Administration Of Recombinant Protein Research Articles

Fibroblast Growth Factors in Cardiovascular Disease.

Despite advancements in managing traditional cardiovascular risk factors, many cardiovascular diseases (CVDs) persist. Fibroblast growth factors (FGFs) have emerged as potential diagnostic markers and therapeutic targets for CVDs. FGF1, FGF2, and FGF4 are primarily used for therapeutic angiogenesis. Clinical applications are being explored based on animal studies using approaches such as recombinant protein administration and adenovirus-mediated gene delivery, targeting patients with coronary artery disease and lower extremity arterial disease. Although promising results have been observed in animal models and early-stage clinical trials, further studies are required to assess their therapeutic potential. The FGF19 subfamily, consisting of FGF19, FGF21, and FGF23, act via endocrine signaling in various organs. FGF19, primarily expressed in the small intestine, plays important roles in glucose, lipid, and bile acid metabolism and has therapeutic potential for metabolic disorders. FGF21, found in various tissues, improves glucose metabolism and insulin sensitivity, suggesting potential for treating obesity and diabetes. FGF23, primarily secreted by osteocytes, regulates vitamin D and phosphate metabolism and serves as an important biomarker for chronic kidney disease and CVDs. Thus, FGFs holds promise for both therapeutic and diagnostic applications in metabolic and cardiovascular diseases. Understanding the mechanisms of FGF may pave the way for novel strategies to prevent and manage CVDs, potentially addressing the limitations of current treatments. This review explores the roles of FGF1, FGF2, FGF4, and the FGF19 subfamily in maintaining cardiovascular health. Further research and clinical trials are crucial to fully understand the therapeutic potential of FGFs in managing cardiovascular health.

Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway

The therapeutic administration of recombinant proteins is utilized in a multitude of research studies for treating various diseases. In this study, we investigate the therapeutic potential of Orosomucoid 2 (Orm2), an acute phase protein predominantly secreted by hepatocytes, for treating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Our results show that high Orm2 expression prevents high-fat-diet (HFD)-induced obesity in mice. Pharmacological administration of recombinant ORM2 protein ameliorates hepatic steatosis, inflammation, hepatocyte injury, and fibrosis in mouse livers afflicted by NAFLD and NASH under dietary stress. Orm2 knockout mice develop spontaneous obesity under a regular diet and exacerbate HFD-induced steatosis, steatohepatitis, and fibrosis. Mechanistically, Orm2 deletion activates the Erk1/2-PPARγ-Cd36 signaling pathway, increasing fatty acid uptake and absorption in hepatocytes and mice. Overall, our findings underscore the critical role of Orm2 in preventing NASH and associated NAFLD in the context of obesity.

Bioinformatics design of recombinant chimeric protein containing SipD and LptD immunogens and evaluation of its immunogenicity against Salmonella Typhimurium

The growing emergence of resistant bacteria is the current global concern for the humans and animals. Vaccination could be the desirable method to preventing such infectious diseases. Safe and effective vaccines are urgently needed to manage and prevent Salmonella contamination. Subunit vaccines are safe approaches for the protection against Salmonella spp. The bioinformatics methods were performed to determine the gene structure. Gene cassette (rLPSI) was ordered in pET28a (+), and cloned into E.coli BL21 (DE3), and the recombinant protein was expressed using IPTG (1mM). Mice were immunized by subcutaneous administration of recombinant protein. Then, the mice were challenged by oral administration of 100LD50 of live S. Typhimurium. The Codon adaptation index of the chimeric gene was multiplied by 0.92. Validation results showed that >90% of residues lie in the desired or extra allowed area of the Ramachandran plot. The recombinant protein (65.9kDa) was expressed in E.coli. Antibody titers in vaccinated mice were significantly different from those in the control groups. Recombinant protein immunization of the mice provided 90% and 70% protection against 10LD50 and 100LD50 of S. Typhimurium, respectively. In general, the results showed the high efficiency of rLPSI chimeric protein as a protective antigen against S. Typhimurium infection. The rLPSI chimeric protein could be an effective recombinant vaccine candidate against S. Typhimurium infection, but more research is needed.

Impact of Secretion-Active Osteoblast-Specific Factor 2 in Promoting Progression and Metastasis of Head and Neck Cancer.

Simple SummaryHead and neck cancers (HNC) exhibit poor survival due to metastases. Our study identified osteoblast-specific factor 2 (OSF-2) as overexpressed in primary tumors, lymph node metastases, and the tumor microenvironment. High OSF-2 levels correlate with metastatic disease and reduced survival of HPV-negative HNC patients. OSF-2’s active secretion signal seems to promote metastases by supporting the tumor microenvironment via the ß1 integrin-induced PI3K and Akt/PKB signaling pathway. We suggest OSF-2 as a potential biomarker and drug target to control (HPV-negative) HNC metastasis and disease management.Treatment success of head and neck cancer (HNC) is still hampered by tumor relapse due to metastases. Our study aimed to identify biomarkers by exploiting transcriptomics profiles of patient-matched metastases, primary tumors, and normal tissue mucosa as well as the TCGA HNC cohort data sets. Analyses identified osteoblast-specific factor 2 (OSF-2) as significantly overexpressed in lymph node metastases and primary tumors compared to normal tissue. High OSF-2 levels correlate with metastatic disease and reduced overall survival of predominantly HPV-negative HNC patients. No significant correlation was observed with tumor localization or therapy response. These findings were supported by the fact that OSF-2 expression was not elevated in cisplatin-resistant HNC cell lines. OSF-2 was strongly expressed in tumor-associated fibroblasts, suggesting a tumor microenvironment-promoting function. Molecular cloning and expression studies of OSF-2 variants from patients identified an evolutionary conserved bona fide protein secretion signal (1MIPFLPMFSLLLLLIVNPINA21). OSF-2 enhanced cell migration and cellular survival under stress conditions, which could be mimicked by the extracellular administration of recombinant protein. Here, OSF-2 executes its functions via ß1 integrin, resulting in the phosphorylation of PI3K and activation of the Akt/PKB signaling pathway. Collectively, we suggest OSF-2 as a potential prognostic biomarker and drug target, promoting metastases by supporting the tumor microenvironment and lymph node metastases survival rather than by enhancing primary tumor proliferation or therapy resistance.

Stimulation of Osteoclast Formation by Oncostatin M and the Role of WNT16 as a Negative Feedback Regulator.

Oncostatin M (OSM), which belongs to the IL-6 family of cytokines, is the most potent and effective stimulator of osteoclast formation in this family, as assessed by different in vitro assays. Osteoclastogenesis induced by the IL-6 type of cytokines is mediated by the induction and paracrine stimulation of the osteoclastogenic cytokine receptor activator of nuclear factor κ-B ligand (RANKL), expressed on osteoblast cell membranes and targeting the receptor activator of nuclear factor κ-B (RANK) on osteoclast progenitor cells. The potent effect of OSM on osteoclastogenesis is due to an unusually robust induction of RANKL in osteoblasts through the OSM receptor (OSMR), mediated by a JAK–STAT/MAPK signaling pathway and by unique recruitment of the adapter protein Shc1 to the OSMR. Gene deletion of Osmr in mice results in decreased numbers of osteoclasts and enhanced trabecular bone caused by increased trabecular thickness, indicating that OSM may play a role in physiological regulation of bone remodeling. However, increased amounts of OSM, either through administration of recombinant protein or of adenoviral vectors expressing Osm, results in enhanced bone mass due to increased bone formation without any clear sign of increased osteoclast numbers, a finding which can be reconciled by cell culture experiments demonstrating that OSM can induce osteoblast differentiation and stimulate mineralization of bone nodules in such cultures. Thus, in vitro studies and gene deletion experiments show that OSM is a stimulator of osteoclast formation, whereas administration of OSM to mice shows that OSM is not a strong stimulator of osteoclastogenesis in vivo when administered to adult animals. These observations could be explained by our recent finding showing that OSM is a potent stimulator of the osteoclastogenesis inhibitor WNT16, acting in a negative feedback loop to reduce OSM-induced osteoclast formation.

Galectin-3 enhances atrial remodelling and arrhythmogenesis through CD98 signalling.

Galectin-3 (Gal-3) is a biomarker of atrial fibrillation (AF) that mediates atrial inflammation. CD98 is the membrane surface receptor for Gal-3. Nevertheless, the role of the Gal-3/CD98 axis in atrial arrhythmogenesis is unclear. In this study, we investigated the effects of Gal-3/CD98 signalling on atrial pathogenesis. Whole cell patch clamp and western blotting were used to analyse calcium/potassium homeostasis and calcium-related signalling in Gal-3-administrated HL-1 atrial cardiomyocytes with/without CD98 neutralized antibodies. Telemetry electrocardiographic recording, Masson's trichrome staining and immunohistochemistry staining of atrium were obtained from mice having received tail-vein injections with Gal-3. Gal-3-treated HL-1 myocytes had a shorter action potential duration, smaller L-type calcium current, increased sarcoplasmic reticulum (SR) calcium content, Na+ /Ca2+ exchanger (NCX) current, transient outward potassium current, and ultrarapid delayed rectifier potassium current than control cells had. Gal-3-treated HL-1 myocytes had greater levels of SR Ca2+ ATPase, NCX, Nav1.5, and NLR family pyrin domain containing 3 (NLRP3) expression and increased calcium/calmodulin-dependent protein kinase II (CaMKII), ryanodine receptor 2 (RyR2), and nuclear factor kappa B (NF-κB) phosphorylation than control cells had. Gal-3-mediated activation of CaMKII/RyR2 pathway was diminished in the cotreatment of anti-CD98 antibodies. Mice that were injected with Gal-3 had more atrial ectopic beats, increased atrial fibrosis, and activated NF-κB/NLRP3 signalling than did control mice (nonspecific immunoglobulin) or mice treated with Gal-3 and anti-CD98 antibodies. Gal-3 recombinant protein administration increases atrial fibrosis and arrhythmogenesis through CD98 signalling. Targeting Gal-3/CD98 axis might be a novel therapeutic strategy for patients with AF and high Gal-3 levels.

Recombinant flagellin protein can efficiently protect mice against salmonella typhi

Introduction: Globally, Salmonella enterica serotype Typhi is responsible for more than 10 million enteric fever cases, annually. Because of the emergence of multidrug resistance strains of many bacteria, including Salmonella Typhi, vaccination may be a preferred strategy to combat infectious diseases. In the present study, the efficiency of flagellin protein as a recombinant vaccine candidate was evaluated in BALB/c mice. Materials and Methods: For this aim, flagellin protein was expressed in E. coli BL21 (DE3). Mice were grouped into two groups: test and control. Test group were immunized by the intraperitoneal administration of recombinant protein in combination with Freund�s adjuvant. Following the completion of the immunization period, the mice were challenged by IP injection of 10 LD50 of live Salmonella Typhi and subsequent culture of their spleens and livers. Results: Flagellin protein expression was confirmed by SDS-PAGE and Western blotting. ELISA showed the proper stimulation of the humoral immunity of the immunized mice. The bacterial count decreased significantly in the spleens and livers of the immunized animals in comparison to the control ones. Conclusions: Findings of this study show the efficiency of flagellin recombinant protein in protecting mice against Salmonella Typhi. © 2021 The Author(s).

Controlled Non‐Viral Gene Delivery in Cartilage and Bone Repair: Current Strategies and Future Directions

AbstractRecent advances in the approval and commercialization of gene therapeutics have fostered the return of gene therapy to center stage. But despite new optimism, no Food and Drug Administration approved product exists for the treatment of orthopedic disorders. Non‐viral gene delivery is a promising alternative to recombinant protein administration and viral gene transduction for orthopedic tissue engineering. When applied using appropriately designed systems, it enables temporal control of the overexpression of therapeutic genes, leading to local production of regulatory factors at physiologically relevant levels. Incorporating genetic material into 3D scaffold biomaterials, that is, gene activated scaffolds or hydrogels, presents a particular opportunity to utilize non‐viral gene therapy for in situ transfection of host cells and the regeneration of damaged tissues and organs. But controlled non‐viral gene delivery for musculoskeletal regeneration depends on a multifactorial design in which the choice of gene delivery method, therapeutic gene, and supportive biomaterial play a central role for the success of this strategy. This paper reviews the different modalities of non‐viral gene delivery used for the repair of bone and cartilage, and explores the current challenges and opportunities for the engineering of functional orthopedic tissues using gene activated scaffolds.

Lactic Acid Bacteria as Vectors: A Novel Approach for Mucosal Vaccine Delivery

Lactic Acid Bacteria (LAB) has been used in food industry due to its classification as food grade microorganism. It has been used for food production as well as preservation on large scale. It is also considered as promising bacterial strain due to its probiotic activity that confirms human health. Moreover, it also shows resistance regarding its survival in Gastrointestinal Tract (GIT). Therefore, use of LAB as delivery platform for drugs as well as production of recombinant protein is a challenging approach for researchers now a day. As, it not only reduces the production cost of drug, but also act as live vector to synthesize and deliver target or therapeutic protein of interest. Moreover, it is possible to produce different proteins from same bacteria simultaneously. Thus altogether, this approach has not only provided an alternative option for intravenous administration of recombinant protein but also gives an alternative insight for delivery system of mucosal vaccine. This review aims to provide an overview in order to use specific species of LAB such as lactococci lactis and Lactobacillus as vector for transfer of vaccine for mucosal as well as in recombinant form. Moreover, use of intron for desired genetic variation into target sites is explained to give directional insight for future studies.

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

Rhinacanthin-C, A Fat-Soluble Extract from Rhinacanthus nasutus, Modulates High-Mobility Group Box 1-Related Neuro-Inflammation and Subarachnoid Hemorrhage-Induced Brain Apoptosis in a Rat Model

High-mobility group box 1 (HMGB1) was shown to be a major extracellular mediator involved in relayed neuro-inflammation in animals after subarachnoid hemorrhage (SAH). It is of interest to examine the effect of rhinacanthin-C (RCT-C, C25H30O5) on pro-inflammatory cytokines/HMGB1 in an SAH-related early brain injury model. A rodent double SAH model was used. RCT-C was administered orally at 100, 200, and 400 μmol/kg/day. Cerebral spinal fluid samples were obtained to assess interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor α using a real-time polymerase chain reaction. Basilar arteries were harvested and cerebral cortex was examined for HMGB1 mRNA and protein expression (western blot) and caspases (real-time polymerase chain reaction). An intrathecal injection of 1 ng of HMGB-1 recombinant protein was given in the 400 μmol/kg/day RCT-C plus SAH groups. The levels of IL-1β, IL-6, and tumor necrosis factor α mRNA were significantly increased in animals subject to SAH, compared with the healthy controls, but were absent in the RCT-C groups. Cleaved caspase-9a as well as HMGB-1 mRNA and protein were significantly reduced in the 400 μmol/kg/day RCT-C treatment groups. Similarly, administration of RCT-C reduced HMGB-1 mRNA and protein expression (P <0.01). RCT-C exerts a neuroprotective effect by reducing cleaved caspase-3- and caspase-9a-related apoptosis. Decreased HMGB-1 mRNA and protein expression in the RCT-C groups corresponds to its anti-inflammatory effect. HMGB-1 recombinant protein administration impaired the neuroprotective and immunosuppressive effect of RCT-C. This finding lends credence that RCT-C modulates the HMGB-1-related pathway and attenuates brain apoptosis in the pathogenesis of SAH.

C1q/TNF-related Protein 4 (CTRP4) Is a Unique Secreted Protein with Two Tandem C1q Domains That Functions in the Hypothalamus to Modulate Food Intake and Body Weight

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance.

A high-capacity cell macroencapsulation system supporting the long-term survival of genetically engineered allogeneic cells

The rapid increase in the number of approved therapeutic proteins, including recombinant antibodies, for diseases necessitating chronic treatments raises the question of the overall costs imposed on healthcare systems. It is therefore important to investigate alternative methods for recombinant protein administration. The implantation of genetically engineered cells is an attractive strategy for the chronic long-term delivery of recombinant proteins. Here, we have developed a high-capacity cell encapsulation system for the implantation of allogeneic myoblasts, which survive at high density for at least one year. This flat sheet device is based on permeable polypropylene membranes sealed to a mechanically resistant frame which confine cells seeded in a tailored biomimetic poly(ethylene glycol) (PEG)-based hydrogel matrix. In order to quantitate the number of cells surviving in the device and optimize initial conditions leading to high-density survival, we implant devices containing C2C12 mouse myoblasts expressing a luciferase reporter in the mouse subcutaneous tissue. We show that initial cell load, hydrogel stiffness and permeable membrane porosity are critical parameters to achieve long-term implant survival and efficacy. Optimization of these parameters leads to the survival of encapsulated myogenic cells at high density for several months, with minimal inflammatory response and dense neovascularization in the adjacent host tissue. Therefore, this encapsulation system is an effective platform for the implantation of genetically engineered cells in allogeneic conditions, which could be adapted to the chronic administration of recombinant proteins.

Antibody Gene Therapy: Getting Closer to Clinical Application?

Monoclonal antibodies are successfully used in the clinic. However, repeated high-dose bolus injections imply high costs. As an alternative to recombinant protein administration, gene therapy may provide a novel mean for systemic delivery of monoclonal antibodies. This strategy has been used in preclinical studies of a wide variety of pathological conditions, including cancer, infectious diseases, drug addiction, retinal neovascularisation and Alzheimer's disease. The two main gene therapy approaches are based on direct gene delivery (using viral or non-viral vectors) or on inoculation of ex vivo genetically modified cells (autologous or allogenic). Viral vectors are highly efficient as gene delivery vehicles and have been tested in numerous clinical trials, but still raise concerns about safety and limitation of the therapeutic effect due to immune responses against viral antigens. On the other hand, use of standard non-viral vectors has been limited by their low transduction efficiency. Practical application of ex vivo genetically modified cells would imply the availability of stocks of "ready-to-use" gene-modified allogenic cells that should be protected from the host immune system. Actually, this field did not meet the expectation raised initially, mainly because of difficulties with obtaining sustained therapeutic plasma levels in animal models. However, this situation is changing rapidly and the therapeutic potential of these approaches is getting closer to clinical applications. This review focuses on the current achievements in preclinical studies, as well as the challenges and future prospects of antibody gene therapy.

BAFF Controls Neural Cell Survival through BAFF Receptor

Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell–activating factor (BAFF) and its receptor (BAFF-R) are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.

Growth Differentiation Factor 5 Regulation in Bone Regeneration

Growth differentiation factor 5 (GDF5) is a member of the bone morphogenic protein (BMP) family and plays critical roles in organ development processes including bone, cartilage, ligament, and joint formation. GDF5 is expressed in the cartilage primordium in the early limb development, and in the interzone of joint formation sites. GDF5 is also observed in adult tissue and cell lines. This spatialtemporal expression pattern of GDF5 proves its essential role in the formation of bone and cartilage. Similar to other members of BMPs, the signaling cascade of GDF5 is originated through binding to type I and type II receptors and thus regulating the downstream intracellular biochemical processes. Mutations of GDF5 are associated with several human and animal diseases that are characterized by skeletal deformity such as short digits and short limbs. In vitro and in vivo studies demonstrated that overexpression of GDF5 or administration of recombinant protein promotes chondrogenesis and osteogenesis. Moreover, a promising feature of GDF5 is osteoinduction, which is used in tissue engineering for bone repair with or without a carrier in animal platforms and in human preclinical settings. The exciting results signify that GDF5 is a compelling candidate for bone tissue engineering by enhancing osteogenesis and angiogenesis. In this review, we will focus the discussion on the basic structure, signaling pathways, function in cartilage and bone formation, and potential clinical application of GDF5 in bone tissue regeneration.

Expression of Cholera Toxin B–Lumbrokinase Fusion Protein in Pichia pastoris—The Use of Transmucosal Carriers in the Delivery of Therapeutic Proteins to Protect Rats Against Thrombosis

Cholera toxin B-subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. However, whether CTB can be used as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins has not been widely studied. Thus, we investigate here the concept of receptor-mediated oral delivery of lumbrokinase (LK) proteins which is an important fibrinolytic enzyme derived from earthworm. CTB and LK, separated by a furin cleavage site, was expressed via Pichia pastoris. The activity and proper folding of recombinant protein in yeast were confirmed by Western blot analysis, fibrin plate assays, and G(M1)-ganglioside ELISA. Following oral administration of recombinant protein, the thrombosis model of rats and mice revealed that the oral treatment of rCTB-LK has a more significant anti-thrombotic effect on animals compared with rLK. It is possible to conclude that CTB can successfully enhance its fusion protein LK to be absorbed. The use of CTB as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins was supported.

C1q/TNF-related Protein-12 (CTRP12), a Novel Adipokine That Improves Insulin Sensitivity and Glycemic Control in Mouse Models of Obesity and Diabetes

Despite the prevalence of insulin resistance and type 2 diabetes mellitus, their underlying mechanisms remain incompletely understood. Many secreted endocrine factors and the intertissue cross-talk they mediate are known to be dysregulated in type 2 diabetes mellitus. Here, we describe CTRP12, a novel adipokine with anti-diabetic actions. The mRNA and circulating levels of CTRP12 were decreased in a mouse model of obesity, but its expression in adipocytes was increased by the anti-diabetic drug rosiglitazone. A modest rise in circulating levels of CTRP12 by recombinant protein administration was sufficient to lower blood glucose in wild-type, leptin-deficient ob/ob, and diet-induced obese mice. A short term elevation of serum CTRP12 by adenovirus-mediated expression improved glucose tolerance and insulin sensitivity, normalized hyperglycemia and hyperinsulinemia, and lowered postprandial insulin resistance in obese and diabetic mice. CTRP12 improves insulin sensitivity in part by enhancing insulin signaling in the liver and adipose tissue. Further, CTRP12 also acts in an insulin-independent manner; in cultured hepatocytes and adipocytes, CTRP12 directly activated the PI3K-Akt signaling pathway to suppress gluconeogenesis and promote glucose uptake, respectively. Collectively, these data establish CTRP12 as a novel metabolic regulator linking adipose tissue to whole body glucose homeostasis through insulin-dependent and independent mechanisms.

Induction of erythropoiesis using human vascular networks genetically engineered for controlled erythropoietin release

AbstractFor decades, autologous ex vivo gene therapy has been postulated as a potential alternative to parenteral administration of recombinant proteins. However, achieving effective cellular engraftment of previously retrieved patient cells is challenging. Recently, our ability to engineer vasculature in vivo has allowed for the introduction of instructions into tissues by genetically modifying the vascular cells that build these blood vessels. In the present study, we genetically engineered human blood–derived endothelial colony-forming cells (ECFCs) to express erythropoietin (EPO) under the control of a tetracycline-regulated system, and generated subcutaneous vascular networks capable of systemic EPO release in immunodeficient mice. These ECFC-lined vascular networks formed functional anastomoses with the mouse vasculature, allowing direct delivery of recombinant human EPO into the bloodstream. After activation of EPO expression, erythropoiesis was induced in both normal and anemic mice, a process that was completely reversible. This approach could relieve patients from frequent EPO injections, reducing the medical costs associated with the management of anemia. We propose this ECFC-based gene-delivery strategy as a viable alternative technology when routine administration of recombinant proteins is needed.

Large-volume Intrathecal Enzyme Delivery Increases Survival of a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative lysosomal storage disorder caused by mutations in TPP1, the gene encoding the lysosomal protease tripeptidyl-peptidase (TPP1). LINCL primarily affects children, is fatal and there is no effective treatment. Administration of recombinant protein has proved effective in treatment of visceral manifestations of other lysosomal storage disorders but to date, only marginal improvement in survival has been obtained for neurological diseases. In this study, we have developed and optimized a large-volume intrathecal administration strategy to deliver therapeutic amounts of TPP1 to the central nervous system (CNS) of a mouse model of LINCL. To determine the efficacy of treatment, we have monitored survival as the primary endpoint and demonstrate that an acute treatment regimen (three consecutive daily doses started at 4 weeks of age) increases median lifespan of the LINCL mice from 16 (vehicle treated) to 23 weeks (enzyme treated). Consistent with the increase in life-span, we also observed significant reversal of pathology and improvement in neurological phenotype. These results provide a strong basis for both clinical investigation of large-volume/high-dose delivery of TPP1 to the brain via the cerebrospinal fluid (CSF) and extension of this approach towards other neurological lysosomal storage diseases.