Development and Utility of an Internal Threshold Control (ITC) Real-Time PCR Assay for Exogenous DNA Detection

Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA

Gait characterization in golden retriever muscular dystrophy dogs using linear discriminant analysis

Background: Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs isof limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler andmore sensitive evaluation of treatment benefits in this important preclinical model.Methods: Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulationof the dependent and independent variables produced three distinct models. The ability of each model to detect gaitalterations and their pattern change with age was tested using a leave-one-out cross-validation approach.Results: Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing agood discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficientlyrepresentative of the disease progression. In Model 2, age in months was added as a supplementary dependentvariable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%).To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable.This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertainingto 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actualgenotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects ofimmunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect ofimmunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages ofdirect statistical analysis with greater sensitivity and more comprehensible data representation.Conclusions: Gait analysis using LDA allows for improved analysis of accelerometry data by applying adecision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs

Cell therapy of Duchenne muscular dystrophy: preclinical trial in GRMD dogs

Duchenne muscular dystrophy (DMD), a genetic progressive X-linked muscular dystrophy, is the most common genetic disease in humans. Cell therapy based on the use of somatic stem cells is a very promising approach. In a dog myopathy model, we isolated a muscle stem cell (MuStem) with the essential requirements for therapeutic use: high amplification capacity, ability to fuse with muscle fibers, renewal of the satellite cell population, dispersion in the whole body after vascular administration, persistence of long-term effect, and dramatic clinical improvement of treated animals. These preclinical results pave the way for a therapeutic trial in children with Duchenne muscular dystrophy.La dystrophie musculaire de Duchenne (DMD) est une maladie génétique progressive du muscle liée au chromosome X. Elle est la maladie génétique la plus fréquente chez l'homme. La thérapie cellulaire basée sur l'utilisation de cellules souches somatiques est une voie thérapeutique riche d'intérêt. Nous avons isolé, chez un modèle de chien myopathe, une cellule souche musculaire (MuStem) qui présente les qualités indispensables à une utilisation thérapeutique: forte capacité d'amplification, capacité à fusionner avec les fibres musculaires, renouvellement du contingent de cellules satellites, dispersion dans l'organisme après administration vasculaire, persistance de l'effet à long terme, spectaculaire amélioration clinique des animaux traités. Ces résultats précliniques ouvrent la voie à un essai thérapeutique chez l'enfant atteint de dystrophie musculaire de Duchenne

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and β1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD

Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model

Étude de la régulation de l'épissage des exons alternatifs K-SAM et BEK du gène codant pour le FGFR-2 (affinement du rôle nucléaire et du mode de régulation des protéines TIA-1 et TIAR)

L'épissage alternatif est un mécanisme fondamental de régulation de l'expression des gènes. Ce mécanisme permet d'obtenir, à partir d'un même ARN pré-messager, plusieurs ARN messagers matures différents, et il participe ainsi activement à la diversification du protéome. Dans cet épissage, des sites d'épissage sont utilisés ou non, en fonction du stade de développement, du type cellulaire ou de l'état physiologique des cellules. L'épissage alternatif est impliqué dans un grand nombre de maladies humaines, et il est donc essentiel de mieux connaître les facteurs intervenant dans la régulation de ce phénomène...NANTES-BU Sciences (441092104) / SudocSudocFranceF

Immune Responses to Gene Product of Inducible Promoters

Efficient gene transfer has been achieved in several animal models using different vector systems, leading to stable transgene expression. The tight control of this expression is now an important outcome for the field of gene therapy. Such regulation is likely to be required for therapeutic applications and in some instances for safety reasons. For this purpose, several regulatable systems depending on small molecules have been developed. Among these, the tetracycline and the rapamycin dependent systems have been largely used. However, if long-term regulation of the transgene has been obtained in small animal models using these inducible systems, when translational studies were initiated in larger animals, the development of an immune response against proteins involved in transgene regulation were often observed. Such immune response was especially documented when using the TetOn tetracycline regulatable system in nonhuman primates (NHP). Humoral and destructive cellular immune responses against the transactivator involved in this regulation system were documented in a large majority of NHP leading to the complete loss of the transgene regulation and expression. This review will describe the immune responses observed in these different model systems applied for transgene regulation. Focus will be finally given on future directions in which such immune responses might be surmounted, enabling long-term transgene regulation in future clinical developments of gene transfer

Individual assay sensitivity.

*<p>From fifteen replicates of plasmid (pDNA) or ITC template. Standard error, s.e., (in parenthesis).</p