Takis Athanasopoulos
Research interests
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InterestsVaccine Development, Gene Therapy
Other
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LanguagesGreek and English
Publications
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5.95Impact points
Highly potent delivery method of gp160 envelope vaccine combining lentivirus-like particles and DNA electrotransfer.
Journal of controlled release : official journal of the Controlled Release Society. 01/2012;
Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like parti... [more] Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like particles (VLPs) and the simplicity of use of DNA vaccines. Characterisation of lentivirus-like particles (lentiVLPs) by western blot, dynamic light scattering and electron microscopy revealed that their protein pattern, size and structure make them promising candidates for HIV-1 vaccines. Although all particles were similar with regard to size and distribution, they clearly differed in p24 capsid protein content suggesting that Rev may be required for particle maturation and Gag processing. In vivo, lentiVLP pseudotyping with the gp160 envelope or with a combination of gp160 and VSV-G envelopes did not influence the magnitude of the immune response but the combination of lentiVLPs with Alum adjuvant resulted in a more potent response. Interestingly, the strongest immune response was obtained when plasmids encoding lentiVLPs were co-delivered to mice muscles by electrotransfer, suggesting that lentiVLPs were efficiently produced in vivo or the packaging genes mediate an adjuvant effect. DNA electrotransfer of plasmids encoding lentivirus-like particles offers many advantages and appears therefore as a promising delivery method of HIV-1 vaccines.
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2.97Impact points
Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.
The journal of gene medicine. 09/2011; 13(9):497-506.
Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of micro... [more] Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8(+) T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8 weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD.
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2.59Impact points
Adeno-associated virus serotypes 7 and 8 outperform serotype 9 in expressing atheroprotective human apoE3 from mouse skeletal muscle.
Metabolism: clinical and experimental. 04/2011; 60(4):491-8.
Intramuscular injection of adeno-associated viral (AAV) vectors is potentially a safe, minimally invasive procedure for the long-term gene expression of circulating antiatherogenic proteins. Here, we compare secretion and atheroprotective effects of human apoE3 after injection of 3 pseudotyped AAV v... [more] Intramuscular injection of adeno-associated viral (AAV) vectors is potentially a safe, minimally invasive procedure for the long-term gene expression of circulating antiatherogenic proteins. Here, we compare secretion and atheroprotective effects of human apoE3 after injection of 3 pseudotyped AAV vectors (AAV2/7, AAV2/8, or AAV2/9), driven by the CMV enhancer/chicken β-actin (CAG) promoter, into skeletal muscle of hyperlipidemic apolipoprotein E-deficient (apoE⁻/⁻) mice. Vector viabilities were verified by transducing cultured C2C12 mouse myotubes and assessing secretion of human apoE3 protein. Both hind limb tibialis anterior muscles of female C57BL/6 apoE⁻/⁻ mice, 2 months old and fed a high-fat diet, were each injected with 1 x 10¹⁰ vector genomes of AAV vector. Identical noninjected mice served as controls; and blood was collected at weeks 0, 1, 2, 4, and 13. At termination (13 weeks), the brachiocephalic artery was excised; and after staining sections, plaque morphometry and fractional lipid content were quantified by computerized image analysis. Intramuscular injection of AAV2/7 and AAV2/8 vectors produced up to 2 μg human apoE3 per milliliter plasma, just below the threshold to reverse dyslipoproteinemia. AAV2/9 was notably less effective, mice having a 3-fold lower level of plasma apoE3 at 13 weeks and a 50% greater burden of atherosclerotic plaque lipid in their brachiocephalic arteries. We conclude that although vector refinement is needed to exploit fully apoE3 atheroprotective functions, AAV2/7 and AAV2/8 are promising gene transfer vectors for muscle-based expression of antiatherogenic circulating proteins.
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4.20Impact points
Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice.
Human gene therapy. 03/2011; 22(11):1379-88.
Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging cap... [more] Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.
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4.72Impact points
β-catenin as a potential key target for tumor suppression.
International journal of cancer. Journal international du cancer. 03/2011; 129(7):1541-51.
β-catenin is a multifunctional protein identified to be pivotal in embryonic patterning, organogenesis and adult homeostasis. It plays a critical structural role in mediating cadherin junctions and is also an essential transcriptional co-activator in the canonical Wnt pathway. Evidence has been docu... [more] β-catenin is a multifunctional protein identified to be pivotal in embryonic patterning, organogenesis and adult homeostasis. It plays a critical structural role in mediating cadherin junctions and is also an essential transcriptional co-activator in the canonical Wnt pathway. Evidence has been documented that both the canonical Wnt pathway and cadherin junctions are deregulated or impaired in a plethora of human malignancies. In the light of this, there has been a recent surge in elucidating the mechanisms underlying the etiology of cancer development from the perspective of β-catenin. Here, we focus on the emerging roles of β-catenin in the process of tumorigenesis by discussing novel functions of old players and new proteins, mechanisms identified to mediate or interact with β-catenin and the most recently unraveled clinical implications of β-catenin regulatory pathways toward tumor suppression.
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Codon optimization of the microdystrophin gene for Duchene muscular dystrophy gene therapy.
Methods in molecular biology (Clifton, N.J.). 01/2011; 709:21-37.
Duchenne muscular dystrophy (DMD) is a severe muscle wasting X-linked genetic disease caused by dystrophin gene mutations. Gene replacement therapy aims to transfer a functional full-length dystrophin cDNA or a quasi micro/mini-gene into the muscle. A number of AAV vectors carrying microdystrophin g... [more] Duchenne muscular dystrophy (DMD) is a severe muscle wasting X-linked genetic disease caused by dystrophin gene mutations. Gene replacement therapy aims to transfer a functional full-length dystrophin cDNA or a quasi micro/mini-gene into the muscle. A number of AAV vectors carrying microdystrophin genes have been tested in the mdx model of DMD. Further modification/optimization of these microgene vectors may improve the therapeutic potency. In this chapter, we describe a species-specific, codon optimization protocol to improve microdystrophin gene therapy in the mdx model.
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6.24Impact points
Transcription factor rational design improves directed differentiation of human mesenchymal stem cells into skeletal myocytes.
Molecular therapy : the journal of the American Society of Gene Therapy. 01/2011; 19(7):1331-41.
There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a pred... [more] There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.
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3.76Impact points
Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization.
BMC genomics. 01/2010; 11:345.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystroph... [more] Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology. Using RNA interference (RNAi) to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (Pvalb and Casq1), or cardiomyopathy (Obscurin, Tcap). In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels in vivo and in vitro. Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage. Therefore, it becomes apparent that any gene therapy approaches for DMD should target early stages in muscle development to attain a maximum clinical benefit. With a clear and specific definition of the transcriptome of dystrophin deficiency, manipulation of identified dysregulated molecules downstream of dystrophin may lead to novel ameliorative approaches for DMD.
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9.43Impact points
Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1.
Proceedings of the National Academy of Sciences of the United States of America. 11/2009;
In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memor... [more] In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing alpha(4)beta(7) integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-gamma production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.
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3.22Impact points
Gene therapy for muscular dystrophy: current progress and future prospects.
Expert opinion on biological therapy. 08/2009; 9(7):849-66.
Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there is no effective treatment but new gene-based therapies are currently being developed with particular noted advances in using conventional gene replacemen... [more] Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there is no effective treatment but new gene-based therapies are currently being developed with particular noted advances in using conventional gene replacement strategies, RNA-based approaches, or cell-based gene therapy with a main focus on Duchenne muscular dystrophy (DMD). DMD is the most common and severe form of muscular dystrophy and current treatments are far from adequate. However, genetic and cell-based therapies, in particular exon skipping induced by antisense strategies, and corrective gene therapy via functionally engineered dystrophin genes hold great promise, with several clinical trials ongoing. Proof-of-concept of exon skipping has been obtained in animal models, and most recently in clinical trials; this approach represents a promising therapy for a subset of patients. In addition, gene-delivery-based strategies exist both for antisense-induced reading frame restoration, and for highly efficient delivery of functional dystrophin mini- and micro-genes to muscle fibres in vivo and muscle stem cells ex-vivo. In particular, AAV-based vectors show efficient systemic gene delivery to skeletal muscle directly in vivo, and lentivirus-based vectors show promise of combining ex vivo gene modification strategies with cell-mediated therapies.
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6.24Impact points
Codon and mRNA Sequence Optimization of Microdystrophin Transgenes Improves Expression and Physiological Outcome in Dystrophic mdx Mice Following AAV2/8 Gene Transfer.
Molecular therapy : the journal of the American Society of Gene Therapy. 10/2008;
Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscl... [more] Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (DeltaAB/R3-R18/DeltaCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (DeltaAB/R3-R18/DeltaCT and DeltaR4-R23/DeltaCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of DeltaAB/R3-R18/DeltaCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized DeltaAB/R3-R18/DeltaCT. However, codon-optimized microdystrophin DeltaR4-R23/DeltaCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.Molecular Therapy (2008); doi:10.1038/mt.2008.186.
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4.29Impact points
Preliminary evaluation of a self-complementary AAV2/8 vector for hepatic gene transfer of human apoE3 to inhibit atherosclerotic lesion development in apoE-deficient mice.
Atherosclerosis. 09/2008;
Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE(-/-)) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus... [more] Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE(-/-)) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (scAAV) serotype 8 vector, driven by a hepatocyte-specific promoter (LP1), for liver-directed gene delivery of human apoE3. Vector viability was validated by transducing cultured HepG2 cells and measuring secretion of apoE3 protein. Male and female apoE(-/-) mice, 6-month old and fed on normal chow, were intravenously injected with 1x10(11)vg (vector genomes) of scAAV2/8.LP1.apoE3; age-matched untreated mice served as controls. In male mice, plasma apoE3 levels were sufficiently high (up to 17mug/ml) to normalize plasma total cholesterol and ameliorate their proatherogenic lipoprotein profile, by reducing VLDL/LDL and increasing HDL 5-fold. At termination (12 weeks) development of aortic atherosclerosis was significantly retarded by 58% (aortic lesion area 8.2+/-1.4% vs. 19.3+/-2.4% in control males; P<0.001). Qualitatively similar anti-atherogenic effects were noted when female mice were treated, but the benefits were less marked and aortic lesions, for example, were reduced by only 33% (15.7+/-3.7% vs. 23.6+/-6.9%). Although group numbers were small (n=4/5), this gender-specific difference reflected two to three times less apoE3 in plasma of female mice at weeks 3 and 6, implying that gene transfer to female liver using scAAV vectors may require additional optimization, despite their established superior potency to conventional single-stranded (ssAAV) vectors.
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4.20Impact points
Human apolipoprotein E expression from mouse skeletal muscle by electrotransfer of nonviral DNA (plasmid) and pseudotyped recombinant adeno-associated virus (AAV2/7).
Human gene therapy. 06/2008; 19(6):569-78.
Plasma apolipoprotein E (apoE) has multiple atheroprotective actions. However, although liver-directed adenoviral gene transfer of apoE reverses hypercholesterolemia and inhibits atherogenesis in apoE-deficient (apoE(-/-)) mice, safety considerations have revived interest in nonviral DNA (plasmid) a... [more] Plasma apolipoprotein E (apoE) has multiple atheroprotective actions. However, although liver-directed adenoviral gene transfer of apoE reverses hypercholesterolemia and inhibits atherogenesis in apoE-deficient (apoE(-/-)) mice, safety considerations have revived interest in nonviral DNA (plasmid) and nonpathogenic adeno-associated viral (AAV) vectors. Here, we assess the effectiveness of these two delivery vehicles by minimally invasive intramuscular injection. First, we constructed AAV2-based expression plasmids harboring human apoE3 cDNA, driven by two muscle-specific promoters (CK6 and C5-12) and one ubiquitous promoter (CAG); each efficiently expressed apoE3 in transfected cultured C2C12 mouse myoblasts, although muscle-specific promoters were active only in differentiated multinucleate myotubes. Second, a pilot study verified that electrotransfer of the CAG-driven plasmid (p.CAG.apoE3) into tibialis anterior muscles, pretreated with hyaluronidase, of apoE(-/-) mice significantly enhanced (p < 0.001) local intramuscular expression of apoE3. However, in a 7-day experiment, the CK6- and C5-12-driven plasmids produced less apoE3 in muscle than did p.CAG.apoE3 (0.61 +/- 0.38 and 0.45 +/- 0.38 vs. 13.38 +/- 7.46 microg of apoE3 per muscle, respectively), but plasma apoE3 levels were below our detection limit (<15 ng/ml) in all mice and did not reverse the hyperlipidemia. Finally, we showed that intramuscular injection of a cross-packaged AAV serotype 7 viral vector, expressing human apoE3 from the CAG promoter, resulted in increasing levels of apoE3 in plasma over 4 weeks, although the concentration reached (1.40 +/- 0.35 microg/ml) was just below the threshold level needed to reduce the hypercholesterolemia. We conclude that skeletal muscle can serve as an effective secretory platform to express the apoE3 transgene, but that improved gene transfer vectors are needed to achieve full therapeutic levels of plasma apoE3 protein.
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7.39Impact points
RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology.
Human molecular genetics. 06/2008;
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice... [more] Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice, the animal model of DMD. Accordingly, we hypothesized that the pathology observed with dystrophin deficiency may be developmentally regulated. Initially, we demonstrated that profound siRNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus (AAV) vectors to express shRNAs targeting dystrophin in skeletal muscle in vivo yielded a potent and specific dystrophin knockdown, but only after approximately 5 months, indicating the very long half-life of dystrophin. Interestingly, and in contrast to what is observed in congenital dystrophin deficiency, long-term ( approximately 1 year) dystrophin knockdown in adult mice did not result, per se, in overt dystrophic pathology or up-regulation of utrophin. This supports our hypothesis and suggests new pathophysiology for the disease. Furthermore, taking into account the rather long half-life of dystrophin, and the notion that the development of pathology is age-dependent, it indicates that a single gene therapy approach before the onset of pathology might convey a long term cure for DMD.
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2.91Impact points
Activity of different vaccine-associated promoter elements in human dendritic cells.
Immunology letters. 02/2008; 115(2):117-25.
Vaccine design approaches that target dendritic cells (DC) aim at achieving high levels of transgene expression. Careful selection of the promoter element driving the foreign gene is therefore important. We have constructed adenovirus vectors carrying the gene for enhanced green fluorescent protein ... [more] Vaccine design approaches that target dendritic cells (DC) aim at achieving high levels of transgene expression. Careful selection of the promoter element driving the foreign gene is therefore important. We have constructed adenovirus vectors carrying the gene for enhanced green fluorescent protein (eGFP) driven by three different promoters, CMV, CMV5 and ubiquitin C (UbC) promoter, and analysed their activity in different populations of human DC, namely blood plasmacytoid (pDC) and myeloid DC (mDC), monocyte-derived DC (moDC), Langerhans (LC) and dermal type DC (dDC). Although the CMV5 promoter was more active than the other two promoters in the HeLa and 911HER cell lines, in human DC the highest level of transgene expression was seen with the CMV promoter. There was very low-level eGFP expression in all cell types transduced with the UbC promoter. Highest eGFP expression levels were observed in moDC, cultured mDC and LC and the lowest levels in pDC. Expression of eGFP was augmented in all DC populations upon stimulation with CD40 ligand (CD40L). These findings demonstrate that the CMV promoter is the most effective of the three promoters tested in a range of different human DC populations.
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Preliminary evaluation of a self-complementary AAV2/8 vector for hepatic gene transfer of human apoE3 to inhibit atherosclerotic lesion development in apoE-deficient mice
Atherosclerosis.
Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE−/−) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (... [more] Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE−/−) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (scAAV) serotype 8 vector, driven by a hepatocyte-specific promoter (LP1), for liver-directed gene delivery of human apoE3. Vector viability was validated by transducing cultured HepG2 cells and measuring secretion of apoE3 protein. Male and female apoE−/− mice, 6-month old and fed on normal chow, were intravenously injected with 1 × 1011 vg (vector genomes) of scAAV2/8.LP1.apoE3; age-matched untreated mice served as controls. In male mice, plasma apoE3 levels were sufficiently high (up to 17 μg/ml) to normalize plasma total cholesterol and ameliorate their proatherogenic lipoprotein profile, by reducing VLDL/LDL and increasing HDL 5-fold. At termination (12 weeks) development of aortic atherosclerosis was significantly retarded by 58% (aortic lesion area 8.2 ± 1.4% vs. 19.3 ± 2.4% in control males; P < 0.001). Qualitatively similar anti-atherogenic effects were noted when female mice were treated, but the benefits were less marked and aortic lesions, for example, were reduced by only 33% (15.7 ± 3.7% vs. 23.6 ± 6.9%). Although group numbers were small (n = 4/5), this gender-specific difference reflected two to three times less apoE3 in plasma of female mice at weeks 3 and 6, implying that gene transfer to female liver using scAAV vectors may require additional optimization, despite their established superior potency to conventional single-stranded (ssAAV) vectors.
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Activity of different vaccine-associated promoter elements in human dendritic cells
Immunology Letters.
Vaccine design approaches that target dendritic cells (DC) aim at achieving high levels of transgene expression. Careful selection of the promoter element driving the foreign gene is therefore important. We have constructed adenovirus vectors carrying the gene for enhanced green fluorescent protein ... [more] Vaccine design approaches that target dendritic cells (DC) aim at achieving high levels of transgene expression. Careful selection of the promoter element driving the foreign gene is therefore important. We have constructed adenovirus vectors carrying the gene for enhanced green fluorescent protein (eGFP) driven by three different promoters, CMV, CMV5 and ubiquitin C (UbC) promoter, and analysed their activity in different populations of human DC, namely blood plasmacytoid (pDC) and myeloid DC (mDC), monocyte-derived DC (moDC), Langerhans (LC) and dermal type DC (dDC). Although the CMV5 promoter was more active than the other two promoters in the HeLa and 911HER cell lines, in human DC the highest level of transgene expression was seen with the CMV promoter. There was very low-level eGFP expression in all cell types transduced with the UbC promoter. Highest eGFP expression levels were observed in moDC, cultured mDC and LC and the lowest levels in pDC. Expression of eGFP was augmented in all DC populations upon stimulation with CD40 ligand (CD40L). These findings demonstrate that the CMV promoter is the most effective of the three promoters tested in a range of different human DC populations.
Following (7)
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Jennifer Pereira
Institute for International Research -
Alberto Malerba
Royal Veterinary College -
Peter Hayes
Imperial College London -
Linda J Popplewell
Royal Holloway, University of London -
Remi Mollicone
CFAR-m
Topics (1)
