Incurable Genetic Disease Research Articles

ATM splicing variants as biomarkers for low dose dexamethasone treatment of A-T

BackgroundAtaxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1.ResultsIn the present study, the new ATMdexa1 transcript was also identified in vivo, specifically in the PMBCs of AT patients treated with intra-erythrocyte Dexamethasone (EryDex). In these patients it was also possible to isolate new “ATMdexa1 variants” originating from canonical and non-canonical splicing, each containing the coding sequence for the ATM kinase domain. The expression of the ATMdexa1 transcript family was directly related to treatment and higher expression levels of the transcript in patients’ blood correlated with a positive response to Dexamethasone therapy. Neither untreated AT patients nor untreated healthy volunteers possessed detectable levels of the transcripts. ATMdexa1 transcript expression was found to be elevated 8 days after the drug infusion, while it decreased 21 days after treatment.ConclusionsFor the first time, the expression of ATM splicing variants, similar to those previously observed in vitro, has been found in the PBMCs of patients treated with EryDex. These findings show a correlation between the expression of ATMdexa1 transcripts and the clinical response to low dose dexamethasone administration.

Inflammatory predisposition predicts disease phenotypes in muscular dystrophy.

Duchenne muscular dystrophy is an incurable genetic disease that presents with skeletal muscle weakness and chronic inflammation and is associated with early mortality. Indeed, immune cell infiltration into the skeletal muscle is a notable feature of the disease pathophysiology and is strongly associated with disease severity. Interleukin (IL)-10 regulates inflammatory immune responses by reducing both M1 macrophage activation and the production of pro-inflammatory cytokines, thereby promoting the activation of the M2 macrophage phenotype. We previously reported that genetic ablation of IL-10 in dystrophic mice resulted in more severe phenotypes, in regard to heart and respiratory function, as evidenced by increased macrophage infiltration, high levels of inflammatory factors in the muscle, and progressive cardiorespiratory dysfunction. These data therefore indicate that IL-10 comprises an essential immune-modulator within dystrophic muscles. In this review, we highlight the pivotal role of the immune system in the pathogenesis of muscular dystrophy and discuss how an increased understanding of the pathogenesis of this disease may lead to novel therapeutic strategies.

347. CRISPR/Cas9-Based Gene Correction of Arginase-Deficient Human Induced Pluripotent Stem Cells to Recover Enzyme Function

Urea cycle disorders (UCDs) are incurable genetic diseases that affect the body's ability to produce urea, leading to hyperammonemia due to a deficiency in any one of six enzymes in the cycle. For arginase deficiency, a mutation in the ARG1 gene, the final step of the cycle, results in hyperargininemia, developmental delays and disabilities, seizures, psychomotor function loss, and in serious cases, death. There is currently no completely effective treatment available. Advances in human induced pluripotent stem cell (hiPSC) research and genome-editing technologies have enabled the genetic modification of stem cells for potential cellular replacement therapies. In this study, we applied such technology to develop a stem cell-based approach for treating arginase deficiency applicable to all arginase deficient patients regardless of their mutation. Methods: Fibroblasts from three patients with arginase deficiency were obtained, defined for their mutation, and reprogrammed into hiPSCs. Selectable, full-length codon optimized human arginase cDNA (coARG) expression cassettes were then developed for site-specific integration into either the HPRT or albumin (ALB) locus. After confirming specificity by Sanger sequencing, genetically corrected hiPSCs were differentiated to hepatocyte-like cells. Results: Fibroblasts were reprogrammed into hiPSCs by applying a STEMCCA lentivirus-based method and were characterized for pluripotency by immunophenotyping for common stemness markers via ICC, alkaline phosphatase staining, and in vivo teratoma formation. Using a site-specific CRISPR/Cas9 nickase-mediated gene transferring system, we inserted coARG by two approaches. First, coARG was inserted into the HPRT locus under the control of constitutive hEF1α promoter (LEAPR); targeting HPRT allowed for positive clonal selection of successful on-target integration by 6-thioguanine treatment. Second, we inserted coARG into the ALB locus for expression under the endogenous ALB promoter (ALB-coARG) as ALB is highly expressed in human liver. After LEAPR and ALB-coARG modification and sequence confirmation, hiPSCs were differentiated to hepatocyte-like cells and characterized by immunophenotyping via ICC and RNA expression via RT-PCR for common hepatic markers; cells demonstrated more fetal-like characteristics. Moreover, LEAPR- and ALB-coARG-modified hepatocyte-like cells demonstrated 41% and 1% functional arginase activity recovery compared to human fetal liver, respectively. Discussion: In this study, we demonstrated the ability to genetically correct mutated ARG1 gene expression in hiPSCs derived from patients with hyperargininemia and restored arginase function in hiPSCs and hepatocyte derivatives by CRISPR/Cas9-based gene addition. As the LEAPR construct demonstrated marked coARG expression, recovery of arginase activity of ALB-ARG-modified hepatocyte-like cells was low; we expect significant arginase recovery after in vivo maturation of transplanted cells as ALB expression increases with hepatocyte maturation. Also, to demonstrate potential in vivo recovery of arginase deficiency pathogenesis, ongoing studies aim to transplant both cohorts of gene-corrected hepatocyte-like cells into an established arginase deficient immunosuppressed mouse model. Successful restoration of enzyme function in patient-specific hiPSCs and relevant cellular derivatives will highlight hiPSCs as a valuable tool in cell replacement therapies and advance applications of genetically modified hiPSCs to treat UCDs and other single enzyme liver deficiencies.

238. Cell Therapeutic Approach Using Dental Pulp Stromal Cells for Duchenne Muscular Dystrophy

Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality that exhibits skeletal muscle weakness with chronic inflammation. Dental pulp stromal cells (DPCs) could be potential therapeutics because of their immunosuppressive properties and multipotency. In the present study, we examined the strategies for effective cell transplantation to develop a novel approach for functional recovery of the skeletal as well as cardiac muscles using a dog model of Duchenne muscular dystrophy. Methods: DPCs were intravenously injected into two littermates of canine X-linked muscular dystrophy in Japan (CXMDJ) at weekly interval for 8 weeks without immunosuppression. Clinical phenotypes in the transplanted dogs were analyzed by using blood exams, physical capacity, magnetic resonance imaging (MRI) analysis compared with non-injected littermates as controls. Results: The downregulation of inflammation in the lower legs of DPCs-treated CXMDJ was confirmed by MRI analysis. Serum levels of pro-inflammatory cytokine IL-6 were well regulated in the treated CXMDJ during experiments, unlike non-injected dogs. Although CXMDJ showed progressive muscle atrophy in the all four limbs, exercise intolerance and abnormal locomotion, we observed improved phenotypes in the DPCs-treated CXMDJ along with the improved pace of walking and running. Conclusion: We suggested that the systemic injection of DPCs ameliorated the progressive phenotype in CXMDJ. The therapeutic effects might be associated with the production of paracrine or endocrine factors that regulate inflammation, and might also stimulate the proliferation of endogenous stem cells at the injured muscle tissue. This strategy of DPCs treatment would be promising for the future DMD cell therapy.

Enforced Exercise Induces Rapid Exhaustion in C. elegans Dystrophin Mutants

Duchene Muscular Dystrophy (DMD) is an incurable genetic disease and those who suffer from it typically die in their second decade. The disease is currently incurable. The objective of this study was to facilitate cost effective high throughput drug screening for DMD. In order to achieve that we developed a worm treadmill platform. Using electrotaxis, the worm treadmill reveals a unique phenotype for dys‐1 (dystrophin) mutants comparable to DMD mouse model. Briefly, the nematode, C. elegans, displays a specific locomotion response to various stimuli including food, chemicals, and electricity. C. elegans’ response to electricity has been previously well described and dubbed electrotaxis ‐ the repeatable behavior of crawling toward the negative pole when subjected to an electric field. For the first time, we used this behavior to initiate and maintain locomotion in order to perform exhaustion tests.Materials and methodsThe “worm treadmill” consists of a specially formulated gel and buffer placed in a classic DNA‐electrophoresis box, a power source, a microscope camera, and an infrared thermometer to monitor gel temperature. 50–150 hypochlorite synchronized worms were transferred to the gel, and a voltage of ≥4 v/cm was applied. The poles of the electric field were alternated as the nematodes reached the end of the gel. The distance and disposition of crawling C. elegans was continuously tracked. The goal was to run the worms to a state of exhaustion – defined as a state when at least 75% of the worms ceased locomotion toward the anode. In order to assess the effects of therapeutics on exercised worms, hypochlorite synchronized eggs were placed on plates with 0.5mg/mL Prednisone, 0.05mg/mL Fluoxetine, or a combination of the two.ResultsWild type worms ran significantly longer and farther than dys‐1 mutants. In addition, we can report that dys‐1 mutants treated with prednisone, fluoxetine, and a combination of both drugs ran statistically significantly longer and farther than untreated dys‐1 mutants. The greatest improvement was observed in the dys‐1 mutants exposed to prednisone and fluoxetine; these worms ran significantly longer than all other groups, as well as significantly farther than untreated dys‐1 mutants or dys‐1 mutants exposed to prednisone or fluoxetine alone.ConclusionOur platform provides a unique phenotype for dys‐1 mutants comparable to DMD mouse model that was previously lacking. It offers a cost effective and simple way for initial screening of novel pharmacological compounds in order to find a cure for this currently incurable disease. In addition, as expected, based on animal models and previously established prednisone effects in other C. elegans mutants, Prednisone has shown therapeutic effects. However, this was the first time this has been shown in forcefully exercised dys‐1 (dystrophin) mutants. The greatest improvement observed with a single drug treatment was with fluoxetine, indicating serotonergic transmission may represent a novel therapeutic target for DMD. The greatest overall improvement was observed with simultaneous fluoxetine and prednisone treatment. This finding provides initial data suggesting that synergistic drug effects should be strongly considered in future screenings for potential DMD therapeutics.Support or Funding InformationThis work was funded by WVSOM intramural grant

Activating frataxin expression by repeat-targeted nucleic acids.

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

402. Mesenchymal Stromal Cells Can Ameliorate the Progressive Phenotype of Dog With Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality that exhibits skeletal muscle weakness with chronic inflammation. Multipotent mesenchymal stromal cells (MSCs) could be a potential therapeutics because of their immunosuppressive properties and ability of differentiation to myogenic cells in situ. In the present study, we examined the strategies for effective cell transplantation to develop a novel approach for functional recovery of the skeletal muscles using a dog model of Duchenne muscular dystrophy.

Epidemiology of inherited epidermolysis bullosa in Romania and genotype-phenotype correlations in patients with dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a rare and so far incurable genetic disease, affecting mainly the skin and mucosal membranes, manifesting with blisters triggered by minor mechanical trauma. Since only few epidemiological data on EB are available, we established a Registry for EB and implemented molecular diagnostic methods. We present epidemiologic data from the EB Registry and genotype-phenotype correlations. In 2006, a registry of patients with EB was initiated in the Department of Dermatology of the University of Medicine, as well as molecular diagnostic tools. The patients were diagnosed on clinical bases, and whenever possible, immunofluorescence mapping and molecular analysis were performed. 89 EB patients were enrolled in the study from 2006 to 2012: 58 patients with dystrophic EB (DEB), 20 with EB simplex, one patient was diagnosed with Kindler syndrome; in 10 patients, the type of EB could not be determined. We have estimated, the total number of EB patients in Romania and we have estimated the incidence and the prevalence of EB. We have also managed to approximate the distribution of EB types in Romania. Moreover, we performed a phenotypic and genotypic characterization in some of the patients included in the EB register.

Dietary n-3 polyunsaturated fatty acids or soy protein isolate did not attenuate disease progression in a female rat model of autosomal recessive polycystic kidney disease

Polycystic kidney disease (PKD) is an incurable genetic disorder that is characterized by multiple benign cysts. As PKD advances, cyst growth increases kidney volume, decreases renal function, and may lead to end-stage renal disease; however, in a PKD rat model, feeding soy protein isolate (SPI) reduced cyst proliferation and growth. The n-3 polyunsaturated fatty acids (PUFAs) are noted for their anti-inflammatory actions. Therefore, diet therapy could offer a potentially efficacious, safe, and cost-effective strategy for treating PKD. The objective of this study was to investigate the role of soy protein and/or n-3 PUFAs on PKD progression and severity in the rat model of autosomal recessive PKD. We hypothesized that the antiproliferative and anti-inflammatory actions associated with soy protein and n-3 PUFA supplementation will attenuate PKD progression in female PCK rats. For 12weeks, young (age, 28 days) female PCK rats were randomly assigned (n=12/group) to 4 different diets: casein±corn oil, casein±soybean oil, SPI±soybean oil, or SPI±1:1 soybean/salmon oil (SPI±SB). The feeding of the different protein and lipid sources had no significant effect on relative kidney weight. Histologic evaluation showed no significant differences in cortical or medullary cyst size, interstitial inflammation, and fibrosis among diet groups. However, rats fed SPI±SB diet had cortical cyst obstruction and the highest (P<.01) serum blood urea nitrogen concentration. Rats fed SPI±SB diet had the highest (P<.001) renal docosahexaeonic acid, but there were no significant differences in renal tissue inflammation and proliferation gene expression among the diet groups. Based on these results, dietary soy protein and/or n-3 PUFAs did not attenuate disease progression or severity in the female PCK rat model of autosomal recessive PKD.

Epigenetic Mechanisms of Rubinstein–Taybi Syndrome

Rubinstein-Taybi syndrome (RTS) is an incurable genetic disorder with combination of mental retardation and physical features including broad thumbs and toes, craniofacial abnormalities, and growth deficiency. While the autosomal dominant mode of transmission is limitedly known, the majority of cases are attributable to de novo mutations in RTS. The first identified gene associated with RTS is CREB-binding protein (CREBBP/CBP). Alterations of the epigenetic 'histone code' due to dysfunction of the CBP histone acetyltransferase activity deregulate gene transcriptions that are prominently linked to RTS pathogenesis. In this review, we discuss how CBP mutation contributes to modifications of histone and how histone deacetylase inhibitors are therapeutically applicable to epigenetic conditioning in RTS. Since most genetic mutations are irreversible and therapeutic approaches are limited, therapeutic targeting of reversible epigenetic components altered in RTS may be an ideal strategy. Expeditious further study on the role of the epigenetic mechanisms in RTS is encouraged to identify novel epigenetic markers and therapeutic targets to treat RTS.

Human Genome Sequencing Project and the implications for international primary healthcare programme in Sub-Sahara African nations

This study has critically examined the impact of the Human Genome Sequencing Project on the WHO-championed primary healthcare programme. The investigators outlined how genomic-based research studies have provided relevant cues about curing many of these hitherto incurable genetic diseases. The need for African inter-regional bodies to set aside between 3%–5% of their annual budget to provide funding for the establishment of SSA-African genomic centres of excellence was emphasised. Innovative public health genomics can play a monumental role not only in eliminating most of the disease burden in the region but also enhance the inadequate, current life expectancy in Sub-Sahara-Africa. Strategies for establishing and sustaining the development of public health genomics and innovative genomic medicine were outlined. Finally, the ethical legal and social implication of genomics research in the context of socio-economic development was presented.

Decrease of α-Chains in β-Thalassemia

In the pathophysiology of β-thalassemia, globin chain imbalance plays a central role in predicting red blood cell (RBC) life span and disease severity. Strategies to improve globin chain imbalance are therefore a legitimate target in the management of this incurable genetic disorder. Classical gene addition with the available retroviral vectors can alter one of the two variables while combined reduction of achains could provide a more potent therapeutic effect. We developed foamy virus (FV) vectors for the production of β-globin and vectors targeting the a-globin transcript using the shRNA technology. Using FVderived vectors, we expressed human anti-a-globin short hairpin RNAs, off a potent PolIII promoter (H1); of the 4 different shRNAs tested, α-globin mRNA reduction varied from 6.3 to 54% of the control CD34+ cells. Similarly, vectors developed for the mouse α-globin, resulted in a significant reduction (range 15-28% of the control) of aglobin in erythroid colonies of Lin- cells. To assay vector performance in vivo, we investigated the hematological parameters in thal3+/- mice transpalnted with FV-transduced Lin- cells, transduced with anti-alpha-globin shRNA. Despite low chimerism and low vector copy numbers (&lt;0.5 per cell), we observed a 10% reduction in red cell distribution width, a marker for distorted erythropoiesis. We finally developed a combination FV vector expressing β-globin off a HS40 enhancer and anti-α-globin shRNA and tested its performance in human CD34+ cells from a thalassemic patient. Globin chain imbalance was ameliorated from a β/α ratio of 0.12 to the level of 0.5, clearly indicating a therapeutic benefit. Overall, shRNA control of α-globin excess is a feasible target but requires improvements since the RNAi effect is tough to predict and should ideally be combined with controllable elements.

Fabry Disease

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by accumulation of Gb-3 (globotriaosylceramide) in cellular lysosomes of tissues throughout the body. With advancing age, lysosomal Gb-3 accumulates in blood vessel walls, nerve cells, smooth muscle, and vital organs. Premature death commonly results from renal failure, heart attack, and stroke when the diagnosis is delayed or overlooked. One of the earliest and most distinctive physical features of FD is a whorl-like keratopathy. This finding is easily identifiable during a routine eye examination with a slit lamp, making eye care practitioners uniquely postured to identify patients and families with this incurable genetic disorder. Much of the pain, suffering, and adverse impact of FD can be avoided if an alert eye care expert sees the patient at an early age, identifies the condition, and makes the appropriate referral. The importance of obtaining a thorough medical history, ancestral health history, and review of systems to correlate ocular and systemic manifestations is emphasized. This report reviews the multisystem involvement of FD and describes the clinical characteristics and expected chronological appearance of ophthalmic and systemic manifestations. The discoveries of late-onset variants, increased prevalence, and modified inheritance pattern of FD are discussed. The profound therapeutic effects of recombinant enzyme replacement therapy (ERT) on multiple organ systems are detailed and demonstrated in a Fabry proband. Improved quality and quantity of life after initiation of ERT underscore the importance of early recognition and correlation of FD symptoms and clinical signs. Treatment strategies and the effectiveness of new adjunctive chaperone therapy are addressed.

Atomic Force Microscopy reveals Diversity of Serpin Polymers

Polymerization of a mutated form of antitrypsin is postulated to cause serpinopathy, a rare and incurable genetic disease. Antitrypsin is the canonical member of the serpin superfamily of structurally homologous proteins with metastable native structures. Most of serpins, including antitrypsin, are regulators of serine and cysteine protease cascades, and protect tissues from excessive actions of extracellular proteolytic enzymes. Polymerization of the mutant Z variant of antitrypsin results in the “loss of function” proteolytic damage of lungs, or in the “gain of function” degeneration of liver cells, loaded with toxic polymers. The understanding of polymerization mechanism holds the key to disease prevention, diagnostics and cures. However, metastability of serpins and complexity of their polymers makes structural studies a difficult task. There are several models of polymerization, based on in vitro studies and molecular modeling, however their relevance to in vivo polymerization and disease etiology has not been established. Here we employed atomic force microscopy (AFM) to compare topography of in vitro and in vivo formed antitrypsin polymers and oligomers. We found that commonly used in vitro methods to induce polymerization result in formation of topographically a very diverse population of oligomers. Most, if not all classes of the in vitro formed oligomers corresponded to similar classes of oligomers isolated from liver. The in vitro polymerization, however did not produce long strands of polymerized antitrypsin. The presence of such strands is the most striking feature in liver cells of human serpinopathy patients and in a mouse model. Here, we report results of our scanning probe microscopy structural analysis of long serpin polymers and propose the mechanism of their formation.

End-stage Posttraumatic Osteoarthritis Treated With THA in Osteogenesis Imperfecta

Osteogenesis imperfecta is an incurable genetic disorder manifested with altered bone quality that predisposes patients to a multitude of fractures throughout their lives, including acetabular fractures. The management of acetabular fractures in patients with osteogenesis imperfecta remains a challenging clinical problem, with a paucity of literature supporting treatments and their outcomes. Limited reports in the literature validate the use of total hip arthroplasty (THA) in patients with osteogenesis imperfecta, and they describe the adult population only.This article describes a case of delayed diagnosis of a transverse acetabular fracture and femoral head impaction fracture that led to posttraumatic end-stage hip osteoarthritis in a 16-year-old boy with osteogenesis imperfecta (Sillence Type I) that was sustained after minimal trauma. Clinical examination 3 months postinjury revealed a significant pelvic obliquity, severe pain with hip range of motion, and limited hip range of motion. Imaging studies revealed a complete loss of articular cartilage and significant joint effusion.The patient underwent THA. No postoperative complications occurred. Two-year follow-up showed an excellent clinical result. The patient's hip was pain free, and he was able to walk with no limp.The authors are unaware of any reported cases of children with osteogenesis imperfecta undergoing THA. Based on the reported literature and the authors' experience, THA can be a reliable surgical option for patients with osteogenesis imperfecta.

Bioreducible Crosslinked Polyelectrolyte Complexes for MMP-2 siRNA Delivery into Human Vascular Smooth Muscle Cells

Bioreducible crosslinked polyplexes were prepared via disulfide bond formation after siRNA condensation with polyethylenimine-modified by deoxycholic acid (PEI-DA) to stabilize polyplex structure in an extracellular environment and to promote transfection efficiency in human smooth muscle cells (hSMCs). The PEI-DA/siRNA polyplexes were further modified by crosslinking the primary amines of PEI with thiol-cleavable crosslinkers. The effect of disulfide crosslinked PEI-DA/siRNA (Cr PEI-DA/siRNA) polyplexes on target gene silencing was investigated by transfecting hSMCs with matrix metalloproteinase-2 (MMP-2) siRNA under serum conditions. The MMP-2 levels in the conditioned medium were examined using gelatin zymography. The Cr PEI-DA/siRNA polyplexes showed increased stability against heparin exchange reactions, while their disulfide linkages were successfully cleaved under reducing conditions. The polyplex crosslinking reaction led to a slight decrease in MMP-2 gene silencing activity in hSMCs due to the insufficient redox potential. However, the gene silencing efficiency of the Cr PEI-DA/siRNA polypexes was gradually improved in response to increasing intracellular reduction potential. The increased serum stability of the Cr PEI-DA/siRNA polyplexes resulted in significant enhancement of the intracellular delivery efficiency especially under serum conditions. The Cr PEI-DA/siRNA polyplex formulation may be a promising siRNA delivery system for the treatment of incurable genetic disorders.

Long-term Engraftment of Multipotent Mesenchymal Stromal Cells That Differentiate to Form Myogenic Cells in Dogs With Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.

Le diagnostic pré-implantatoire couplé au typage HLA : l’expérience française

Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclère Hospital after embryo selection at the Paris preimplantation genetic diagnosis (PGD) center. His birth revived the controversy over "double PGD". This procedure, authorized in France since 2006, allows couples who already have a child with a serious, incurable genetic disease, to opt for PGD in order to select a healthy embryo that is HLA-matched to the affected sibling and who may thus serve as an ombilical cord blood donor. The procedure is particularly complex and the baby take-home rate is still very low. Double PGD is strictly regulated in France, and candidate couples must first receive individual authorization from the Biomedicine Agency. In our experience, these couples have a strong desire to have children, as reflected by the large number of prior spontaneous pregnancies (25% of couples). Likewise, most of these couples request embryo transfer even when there is no HLA-matched embryo, which accounts for more than half of embryo transfers. The controversy surrounding this practice has flared up again in recent weeks, over the concepts of "designer babies" and "double savior siblings" (the baby is selected to be free of the hereditary disease, and may also serve as a stem cell donor for the affected sibling).

Self-Assembled and Nanostructured siRNA Delivery Systems

A wide range of organic and inorganic materials have been used in the development of nano-scale self-assembling gene delivery systems to improve the therapeutic efficacy of nucleic acid drugs. Small interfering RNA (siRNA) has recently been recognized as a promising and potent nucleic acid medicine for the treatment of incurable genetic disorders including cancer; however, siRNA-based therapeutics suffer from the same delivery problems as conventional nucleic acid drugs such as plasmid DNA and antisense oligonucleotides. Many of the delivery strategies developed for nucleic acid drugs have been applied to siRNA therapeutics, but they have not produced satisfactory in vivo gene silencing efficiencies to warrant clinical trials. This review discusses recent progress in the development of self-assembled and nanostructured delivery systems for efficient siRNA-induced gene silencing and their potential application in clinical settings.

ERK Involvement in Resistance to Apoptosis in Keratinocytes with Mutant Keratin

The consequences of cell stress induced by misfolded proteins are an important contributor to many human diseases. One such disease is epidermolysis bullosa simplex (EBS), caused by mutations in the structural proteins (keratins K5 or K14) of the proliferative compartment of the epidermis (basal keratinocyte layer), leading to cell fragility and blistering. In severe EBS, the mutation is associated with aggregates of nonfilamentous keratin protein, and cell lines carrying such mutations show a constitutively activated stress response. Analysis of the cellular mechanisms leading to cell breakdown on physical stress may point the way to mutation-independent therapeutic approaches to these incurable genetic disorders. We therefore subjected EBS cell lines, immortalized from patients with EBS, to an oscillating mechanical stress in assays designed to mimic the physical trauma that leads to cell breakdown in vivo. These experiments show that mechanical stress activates extracellular signal-regulated kinase (ERK) signaling in these cells, and that the keratin mutant cells also show a resistance to apoptosis following mechanical stress that is reversed by inhibiting ERK. The consequences of constitutive expression of large amounts of defective structural protein in a tissue cell must be properly understood for the development of safe and effective therapies for these disorders.