Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.

There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.

Transient suppression of hepatocellular replication in the mouse liver following transduction with recombinant adeno-associated virus.

Recombinant vectors based on adeno-associated virus (AAV) are proving to be powerful tools for genetic manipulation of the liver, for both discovery and therapeutic purposes. The system can be used to deliver transgene cassettes for expression or, alternatively, DNA templates for genome editing via homologous recombination. The replicative state of target cells is known to influence the efficiency of these processes and knowledge of the host-vector interactions involved is required for optimally effective vector deployment. Here we show, for the first time in vivo, that in addition to the known effects of hepatocellular replication on AAV-mediated gene transfer, the vector itself exerts a potent, albeit transient suppressive effect on cell cycle progression that is relieved on a time course that correlates with the known rate of clearance of input single-stranded vector DNA. This finding requires further mechanistic investigation, delineates an excellent model system for such studies and further deepens our insight into the complexity of interactions between AAV vectors and the cell cycle in a clinically promising target tissue.

A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.

X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development.

AAV-encoded OTC activity persisting to adulthood following delivery to newborn spf(ash) mice is insufficient to prevent shRNA-induced hyperammonaemia.

Urea cycle defects presenting in the neonatal period with hyperammonaemia are associated with high morbidity and mortality, and necessitate liver transplantation for long-term management. Gene therapy is therefore an attractive possibility, with vectors based on adeno-associated virus (rAAV) currently showing exciting promise in liver-targeted clinical trials in adults. Successful use of rAAV vectors in infants, however, is more challenging as episomal rAAV genomes will be lost from proliferating hepatocytes during liver growth, leaving stable transgene expression dependent on the subset of vector genomes that undergo genomic integration. To explore this challenge, we exploited the partially ornithine transcarbamylase (OTC)-deficient spf(ash) mouse model and small hairpin RNA-mediated knockdown of residual endogenous OTC enzyme activity in adult mice that had received neonatal treatment with an OTC-encoding rAAV. This leaves mice reliant on vector-encoded OTC activity that has persisted from the newborn period. Despite stable transduction in approximately 8% of hepatocytes and residual vector-encoded OTC activity of up to 33% of wild-type, well above endogenous spf(ash) levels (5-7%), mice were not protected from hyperammonaemia. These data show that the distribution of OTC activity within the liver is critical and that rAAV vector re-delivery after early neonatal treatment is likely to be necessary for stable control of hyperammonaemia into adulthood.

Pancreatic transdifferentiation in porcine liver following lentiviral delivery of human furin-cleavable insulin.

Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic ß-cells. Gene therapy is one strategy being actively explored to cure TID by affording non-ß-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)-diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some ß-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 10(9) transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several ß-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed. Triple immunofluorescence showed extensive insulin expression, as was previously observed in our studies with rodents. Additionally, a small amount of glucagon and somatostatin protein expression was seen. Collectively, these data indicate that pancreatic transdifferentiation of the liver tissue had occurred. Our data suggest that this regimen may ultimately be used clinically to cure TID, however more work is required to replicate the successful reversal of diabetes in increased numbers of pigs.

Comparison of gene transfer to the murine liver following intraperitoneal and intraportal delivery of hepatotropic AAV pseudo-serotypes.

Adeno-associated virus (AAV) vectors are highly efficient for liver-targeted gene delivery in murine models and show promise in early phase human clinical trials. This efficiency is capsid-dependent and was only achieved after discovery that the AAV2 vector genome could be trans-encapsidated into the capsids of other AAV serotypes. This confers novel host-vector biology and target tissue tropism. Optimal exploitation of the growing number of AAV vector pseudo-serotypes, however, requires detailed context-dependent characterisation of transduction performance. In this study, we compared the pattern and efficiency of gene delivery to the adult mouse liver following intraportal and intraperitoneal injection of vectors pseudo-serotyped with known hepatotropic capsids from AAV type 7, 8, 9 and rhesus 10. Vectors pseudo-serotyped with these hepatotropic capsids proved relatively efficient irrespective of administration route, with higher transgene expression in males despite equivalent vector genome delivery in females. Transgene expression was predominantly centrilobular in contrast to the AAV2 capsid, which gave a periportal pattern of expression. Most intriguingly, vector genome performance appeared to be delivery route-dependent, consistent with the possibility of in vivo capsid modification. These data not only inform the experimental use of AAV vectors, but also provide insight into novel aspects of host-vector biology requiring further focused analysis.

In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.

Mutant proteins have the potential to exert dominant-negative effects that might limit the therapeutic efficacy of their wild-type counterparts after gene transfer. For ornithine transcarbamylase (OTC) deficiency, in vitro studies have suggested the presence of dominant-negative effects, however, supporting in vivo studies have not been conducted. In this study, we exploited the capacity of recombinant adeno-associated virus (rAAV) 2/8 vectors to deliver transgenes to the mouse liver with high efficiency to determine whether expression of selected OTC mutant proteins exert inhibitory effects on endogenous wild-type OTC enzymatic activity. Using site-directed mutagenesis we constructed three OTC mutants with a theoretical or reported in vitro capacity to exert dominant-negative effects, and delivered these to the liver using rAAV2/8. Each mutation had been earlier identified in patients with OTC deficiency. Treated mice showed no increase in urinary orotic acid levels or reduction in OTC activity despite supra-physiological expression of the mutant proteins, consistent with an absence of dominant-negative effects. These data have important implications for the development of gene therapy strategies for OTC deficiency and validate a model system in which potential dominant-negative effects of specific mutations in prospective patients can be examined empirically before gene therapy.

Antigen-specific humoral tolerance or immune augmentation induced by intramuscular delivery of adeno-associated viruses encoding CTLA4-Ig-antigen fusion molecules.

This study initially sought to investigate the immunostimulatory properties of recombinant adeno-associated virus (rAAV) with a view to developing a genetic vaccine for malaria using muscle as a target tissue. To augment humoral immunity, the AAV-encoded antigen was genetically fused with CTLA4-Ig, a recombinant molecule that binds B7 costimulatory molecules. At 10(9) vg, CTLA4-Ig fusion promoted the humoral immune response 100-fold and was dependent on CTLA4-Ig binding with B7 costimulatory molecules, confirming plasmid DNA models using this strategy. In distinct contrast, 10(12)-10(13) vg of rAAV1 specifically induced long-lived humoral tolerance through a mechanism that is independent of CTLA4-Ig binding with B7. This finding was unexpected, as rAAV delivery to muscle, unlike liver, has shown that this tissue provides a highly immunogenic environment for induction of humoral immunity against rAAV transgene products. An additional unpredicted consequence of antigen fusion with CTLA4-Ig was the enhancement of antigen expression by approximately one log, an effect mapped to the hinge and Fc domain of IgG(1,) but not involving antigen dimerization or the neonatal Fc receptor. Collectively, these findings significantly advance the potential of rAAV both as a vaccine or immunotherapeutic platform for the induction of antigen-specific humoral immunity or tolerance and as a gene therapeutic delivery system.

Potential of AAV vectors in the treatment of metabolic disease.

Inborn errors of metabolism are collectively common, frequently severe and in many instances difficult or impossible to treat. Accordingly, there is a compelling need to explore novel therapeutic modalities, including gene therapy, and examine multiple phenotypes where the risks of experimental therapy are outweighed by potential benefits to trial participants. Among available gene delivery systems recombinant AAV shows special promise for the treatment of metabolic disease given the unprecedented efficiencies achieved in transducing key target tissues, such as liver and muscle, in small animal models. To date over 30 metabolic disease phenotypes have been investigated in small animal studies with complete phenotype correction being achieved in a substantial proportion. Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.

Regulatory immune cells in kidney disease.

Lymphocytes and macrophages act as effector immune cells in the initiation and progression of renal injury. Recent data have shown that subpopulations of these immune cells (regulatory T lymphocytes and alternately-activated or regulatory macrophages) are potent modulators of tissue injury and repair in renal disease. Recent animal studies examining the therapeutic effect of these cells raise the exciting possibility that strategies targeting these cell types may be effective in treating and preventing kidney disease in humans. This review will describe their biological role in experimental kidney disease and therapeutic potential in clinical nephrology.

Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct.

Gene transfer vectors encoding two or more genes are potentially powerful research tools and are poised to play an increasingly important role in gene therapy applications. Common strategies employed to express more than one transgene per vector include the use of multiple promoters, internal ribosome entry site (IRES) elements, splicing signals and fusion proteins. Of these, the IRES elements and multiple promoters have been most widely used. The use of multiple promoters, however, may be compromised by interference between promoters, promoter silencing and vector rearrangements or deletions. In this study, we demonstrate promoter interference between two internal heterologous promoters in the context of a late-generation lentiviral vector. The interference, involving the human cytomegalovirus-immediate-early promoter and human elongation-factor-1alpha promoter, occurred bidirectionally with both promoters markedly impairing expression of the adjacent transcription unit. The data presented not only highlight the potential for interference between these widely-used promoters, but also the value of a sequential approach to vector construction that allows such effects to be recognized.

Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy.

In an earlier study exploring the potential of gene transfer to repair myocardial conduction defects, we observed that myotubes, generated by forced expression of MyoD, exhibit reduced excitability when also modified to express connexin43 (Cx43). We hypothesized that this effect was caused by gap junction-mediated coupling between myotubes and the underlying fibroblast feeder layer. This intriguing possibility has important implications for ongoing efforts to develop strategies for repairing myocardial conduction defects by gene transfer, and also provides novel insights into the electrophysiological function of naturally occurring heterologous cell coupling within the heart. Although a conductive function for fibroblasts through heterologous coupling has previously been reported, the current study provides novel evidence that fibroblasts can modulate cardiomyocyte excitability in a Cx43-dependent manner. In a co-culture study system, neonatal rat cardiomyocytes were grown on monolayers of mouse fibroblasts with genetically altered Cx43 expression and the effect on intrinsic beat frequency examined. Cardiomyocytes grown on wild-type (WT) fibroblasts expressing native levels of Cx43 beat significantly slower than cells grown on fibroblasts devoid of this molecule (germline knockout) or with dominant-negative functional suppression. Expression of Cx43 in fibroblasts from Cx43 knockout mice restored cardiomyocyte beat frequency, to rates comparable with those observed in co-culture with WT fibroblasts.

Gene therapy: applications and progress towards the clinic.

Gene therapy was originally conceived as an approach to the treatment of genetic disease, to repair or replace a faulty gene. Subsequently, gene therapy clinical trials have been undertaken for a wide range of conditions, particularly cancer and AIDS. Overall, the results from gene therapy have been disappointing. The reasons include the following: (i) low gene transfer efficiencies and (ii) shortcomings in the identification and manipulation of appropriate target cells, including progenitor cell populations required for the maintenance of long-term effects. Today, the immense potential of gene therapy remains, but more basic research is required to improve technical aspects of this form of cellular therapy.

C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

Cardiac gene therapy: therapeutic potential and current progress.

Cardiovascular disease remains a major cause of morbidity and mortality in modern societies. While contemporary treatment modalities are making steady inroads to reduce this disease burden there remains a pressing need to vigorously explore novel therapeutic strategies. Rapid advances in our understanding of molecular pathology and the evolution of increasingly efficient gene transfer technology offer the imminent prospect of gene-based approaches to, at least, a subset of cardiovascular pathophysiologies. Initially envisaged as a treatment strategy for inherited monogenic disorders, it is now apparent that gene therapy has broader potential that encompasses acquired polygenic diseases, including many that affect the cardiovascular system. Extensive in vitro and animal studies are providing an increasingly sound scientific basis for cautious human evaluation. This review focuses on gene therapy of diseases primarily afflicting the heart, and provides an overview of gene and vector delivery systems with particular emphasis on systems suited to individual cardiac conditions. The pathophysiology underlying these conditions and molecular targets for therapeutic intervention are also reviewed.

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

Adeno-associated virus vectors show variable dependence on divalent cations for thermostability: implications for purification and handling.

Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations. Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance. Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile.

Human PBMC-derived dendritic cells transduced with an adenovirus vectorinduce cytotoxic T-lymphocyte responses against a vector-encoded antigen in vitro.

Dendritic cells (DC) are among the most potent antigen-presenting cells known and play an important role in the initiation of antigen-specific T-lymphocyte responses. Several recent studies have demonstrated that DC expressing vector-encoded tumor-associated antigens can induce protective and therapeutic immunity in murine cancer models. In the current study we set out to examine in vitro the utility of adenovirus vectors in the transduction of human DC for the induction of antigen-specific T-lymphocyte responses against a defined vector-encoded antigen. DC were derived from the adherent fraction of PBMC by culture in defined medium containing GM-CSF and IL-4. A replication-defective E1/E3-deleted type 5 adenovirus vector encoding bacterial beta-galactosidase (beta-gal) under the transcriptional control of a CMV promoter was used to transduce DC at multiplicities of infection (MOI) up to 1000. While high MOI were required to achieve efficient transduction there was no significant effect on DC morphology, immunophenotype or potency in allogeneic lymphocyte proliferation assays. Furthermore, transduced DC-induced antigen-specific CTL activity against adenoviral proteins and more significantly, the vector-encoded antigen beta-gal. These data clearly demonstrate the potential of adenovirus vectors in anticancer DC vaccine strategies and provide an important link between existing animal data and human clinical application.