purpose: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo.

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response.

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway.

In Vivo Editing of the Adult Mouse Liver Using CRISPR/Cas9 and Hydrodynamic Tail Vein Injection.

CRISPR/Cas9 technology allows facile modification of the genome in virtually any desired way through the use of easily designed plasmid constructs that express a gRNA targeting a genomic site-of-interest and Cas9. Hydrodynamic tail vein injection, on the other hand, is a simple method to deliver "naked" plasmid DNA to 5-40% of the hepatocytes of the liver of adult mice. Here, we describe how these two techniques can be combined to create a workflow for fast, easy, and cost-efficient in vivo genome editing of the adult mouse liver.

Synchronous delivery of hydroxyapatite and connective tissue growth factor derived osteoinductive peptide enhanced osteogenesis.

In bone tissue engineering, electrospun fibrous scaffolds can provide excellent mechanical support, extracellular matrix mimicking components, such as 3D spacial fibrous environment for cell growth and controlled release of signaling molecules for osteogenesis. Here, a facile strategy comprising the incorporation of an osteogenic inductive peptide H1, derived from the cysteine knot (CT) domain of connective tissue growth factor (CTGF), in the core of Silk Fibroin (SF) was developed for osteogenic induction, synergistically with co-delivering hydroxyapatite (HA) from the shell of poly(l-lactic acid-co-ε-caprolactone) (PLCL). The core-shell nanofibrous structure was confirmed by transmission electron microscopy (TEM).

A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation.

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines.

Up-Regulated FGFR1 Expression as a Mediator of Intrinsic TKI Resistance in EGFR-Mutated NSCLC.

Non-small cell lung carcinoma patients with epidermal growth factor receptor (EGFR) mutations are offered EGFR tyrosine kinase inhibitors (TKI) as first line treatment, but 20-40% of these patients do not respond. High expression of alternative receptor tyrosine kinases, such as Fibroblast growth factor receptor 1 (FGFR1), potentially mediates intrinsic EGFR TKI resistance. To study this in molecular detail, we used CRISPR-dCas9 Synergistic Activation Mediator (SAM) for up-regulation of FGFR1 in physiological relevant levels in the EGFR mutated NSCLC cell lines HCC827 and PC9 thereby generating HCC827 and PC9.

Non-covalent hitchhiking on endogenous carriers as a protraction mechanism for antiviral macromolecular prodrugs.

Albumin is a highly successful tool of drug delivery providing drastically extended body and blood residence time for the associated cargo, but it only traffics single drug copies at a time. In turn, macromolecular prodrugs (MP) are advantaged in carrying a high drug payload but offering only a modest extension of residence time to the conjugated drugs. In this work, we engineer MP to contain terminal groups that bind to albumin via non-covalent association and reveal that this facile measure affords a significant protraction for the associated polymers.

Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein.

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes.

Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement.

Recombinant albumin-drug genetic fusions are an effective technology to prolong the serum half-life of therapeutics that has resulted in marketed products. Indirect evidence suggests albumin fusions' long circulation is controlled by engagement with the cellular recycling neonatal Fc receptor (FcRn) in addition to reduced kidney filtration. In this work, we have used a panel of recombinant fusions, engineered with different human FcRn (hFcRn) affinity, including a novel high binding albumin variant (HBII), to directly define and importantly, control the intracellular mechanism as a half-life extension tuning method.

Theranostic Niosomes for Efficient siRNA/MicroRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells.

RNA interference-mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs [small interfering RNA (siRNA)/microRNA (miRNA)] to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for the in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed "niosome", that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

Long-Acting, Potent Delivery of Combination Antiretroviral Therapy.

Antiretroviral therapy (ART) has revolutionized HIV treatment, yet grand challenges remain: (i) short blood and body residence time of the antiviral drugs, (ii) relative poor antiretroviral drug penetrance into key tissue reservoirs of viral infection, namely, the spleen and lymph nodes, and (iii) obstacles in different pharmacokinetics of the necessary combination drugs. We present a novel drug delivery approach that simultaneously overcomes these limitations. We designed albumin-polymer-drug conjugates where albumin ensures long body residence time as well as lymphatic accumulation of the conjugate.

Fibrogenic and angiogenic commitments of human induced pluripotent stem cells derived mesenchymal stem cells in connective tissue growth factor-delivering scaffold in an immune-deficient mice model.

Compared to terminal differentiated cells, stem cells play important roles in the maintenance and regeneration, and thus have been intensively researched as the most promising cell based therapy. In order to maximize the effectiveness of stem cell based therapies, it is essential to understand the environmental (niche) signals that regulate stem cell behavior. Recent findings suggest that fibroblasts have a mesenchymal origin and that mesenchymal stem cells (MSCs) demonstrate proangiogenic function, where both fibrogenic and angiogenic activities are associated with connective tissue growth factor (CTGF), a matricellular protein that serves as an essential mediator of skeletogenesis in development and vascular remodeling.

CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

Background & Aims: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis.

Synthetic Polymer with a Structure-Driven Hepatic Deposition and Curative Pharmacological Activity in Hepatic Cells.

Synthetic polymers make strong contributions as tools for delivery of biological drugs and chemotherapeutics. The most praised characteristic of polymers in these applications is complete lack of pharmacological function such as to minimize the side effects within the human body. In contrast, synthetic polymers with curative pharmacological activity are truly rare.

A genetically inducible porcine model of intestinal cancer.

Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer.

Analysis of Factor D Isoforms in Malpuech-Michels-Mingarelli-Carnevale Patients Highlights the Role of MASP-3 as a Maturase in the Alternative Pathway of Complement.

Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation.

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1.

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration.