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Metabolism of propionate in human subjects was studied using bolus administration of 1-13C-propionate i.v. or orally. The study population consisted of five patients with propionic acidemia (PA), eight with methylmalonic acidemia (MMA; four responsive to vitamin B12), one each with multiple carboxylase deficiency and transcobalamin-II deficiency, and five healthy volunteers. Concentrations of 1-13C-propionate were measured in blood in three patients with PA, two with MMA, and two controls. Breath samples were obtained at intervals during 3 h after the dose, isotopic enrichment of 13CO2 was measured, and the cumulative percentage of recovery of 13C was calculated from the individual's predicted resting energy expenditure. Recovery of 13CO2 and half-time of 1-13C-propionate in PA were significantly less than normal. The same parameters in MMA were below normal, but significantly greater than in PA. Recovery of 13CO2 was well correlated with clinical severity in PA, but did not correlate in MMA. Differences between MMA and PA may indicate different distribution of propionate pools, differences in inducibility of residual enzyme activities, or an alternate pathway for decarboxylation of propionate available in MMA but not PA. Only one patient with PA demonstrated increased 13CO2 production during biotin treatment. In a B12-responsive MMA patient, no differences were noted within 2 d of initiating treatment with B12, but there was an increase in 13CO2 production after 4 mo. Recovery of 13CO2 was normal in the patient with transcobalamin-II deficiency before and after treatment with vitamin B12.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Champions are widely recognized as playing a key role in the successful implementation of evidence-based interventions within the health care sector; however, little is known about which characteristics and skills enable them to play that role. Furthermore, previous studies have measured only individual champions' responses to personal attributes without incorporating input from other observers. A mixed-methods study was conducted to identify, analyze, and group the behaviors and characteristics of champions who have successfully promoted the adoption of new initiatives within the health care delivery system, taking into consideration self and peer perspectives., Methods: Using a mixed-methods, cross-sectional triangulation design with a convergence model, quantitative data were collected and analyzed from health care champions (n = 30) and their colleagues (n = 58) from 11 countries using a survey. Every champion and a subset of colleagues (n = 14) also participated in in-depth interviews. Descriptive statistics were used to explore the relationship between champion and colleague responses to survey items; chi-squared tests and Kruskal-Wallis tests were used to compare the differences. Thematic content analysis of qualitative data was used to explore champion-like behaviors and features. Characteristics of champions were categorized using the Transformational Leadership Theory framework., Results: Champions exhibited characteristics that facilitated trust and encouraged motivation among their colleagues to adopt innovations, such as being intrinsically motivated, persistent, enthusiastic, and highly effective communicators. Champions were described by their colleagues as empathetic, curious, physically present, approachable, and often soliciting feedback from others. Although there was a high degree of agreement between champion and colleague survey responses, champions were more likely to underrate their skills and abilities to instigate change compared to their colleagues., Conclusion: Both champions and colleagues described key champion-like characteristics, but champions often downplayed the characteristics and behaviors that make champions uniquely effective at facilitating the adoption of evidence-based interventions., Plan Language Abstract: Health care champions are people who promote the adoption of new initiatives to improve the quality of patient care among their colleagues within health care settings. Champions are often viewed by organizational leaders and researchers as critical for the successful implementation of new ideas; however, little is known about what specific skills or characteristics make them effective at promoting the adoption of new ideas among their colleagues. Most studies on champions' behaviors have only included the perspectives of champions, and not perspectives from others within the organization. The goal of our study was to not only explore champions' perspectives of themselves, but also the views of champions' colleagues to understand why and how champions motivated and influenced their colleagues to try new things. Findings from this study could lead to more accurate identification of health care champions, which in turn could lead to more efficient and effective adoption of new initiatives to improve the quality of patient care., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

European Bronze and Iron Age vitrified hillforts have been known since the 1700s, but archaeological interpretations regarding their function and use are still debated. We carried out a series of experiments to constrain conditions that led to the vitrification of the inner wall rocks in the hillfort at Broborg, Sweden. Potential source rocks were collected locally and heat treated in the laboratory, varying maximum temperature, cooling rate, and starting particle size. Crystalline and amorphous phases were quantified using X-ray diffraction both in situ, during heating and cooling, and ex situ, after heating and quenching. Textures, phases, and glass compositions obtained were compared with those for rock samples from the vitrified part of the wall, as well as with equilibrium crystallization calculations. 'Dark glass' and its associated minerals formed from amphibolite or dolerite rocks melted at 1000-1200 °C under reducing atmosphere then slow cooled. 'Clear glass' formed from non-equilibrium partial melting of feldspar in granitoid rocks. This study aids archaeological forensic investigation of vitrified hillforts and interpretation of source rock material by mapping mineralogical changes and glass production under various heating conditions.

Commercial genetic testing laboratories are increasingly employing genetic counselors. As a result, the role of these or many genetic counselors is shifting from primarily direct patient counseling in clinics and hospitals to more laboratory-centered activities that involve case coordination, customer liaison, variant classification, marketing, and sales. Given the importance of these commercial entities to the genetic counseling profession, this commentary examines the current financial situation of four publicly traded, genetic testing companies. It also explores how the various roles of genetic counselors are likely to be affected by the financial pressures these companies face.

The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting., (Copyright © 2015. Published by Elsevier B.V.)

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 μg/g and maximal expression at approximately 400 μg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Duchenne muscular dystrophy (DMD) is a genetic disease affecting about one in every 3,500 boys. This X-linked pathology is due to the absence of dystrophin in muscle fibers. This lack of dystrophin leads to the progressive muscle degeneration that is often responsible for the death of the DMD patients during the third decade of their life. There are currently no curative treatments for this disease but different therapeutic approaches are being studied. Gene therapy consists of introducing a transgene coding for full-length or a truncated version of dystrophin complementary DNA (cDNA) in muscles, whereas pharmaceutical therapy includes the use of chemical/biochemical substances to restore dystrophin expression or alleviate the DMD phenotype. Over the past years, many potential drugs were explored. This led to several clinical trials for gentamicin and ataluren (PTC124) allowing stop codon read-through. An alternative approach is to induce the expression of an internally deleted, partially functional dystrophin protein through exon skipping. The vectors and the methods used in gene therapy have been continually improving in order to obtain greater encapsidation capacity and better transduction efficiency. The most promising experimental approaches using pharmaceutical and gene therapies are reviewed in this article.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Gene therapy of musculoskeletal disorders warrants efficient gene transfer to a wide range of muscle groups. Reengineered adeno-associated viral (AAV) vectors that selectively transduce muscle tissue following systemic administration are attractive candidates for such applications. Here we provide examples of several lab-derived AAV vectors that display systemic tissue tropism in mice. Methods to evaluate the efficiency of gene transfer to skeletal muscle following intravenous or isolated limb infusion of AAV -vectors in mice are discussed in detail.

Positron emission tomography/computed tomography (PET/CT) hybrid imaging can be used to gain insights into a synthetic siRNA delivery system targeted to the liver. Either siRNA or the delivery vehicle was labeled with (64)Cu via 1, 4, 7, 10- tetraazacyclododecane- 1, 4, 7, 10- tetraacetic acid (DOTA) chelation. This study confirmed that the siRNA delivery system was successfully targeted to the liver. Incorporation of the siRNA into the delivery system protected the siRNA from renal filtration long enough so that the siRNA could be delivered to the liver. PET/CT imaging was important for confirming biodistribution and for determining differences in the distribution of labeled siRNA, siRNA incorporated into the delivery system, and the delivery system without siRNA.

The administration route is emerging as a critical aspect of nonviral and viral vector delivery to muscle, so as to enable gene therapy for disorders such as muscular dystrophy. Although direct intramuscular routes were used initially, intravascular routes are garnering interest because of their ability to target multiple muscles at once and to increase the efficiency of delivery and expression. For the delivery of naked plasmid DNA, our group has developed a hydrodynamic, limb vein procedure that entails placing a tourniquet over the proximal part of the target limb to block all blood flow and injecting the gene vector rapidly in a large volume so as to enable the gene vector to be extravasated and to access the myofibers. The present study was conducted in part to optimize the procedure in preparation for a human clinical study. Various injection parameters such as the effect of papaverine preinjection, tourniquet inflation pressure and duration, and rate of injection were evaluated in rats and nonhuman primates. In addition, the safety of the procedure was further established by determining the effect of the procedure on the neuromuscular and vascular systems. The results from these studies provide additional evidence that the procedure is well tolerated and they provide a foundation on which to formulate the procedure for a human clinical study.

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD-positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD(+) muscle fibers in 12-19-month-old mdx mice increased 3-fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra- and interlimb amounts of EBD in 2-7-month-old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse.

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents.

Objective: To evaluate the ability of sapropterin dihydrochloride (pharmaceutical preparation of tetrahydrobiopterin) to increase phenylalanine (Phe) tolerance while maintaining adequate blood Phe control in 4- to 12-year-old children with phenylketonuria (PKU)., Study Design: This international, double-blind, randomized, placebo-controlled study screened for sapropterin response among 90 enrolled subjects in Part 1. In Part 2, 46 responsive subjects with PKU were randomized (3:1) to sapropterin, 20 mg/kg/d, or placebo for 10 weeks while continuing on a Phe-restricted diet. After 3 weeks, a dietary Phe supplement was added every 2 weeks if Phe control was adequate., Results: The mean (+/-SD) Phe supplement tolerated by the sapropterin group had increased significantly from the pretreatment amount (0 mg/kg/d) to 20.9 (+/-15.4) mg/kg/d (P < .001) at the last visit at which subjects had adequate blood Phe control (<360 micromol/L), up to week 10. Over the 10-week period, the placebo group tolerated only an additional 2.9 (+/-4.0) mg/kg/d Phe supplement; the mean difference from the sapropterin group (+/-SE) was 17.7 +/- 4.5 mg/kg/d (P < .001). No severe or serious related adverse events were observed., Conclusions: Sapropterin is effective in increasing Phe tolerance while maintaining blood Phe control and has an acceptable safety profile in this population of children with PKU.

Intravenous hydrodynamic injections into the liver and skeletal muscle have increased the efficacy of naked DNA delivery to a level that makes therapeutically relevant gene transfer attainable. Although there are no concerns about the immunogenicity of the delivered DNA itself, transgene products that are foreign to the host can trigger an immune response and hamper the therapeutic effect. Our goal was to determine whether and to what extent some known preventive measures are applicable to these delivery methods in order to achieve longterm expression of foreign proteins in immunocompetent mice. We designed plasmid DNA vectors that expressed a marker gene under the control of either a ubiquitous or a tissue-specific promoter. We also included microRNA (miR) target sites in the transcripts in order to silence expression in antigen-presenting cells (APCs). The constructs were delivered either into muscle or liver, using outbred ICR and inbred C57BL=6 mice. The data suggest that firefly luciferase, a potent immunogen, triggered a uniform immune response only in outbred ICR mice, and only when expressed from a ubiquitous promoter. This response could not be prevented by including APC-specific miR target sites in the transcript. In contrast, the probability of immune rejection in ICR mice could be significantly diminished by using tissue-specific promoters, and under these circumstances, the silencing of transgene expression in APCs did confer some benefits. After a single hydrodynamic injection, inbred mice did not reject luciferase under any of the tested conditions for at least 8 weeks. To test whether they became tolerized, they were challenged with a second boost of a cytomegalovirus promoter-driven luciferase construct. This triggered a strong immune response, suggesting that luciferase-reactive cells from the animals' T and B cell repertoire had not been eliminated. This secondary reaction could not be prevented by silencing expression in APCs. In conclusion, for the clinical application of hydrodynamic naked DNA delivery the use of tissue-specific promoters in combination with silencing expression in APCs will increase the probability of long-term expression, but the most desirable outcome, the establishment of transgene tolerance, appears unlikely to be achieved by any of these measures.

Background: Phenylketonuria (PKU) requires a lifelong low-phenylalanine diet that provides the majority of protein from a phenylalanine-free amino acid (AA) formula. Glycomacropeptide (GMP), an intact protein formed during cheese production, contains minimal phenylalanine., Objective: The objective was to investigate the effects of substituting GMP food products for the AA formula on acceptability, safety, plasma AA concentrations, and measures of protein utilization in subjects with PKU., Design: Eleven subjects participated in an inpatient metabolic study with two 4-d treatments: a current AA diet (AA diet) followed by a diet that replaced the AA formula with GMP (GMP diet) supplemented with limiting AAs. Plasma concentrations of AAs, blood chemistries, and insulin were measured and compared in AA (day 4) and GMP diets (day 8)., Results: The GMP diet was preferred to the AA diet in 10 of 11 subjects with PKU, and there were no adverse reactions to GMP. There was no significant difference in phenylalanine concentration in postprandial plasma with the GMP diet compared with the AA diet. When comparing fasting with postprandial plasma, plasma phenalyalanine concentration increased significantly with the AA but not with the GMP diet. Blood urea nitrogen was significantly lower, which suggests decreased ureagenesis, and plasma insulin was higher with the GMP diet than with the AA diet., Conclusions: GMP, when supplemented with limiting AAs, is a safe and highly acceptable alternative to synthetic AAs as the primary protein source in the nutritional management of PKU. As an intact protein source, GMP improves protein retention and phenylalanine utilization compared with AAs.

Individuals with phenylketonuria (PKU) must follow a lifelong low-phenylalanine (Phe) diet to prevent neurological impairment. Compliance with the low-Phe diet is often poor owing to restriction in natural foods and the requirement for consumption of a Phe-free amino acid formula or medical food. Glycomacropeptide (GMP), a natural protein produced during cheese-making, is uniquely suited to a low-Phe diet because when isolated from cheese whey it contains minimal Phe (2.5-5 mg Phe/g protein). This paper reviews progress in evaluating the safety, acceptability and efficacy of GMP in the nutritional management of PKU. A variety of foods and beverages can be made with GMP to improve the taste, variety and convenience of the PKU diet. Sensory studies in individuals with PKU demonstrate that GMP foods are acceptable alternatives to amino acid medical foods. Studies in the PKU mouse model demonstrate that GMP supplemented with limiting indispensable amino acids provides a nutritionally adequate source of protein and improves the metabolic phenotype by reducing concentrations of Phe in plasma and brain. A case report in an adult with classical PKU who followed the GMP diet for 10 weeks at home indicates safety, acceptability of GMP food products, a 13-14% reduction in blood Phe levels (p<0.05) and improved distribution of dietary protein throughout the day compared with the amino acid diet. In summary, food products made with GMP that is supplemented with limiting indispensable amino acids provide a palatable alternative source of protein that may improve dietary compliance and metabolic control of PKU.

Expanded newborn screening detects patients with modest elevations in citrulline; however it is currently unclear how to treat these patients and how to counsel their parents. In order to begin to address these issues, we compared the clinical, biochemical, and molecular features of 10 patients with mildly elevated citrulline levels. Three patients presented with clinical illness whereas seven came to attention as a result of expanded newborn screening. One patient presented during pregnancy and responded promptly to IV sodium phenylacetate/sodium benzoate and arginine therapy with no long-term adverse effects on mother or fetus. Two children presented with neurocognitive dysfunction, one of these responded dramatically to dietary protein reduction. ASS enzyme activity was not deficient in all patients with biallelic mutations suggesting this test cannot exclude the ASS1 locus in patients with mildly elevated plasma citrulline. Conversely, all symptomatic patients who were tested had deficient activity. We describe four unreported mutations (p.Y291S, p.R272H, p.F72L, and p.L88I), as well as the common p.W179R mutation. In silico algorithms were inconsistent in predicting the pathogenicity of mutations. The cognitive benefit in one patient of protein restriction and the lack of adverse outcome in seven others restricted from birth, suggest a role for protein restriction and continued monitoring to prevent neurocognitive dysfunction., ((c) 2008 Wiley-Liss, Inc.)

Background: To properly study gene expression in vivo, often long-term expression is desired. Previous studies using plasmid DNA (pDNA) vectors have typically resulted in short-term expression. Here, we evaluated combinations of the albumin promoter with different enhancers and untranslated regions for liver-specific expression in mice., Methods: A series of pDNA secreted alkaline phosphatase (SEAP) reporter gene expression vectors was constructed using the albumin promoter and various other expression cassette elements. Each was evaluated for level and duration of SEAP expression in mice following hydrodynamic tail vein delivery., Results: Sustained liver expression was obtained from vectors combining the albumin promoter with an albumin 3' untranslated region (3'UTR). The level of expression was increased by inclusion of enhancers and a 5' intron. The optimal expression vector consisted of the albumin promoter combined with an alpha-fetoprotein MERII enhancer, 5' intron from the factor IX gene, and the 3'UTR from the albumin gene including intron 14. With this vector, SEAP reporter gene expression levels remained high for 1 year, at levels comparable to those obtained from the cytomegalovirus (CMV) promoter on day 1. Expression of human apolipoprotein E3 (hApoE) in ApoE knockout mice provided a dose-dependent correction of their hypercholesterolemia., Conclusions: Liver-specific sustained transgene expression can be obtained at very high levels from optimized pDNA vectors, without the use of integration systems. Such vectors will further facilitate biological studies of genes in vivo and may find application in gene therapy.

Phenylketonuria (PKU) caused by phenylalanine hydroxylase (PAH) deficiency leads to toxic accumulation of phenylalanine (Phe). PAH is predominantly expressed in liver and its activity requires a supply of tetrahydrobiopterin (BH(4)) cofactor, but we propose that expression of a complete Phe hydroxylating system (PAH plus BH(4) synthetic enzymes) in skeletal muscle will lead to therapeutic reduction of blood Phe levels in Pah(enu2) mice, a model of human PKU. In order to test this hypothesis, we first developed transgenic Pah(enu2) mice that lack liver PAH activity but coexpress, in their skeletal muscle, PAH and guanosine triphosphate cyclohydrolase I (GTPCH). The latter is responsible for the committing enzymatic step in BH(4) biosynthesis. Despite sufficient muscle enzyme expression, these mice remained hyperphenylalaninemic, thereby suggesting that expression of additional BH(4) synthetic enzymes would be necessary. A recombinant triple-cistronic adeno-associated virus-2 (AAV2) pseudotype 1 vector expressing PAH along with GTPCH and 6-pyruvoyltetrahydrobiopterin synthase (PTPS), the next step in BH(4) synthesis, was generated. Injection of this vector into the gastrocnemius muscles of Pah(enu2) mice led to stable and long-term reduction of blood Phe and reversal of PKU-associated coat hypopigmentation. We propose that muscle-directed gene therapy will be a viable alternative treatment approach to PKU and other inborn errors of metabolism.

An acyl-CoA dehydrogenase has been identified as part of the mitochondrial beta-oxidation pathway in the ascomycete fungus Aspergillus nidulans. Disruption of the scdA gene prevented use of butyric acid (C(4)) and hexanoic acid (C(6)) as carbon sources and reduced cellular butyryl-CoA dehydrogenase activity by 7.5-fold. While the mutant strain exhibited wild-type levels of growth on erucic acid (C(22:1)) and oleic acid (C(18:1)), some reduction in growth was observed with myristic acid (C(14)). The DeltascdA mutation was found to be epistatic to a mutation downstream in the beta-oxidation pathway (disruption of enoyl-CoA hydratase). The DeltascdA mutant was also unable to use isoleucine or valine as a carbon source. Transcription of scdA was observed in the presence of either fatty acids or amino acids. When the mutant was grown in medium containing either isoleucine or valine, organic acid analysis of culture supernatants showed accumulation of 2-oxo acid intermediates of branched chain amino acid catabolism, suggesting feedback inhibition of the upstream branched-chain alpha-keto acid dehydrogenase.

The delivery of a variety of nucleic acids such as plasmid DNA (pDNA) and small interfering RNA (siRNA) to mammalian cells is both an important research tool and potential therapeutic approach. Synthetic vehicles (SVs) that include lipoplexes and polyplexes, are widely used for non-viral delivery. A promising method of improving the efficacy of this approach is to create SVs that are chemically dynamic, so that delivery is enabled by the cleavage of chemical bonds upon exposure to various physiological environments or external stimuli. An example of this approach is the use of masked endosomolytic agents (MEAs) that improve the release of nucleic acids from endosomes, a key step during transport. When the MEA enters the acidic environment of the endosome, a pH-labile bond is broken, releasing the agent';s endosomolytic capability. Another challenge has been to develop SVs that enable in vivo delivery. Recently, an MEA that was used within dynamic polyconjugates (DPCs) enabled the efficient delivery of siRNA into hepatocytes in vivo. The use of labile bonds to mask endosomolytic agents, provides a critical design feature, because it enables efficient in vivo delivery without sacrificing endosomolytic function for release into the cytoplasm.

We have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model.

Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.

In vivo phage display is a powerful source of new peptide ligands for specific organ targeting by drugs and gene therapy vectors. Since the introduction of this methodology a decade ago, a number of peptides that preferentially react with organ-specific endothelium and parenchymal markers have been selected. One organ that has been conspicuously missing from these selection studies is the liver, which possesses a multitude of acquired and hereditary disorders and represents a highly important therapeutic target. Herein, we set out to fill this gap by introducing a novel peptide display system containing cloned sequences in the tail fiber protein (p17) of phage T7. The p17 display effectively avoids the innate immune system and is well suited both for selection of new liver-specific ligands and for validation of protein sequences that have been implicated in liver targeting by the use of conventional biochemical methods.

The main barrier to the use of RNAi in mammalian systems is the difficulty in delivering siRNA or shRNA to the appropriate tissues. Although progress has been made in this area, many of the technologies developed require specialized expertise and reagents that are beyond the reach of most investigators. In contrast, the hydrodynamic injection technique is simple to perform and enables highly efficient delivery of naked, unmodified siRNA to a number of tissues, especially the liver. This review describes the development of the technique and explores the possible mechanisms that enable uptake of siRNA to biological effect. Examples of the use of hydrodynamic injection in animal models of disease and for the study of gene function are presented and discussed.

Anemia frequently accompanies chronic diseases such as progressive renal failure, acquired immunodeficiency syndrome, and cancer. Patients are currently treated with erythropoietin (EPO) replacement therapy, using various recombinant human EPO protein formulations. Although this treatment is effective, gene therapy could be more economical and more convenient for the long-term management of the disease. The objective of this study was to develop a naked DNA-based gene therapy protocol that could fill this need. Hydrodynamic limb vein technology has been shown to be an effective and safe procedure for delivering naked plasmid DNA (pDNA) into the skeletal muscles of limbs. Using this method, we addressed the major challenge of an EPO-based gene therapy of anemia: maintaining stable, long-term expression at a level that sufficiently promotes erythropoiesis without leading to polycythemia. The results of our study, using a rat anemia model, provide proof of principle that repeated delivery of small pDNA doses has an additive effect and can gradually lead to the correction of anemia without triggering excessive hematopoiesis. This simple method provides an alternative approach for regulating EPO expression. EPO expression was also proportional to the injected pDNA dose in nonhuman primates. In addition, long-term (more than 450 days) expression was obtained after delivering rhesus EPO cDNA under the transcriptional control of the muscle-specific creatine kinase (MCK) promoter. In conclusion, these data suggest that the repeated delivery of small doses of EPO expressing pDNA into skeletal muscle is a promising, clinically viable approach to alleviate the symptoms of anemia.

The purpose of this work is to investigate the use of magnetic resonance imaging (MRI) to monitor the effects of high-pressure naked plasmid DNA (pDNA) intravascular injections in primate limbs, studying both the distribution of the injected solution in the muscle space, as well as the effects on the vascular system. The distal portion of the four limbs of each of six rhesus monkeys were hydrodynamically injected with naked pDNA, which expressed the luciferase reporter gene. Three-dimensional (3D) T1-weighted gradient echo and 2D multislice T2-weighted fast spin echo (FSE) series were acquired before and immediately after the injection to confirm the volume of solution injected into the limb, and to study the distribution of the injected solution in the individual muscle groups. Time-resolved contrast-enhanced 3D magnetic resonance angiography (MRA) was performed several days before, immediately after, and in a follow-up examination after the pDNA injection to study the effects of the procedure on the primate peripheral vascular system. T1-weighted gradient echo imaging confirmed the delivery of the majority of the solution after successful pDNA injections. T2-weighted FSE imaging demonstrated the distribution of the saline solution in individual muscles in the target limbs, with enhancement showing a weak but significant correlation with the level of gene expression. Time-resolved contrast-enhanced MRA demonstrated effects of the injection procedure on the arterial and venous vascular systems, and the intramuscular compartments; and these effects largely returned to normal on short-term follow-up.

In April 2000, the Wisconsin Newborn Screening Program implemented tandem mass spectrometry (MS/MS) technology to expand the newborn screening panel from 13 to 48 disorders, the majority of which are inborn errors of metabolism. Among other tests, this technology measures the acylcarnitine profile from blood spots collected from infants at 24 to 48 hours of age. During the first 5.75 years of expanded screening, 27 infants were identified with elevated C5-acylcarnitine concentrations, an unexpectedly high number for any inborn error of metabolism. For these infants, elevated C5-acylcarnitines suggested a diagnosis of isovaleric acidemia (IVA), a metabolic defect of leucine metabolism. Subsequent testing showed that the infants did not have isovaleric acidemia, but did have 2-methylbutyryl-CoA dehydrogenase deficiency or 2-MBAD deficiency, a newly described defect of isoleucine metabolism. (An official abbreviation has not been established for this disorder. Other abbreviations include SBCADD, 2-MBG, and 2-MBCD deficiency.) All but 1 of the 27 infants identified with 2-MBAD deficiency are offspring of Hmong parents. To date, those diagnosed with the disorder in the Hmong community have been largely asymptomatic, though further research is needed to determine whether newborns with 2-MBAD deficiency are at risk for neurodevelopmental disorders.

Over the last few years, hydrodynamic tail vein delivery has established itself as a simple, yet very effective method for gene transfer into small rodents. Hydrodynamic delivery of plasmid DNA expression vectors or small interfering RNA allows for a broad range of in vivo experiments, including the testing of regulatory elements, antibody generation, evaluation of gene therapy approaches, basic biology and disease model creation (non-heritable transgenics). The recent development of the hydrodynamic limb vein procedure provides a safe nucleic acid delivery technique with equally high efficiency in small and large research animals and, importantly, the prospects for clinical translation.

This study found that divalent cations induced the further condensation of partially condensed DNA within nonstochiometric polycation complexes. The addition of a few mmol of a divalent cation such as calcium reduced by half the inflection point at which DNA became fully condensed by poly-L-lysine (PLL) and a variety of other polycations. The effect on DNA condensation was initially observed using a new method, which is based on the concentration-dependent self-quenching of fluorescent moieties (e.g., rhodamine) covalently linked to the DNA backbone at relatively high densities. Additional analyses, which employed ultracentrifugation, dynamic light scattering, agarose gel electrophoresis, and atomic force microscopy, confirmed the effect of divalent cations. These results provide an additional accounting of the process by which divalent cations induce greater chromatin compaction that is based on the representation of chromatin fibers as a nonstoichiometric polyelectrolyte complex. They also offer a new approach to assemble nonviral vectors for gene therapy., (Copyright 2006 Wiley Periodicals, Inc.)

Background: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA., Methods: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance., Results: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them., Conclusions: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

A critical step for liver-directed gene therapy is the selective targeting of nucleic acids to hepatocytes. We have previously discovered that the proximal half of the T7 phage tail fiber protein (p17) targeted intact T7 phage and recombinant proteins to hepatocytes in vivo. In the present study, we have localized the targeting activities to a 33 amino acid sequence within the p17 coiled-coil rod domain. Given that the tail fiber domain from which the peptide was derived may form alpha and triple helical structures, biophysical studies (CD spectra and analytical ultracentrifugation) were conducted to determine the secondary and tertiary structures of the peptide. This peptide is able to target proteins, polymers, and siRNA and also particles such as DNA polyplexes and liposomes to hepatocytes. A variety of coupling strategies and chemistries were employed, thus demonstrating that this peptide is a versatile system for delivering cargo. The ability of this hepatocyte-targeting peptide to target DNA-containing particles suggests that it should be useful in the development of both nonviral and viral vectors. However, biological function of delivered cargo has not been demonstrated. This was primarily due to failure of delivered cargo to escape the endosomes. Further studies are in progress to provide functional activity of delivered nucleic acids by enabling their endosomal escape.

Background: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general., Methods: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes., Results: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later., Conclusions: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used to induce antigen-specific antibodies in mice, rats, and rabbits. Both methods allowed the efficient generation of high-titer, antigen-specific antibodies with an overall success rate of Western detectable antibodies of 78% and 92%, respectively. High-titer antibodies were typically present after 3 hydrodynamic tail vein plasmid DNA deliveries, 5 weeks after the initial injection (i.e., prime). For hydrodynamic limb vein plasmid DNA delivery, two deliveries were sufficient to induce high-titer antibody levels. Tail vein delivery was less successful at generating antibodies directed against secreted proteins as compared with limb vein delivery. Material for screening was generated by,transfection of the immunization vector into mammalian cell lines. The cell line (COS-7) that produced the highest level of antigen expression performed best in Western blot analysis screens. In summary, intravenous delivery of antigen-expressing plasmid DNA vectors is an effective genetic immunization method for the induction of antigen-specific antibodies in small and large research animals.

RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara-/- mice. Combining the profiles from mice treated with the PPARalpha agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARalpha regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara-/- mice. In contrast to Ppara-/- mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies.

Cationic membrane disruptive peptides such as melittin would appear to have attributes necessary for DNA delivery: DNA binding via electrostatic interactions and membrane lysis to enable cytoplasmic delivery. However, the relatively small overall charge of membrane disruptive peptides results in weak interactions with DNA. As a model of cationic membrane disruptive peptides, amphiphilic polyvinyl ethers were synthesized. The number of positively charged groups incorporated into these polymers is substantially greater than membrane-active peptides, which enables these polymers to form stable complexes with DNA. By varying the length of the hydrophobic groups incorporated into the polymer from one to four carbons, the dependence of membrane activity on side chain length was established. The ability of these polymers to transfect DNA in tissue culture was tested, and it was found that transfection efficiency is dependent upon the membrane disruptive activity of the polymer. Comparison of melittin and synthetic polymers suggests that transfection and toxicity appear to be dependent upon their affinity for DNA. This demonstration of relationships among membrane lysis, transfection, DNA binding, and polymer side-chain composition establishes a new class of transfection reagents and may guide in the design of polymers and formulations that will enable efficient in vivo transfection.

Extensive use of RNA interference in mammals has been hindered by the inability to effectively deliver small interfering RNAs (siRNAs) or DNA-based constructs designed to express siRNAs. In this chapter, we describe the high-pressure or hydrodynamic intravascular injection technique used to deliver these nucleic acids to mice and nonhuman primates. Emphasis is placed on the use of this technique for delivery to the liver.

The administration of naked nucleic acids into animals is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models (Herweijer and Wolff, 2003; Hodges and Scheule, 2003). It is also being used in several human clinical trials for genetic vaccines, Duchenne muscular dystrophy, peripheral limb ischemia, and cardiac ischemia (Davis et al., 1996; Romero et al., 2002; Tsurumi et al., 1997). Naked DNA is an attractive non-viral vector because of its inherent simplicity and because it can easily be produced in bacteria and manipulated using standard recombinant DNA techniques. It shows very little dissemination and transfection at distant sites following delivery and can be readministered multiple times into mammals (including primates) without inducing an antibody response against itself (i.e., no anti-DNA antibodies generated) (Jiao et al., 1992). Also, contrary to common belief, long-term foreign gene expression from naked plasmid DNA (pDNA) is possible even without chromosome integration if the target cell is postmitotic (as in muscle) or slowly mitotic (as in hepatocytes) and if an immune reaction against the foreign protein is not generated (Herweijer et al., 2001; Miao et al., 2000; Wolff et al., 1992; Zhang et al., 2004). With the advent of intravascular and electroporation techniques, its major restriction--poor expression levels--is no longer limiting and levels of foreign gene expression in vivo are approaching what can be achieved with viral vectors. Direct in vivo gene transfer with naked DNA was first demonstrated when efficient transfection of myofibers was observed following injection of mRNA or pDNA into skeletal muscle (Wolff et al., 1990). It was an unanticipated finding in that the use of naked nucleic acids was the control for experiments designed to assess the ability of cationic lipids to mediate expression in vivo. Subsequent studies also found foreign gene expression after direct injection in other tissues such as heart, thyroid, skin, and liver (Acsadi et al., 1991; Hengge et al., 1996; Kitsis and Leinwand, 1992; Li et al., 1997; Sikes and O'Malley 1994; Yang and Huang, 1996). However, the efficiency of gene transfer into skeletal muscle and these other tissues by direct injection is relatively low and variable, especially in larger animals such as nonhuman primates (Jiao et al., 1992). After our laboratory had developed novel transfection complexes of pDNA and amphipathic compounds and proteins, we sought to deliver them to hepatocytes in vivo via an intravascular route into the portal vein. Our control for these experiments was naked pDNA and we were once again surprised that this control group had the highest expression levels (Budker et al., 1996; Zhang et al., 1997). High levels of expression were achieved by the rapid injection of naked pDNA in relatively large volumes via the portal vein, the hepatic vein, and the bile duct in mice and rats. The procedure also proved effective in larger animals such as dogs and nonhuman primates (Eastman et al., 2002; Zhang et al., 1997). The next major advance was the demonstration that high levels of expression could also be achieved in hepatocytes in mice by the rapid injection of naked DNA in large volumes simply into the tail vein (Liu et al., 1999; Zhang et al., 1999). This hydrodynamic tail vein (HTV) procedure is proving to be a very useful research tool not only for gene expression studies, but also more recently for the delivery of small interfering RNA (siRNA) (Lewis et al., 2002; McCaffrey et al., 2002). The intravascular delivery of naked pDNA to muscle cells is also attractive particularly since many muscle groups would have to be targeted for intrinsic muscle disorders such as Duchenne muscular dystrophy. High levels of gene expression were first achieved by the rapid injection of naked DNA in large volumes via an artery route with both blood inflow and outflow blocked surgically (Budker et al., 1998; Zhang et al., 2001). Intravenous routes have also been shown to be effective (Hagstrom et al., 2004; Liang et al., 2004; Liu et al., 2001). For limb muscles, the ability to use a peripheral limb vein for injection and a proximal, external tourniquet to block blood flow renders the procedure to be clinically viable. This review concerns itself with the mechanism by which naked DNA is taken up by cells in vivo. A greater understanding of the mechanisms involved in the uptake and expression of naked DNA, and thus connections between postulated mechanisms and expression levels, is emphasized. Inquiries into the mechanism not only aid these practical efforts, but are also interesting on their own account with relevance to viral transduction and cellular processes. The delivery to hepatocytes is first discussed given the greater information available for this process, and then uptake by myofibers is discussed.

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response., (Copryright Mary Ann Liebert, Inc.)

Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications., (Copyright The American Society of Gene Therapy)