A Commentary on Commercial Genetic Testing and the Future of the Genetic Counseling Profession.

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.

Protease-triggered siRNA delivery vehicles.

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.

Current status of pharmaceutical and genetic therapeutic approaches to treat DMD.

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.

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA.

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.

Systemic gene transfer to skeletal muscle using reengineered AAV vectors.

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.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

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.

Hybrid PET/CT for noninvasive pharmacokinetic evaluation of dynamic PolyConjugates, a synthetic siRNA delivery system.

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.

Evaluation of hydrodynamic limb vein injections in nonhuman primates.

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.

Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.

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.

Functional efficacy of dystrophin expression from plasmids delivered to mdx mice by hydrodynamic limb vein injection.

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.

Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP).

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.

Efficacy of sapropterin dihydrochloride in increasing phenylalanine tolerance in children with phenylketonuria: a phase III, randomized, double-blind, placebo-controlled study.

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.

Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids.

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.

Effect of tissue-specific promoters and microRNA recognition elements on stability of transgene expression after hydrodynamic naked plasmid DNA delivery.

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.

siRNA and DNA transfer to cultured cells.

Transfection is a powerful non-viral technology used to deliver foreign nucleic acids into eukaryotic cells, and is the method of choice for a variety of applications including studying the functional role of particular genes and the proteins they code for. By over-expressing genes to produce protein of interest and also by knocking down specific genes, researchers are able to accurately define the role of genes and the protein they encode in various cellular processes. Therefore, this powerful technology is a very vital component of the array of scientific research tools.

Safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients with phenylketonuria.

Phenylketonuria (PKU) is an inherited metabolic disease characterized by phenylalanine (Phe) accumulation, which can lead to neurocognitive and neuromotor impairment. Sapropterin dihydrochloride, an FDA-approved synthetic formulation of tetrahydrobiopterin (6R-BH4, herein referred to as sapropterin) is effective in reducing plasma Phe concentrations in patients with hyperphenylalaninemia due to tetrahydrobiopterin (BH4)-responsive PKU, offering potential for improved metabolic control. Eighty patients, > or =8 years old, who had participated in a 6-week, randomized, placebo-controlled study of sapropterin, were enrolled in this 22-week, multicenter, open-label extension study comprising a 6-week forced dose-titration phase (5, 20, and 10 mg/kg/day of study drug consecutively for 2 weeks each), a 4-week dose-analysis phase (10 mg/kg/day), and a 12-week fixed-dose phase (patients received doses of 5, 10, or 20 mg/kg/day based on their plasma Phe concentrations during the dose titration).

The role of molecular testing and enzyme analysis in the management of hypomorphic citrullinemia.

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.

Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system.

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).

Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery.

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.

Breaking the bonds: non-viral vectors become chemically dynamic.

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.

Rapidly reversible hydrophobization: an approach to high first-pass drug extraction.

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.