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

The RPGRIP1-deficient dog, a promising canine model for gene therapy

PURPOSE: To evaluate the RPGRIP1-deficient miniature longhaired dachshund (MLHD) dog as a potential candidate for gene therapy. METHODS: Six RPGRIP1-deficient MLHD dogs from our dog colony have been observed for two years using a variety of noninvasive procedures. These included bilateral full-field electroretinograms (ERG) to evaluate retinal function, fundus photographs to evaluate retinal vascularization, and optical coherence tomographs (OCT) to evaluate retinal thickness. We also performed histological examination of hematoxylin- and eosin-stained retinal sections as well as sections labeled in situ by the terminal dUTP nick end labeling (TUNEL) method. RESULTS: ERG findings showed that as early as 2 months of age, cone function was lost while rod function was preserved. However, by 9 months of age, both cone and rod functions could not be detected. Functional visual assessment based on the ability to avoid obstacles showed that vision was retained up to the age of 11 months. Both OCT and histopathology studies revealed a progressive thinning of the outer nuclear layer (ONL) over the first 2 years of age. TUNEL labeling identified apoptotic photoreceptor cell death as the cause of this thinning of the ONL. CONCLUSIONS: A treatment strategy should consist in initiating gene therapy as early as possible after birth to prevent or delay the loss of rod function. In the MLHD, successful subretinal delivery of a therapeutic vector is feasible at 2 months of age and may prevent or delay the loss of rod function

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

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus–free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 × 1011 particles/ml; 95% confidence interval [CI], 7.89 × 1011 to 1.05 × 1012 particles/ml), vector genomes ({X}, 3.28 × 1010 vector genomes/ml; 95% CI, 2.70 × 1010 to 4.75 × 1010 vector genomes/ml), transducing units ({X}, 5.09 × 108 transducing units/ml; 95% CI, 2.00 × 108 to 9.60 × 108 transducing units/ml), and infectious units ({X}, 4.37 × 109 TCID50 IU/ml; 95% CI, 2.06 × 109 to 9.26 × 109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed

Étude du trans-épissage comme outil de thérapie génique dans le modèle canin de la mucopolysaccharidose de type VII

Parmi les stratégies correctives en thérapie génique, le trans-épissage permet de remplacer, au niveau de l'ARN pré-messager, une séquence mutante par une séquence normale exogène portée par un PTM dans une réaction contrôlée par le spliceosome. Dans cette étude, nous avons évalué la faisabilité du trans-épissage dans le modèle canin de la mucopolysaccharidose de type VII. Nous avons testé différents PTM, de même que différentes molécules adjuvantes, le snRNAU7 et les ARN bifonctionnels. Nos résultats montrent que dans un système minigène, le trans-e pissage permet la reprogrammation de l ARN pré-messager ß-glucuronidase, détectable au niveau moléculaire comme au niveau protéique. Cependant, les choses sont beaucoup plus compliquées dans un environnement naturel. Les PTM sont ici capables d'interagir avec leur cible, mais l'efficacité de la réaction est le facteur limitant le plus critique. Ainsi, s'il a pu être possible de détecter des ARN messagers trans-épissés , notamment en présence du snRNA U7 et des ARN bifonctionnels, nous n'avons jamais été en mesure de détecter de protéine issue de la réaction de trans-épissage dans cet environnement. Cette étude, ainsi que d'autres, pointent la difficulté de manipulation du trans-épissage dans les protocoles de thérapie génique.Among corrective strategies in gene therapy, trans-splicing allows the reprogramming, at the pre-messenger RNA level, of a mutant sequence by an exogenous and normal sequence carried by a PTM in a reaction controlled by the spliceosome. In this study, we evaluated the feasibility of trans-splicing in the canine model of mucopolysaccharidosis VII. We tested different PTM and different adjuvant molecules, the U7 snRNA and the bifunctional RNA. Our results show that in an artificial minigene context, trans-splicing allows the reprogramming of the GusB transcript, as seen at the molecular and at the protein levels. However, things are more difficult in the natural context. In this environment, PTM are able to react with their target, but the efficiency is the major limiting factor. Thus, even if it was possible to detect trans-splicing resulting transcript, particularly in the presence of the U7 snRNA and of the bifunctional RNA, we could not detect trans-splicing resulting protein in this environment. This study, like others, points the difficulty to manipulate trans-splicing in the gene therapy protocols.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Induction de lymphocytes T régulateurs humains par activation de la voie Notch

Les lymphocytes T régulateurs (LTR) sont des cellules immunosuppressives régulant la tolérance périphérique. Notch est une famille de récepteurs transmembranaires exprimés à la surface des lymphocytes jouant un rôle important dans la lymphopoïèse. L'activation de Notch est secondaire à la liaison avec son ligand Jagged1. Nous avons démontré dans 2 modèles de stimulation antigénique in vitro (virale et allogénique) que la surexpression de Jagged1 sur des lymphocytes B humains transformés par l'Epstein Barr virus (EBV) utilisées comme cellules présentatrices d'antigènes et mises en culture avec des lymphocytes T humains permettait l'induction de LTR spécifiques de l'EBV dans le premier modèle et des alloantigènes dans le second. Ces résultats démontrent que l'activation forcée de Notch lors d'une présentation antigénique permet l'induction de LTR spécifiques d antigènes et ajoutent à Notch un rôle possible dans la régulation du système immunitaire périphérique.Regulatory T lymphocytes (Tr) are immunosuppressive cells that regulate the peripheral tolerance. Notch is a family of transmembrane receptors expressed at the surface of lymphocytes. Notch plays a important role in the lymphopoiesis. The ligation of the ligand Jaggedl induces the activation of Notch. We have demonstrated in two models of antigenic stimulation in vitro (viral and allogeneic) that overexpression of Jagged1 on human B cells transformed by the Epstein Barr Virus (EBV) used as antigen presenting cells and cultured with human T lymphocytes induced EBV specific Tr in the first model and allogeneic specific Tr in the second one. These results demonstrate that forced activation of Notch during an antigenic stimulation induce antigen specific Tr and add to Notch a possible role in the regulation of the peripheral immune system.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Développement de méthodes de production et d'analyse de vecteurs recombinants dérivés de l'adéno-associated virus

De nombreuses études in vivo ont montré que les vecteurs recombinants dérivés de l'adéno-associated virus (AAVr) peuvent transduire efficacement de nombreux organes et conduisent à une expression stable et à long terme du gène. Ces résultats encourageants ont conduit au développement rapide des essais cliniques utilisant les vecteurs AAVr-2. Cependant ces essais chez l'homme nécessitent de grandes quantités de vecteurs AAVr. Les techniques conventionnelles de production et purification montrent alors leurs limites. Nous avons donc développé des méthodes de productions alternatives plus simples et reproductibles, en établissant des lignées stables, d'encapsidation, contenant intégré dans leur génome les gènes rep-cap hybrides pour les sérotypes-1 et -5. Nous avons aussi décrit une procédure de purification par chromatographie liquide basée sur des résines échangeuses d'ions. Cette procédure a l'avantage d'être directement applicable à grande échelle, et pourrait s'adapter à différents sérotypes. Cette méthode permet de purifier le sérotype-5, le plus divergent au niveau de la séquence protéique de la capside comparée à 1'AAVr-2. Les stocks d'AAVr obtenus présentent une pureté supérieure aux méthodes traditionnelles et transduisent aussi efficacement les organes cibles. Afin de mieux caractériser et différentier les capsides AAVr des différents sérotypes de plus en plus nombreux, nous avons montré que la digestion protéolytique des virions AAVr-2, donne un profil de digestion unique. Cette analyse protéolytique des capsides AAVr permet de distinguer les AAVr-2 des AAV-1 et -5.Numerous in vivo studies have demonstrated that rAAV vectors can efficiently transduce many tissues and lead to stable gene expression. These encouraging results have led to a rapid development of clinical trials involving the use of rAAV-2 vectors. However, the obtainment of large-scale rAAV-2 vector stocks for clinical assay is still hampered by the conventional production methods that are not efficient and difficult to scale-up. We develop a simple and reproducible alternative method based on stable packaging hybrid cell lines containing integrated into their genome the rep-cap genes of serotypes -1 and -5. Another critical step for the assessment of these vectors in clinical trials is the method used for purification of rAAV particles. We developed a purification method, which comprises two-step chromatography process based on the use of ion- exchange resins. This process can be easily scaled up and could be applied to different AAV serotypes. We purified the serotype-5, the most divergent in terms of capsid composition compared to AAV-2. The rAAV stocks obtained are pure and transduced efficiently target tissues. To characterize rAAV capsid from different serotype, we have shown that proteolytic digestion of rAAV virions is able to generate a characteristic cleavage pattern that can be use to distinguish AAV-2 from AAV-1 and -5.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF