Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies with an estimated prevalence of 1:25.000. Pain is one of the most debilitating symptoms in FSHD patients and it is assumed that it affects negatively both mobility and quality of life (QoL). We aim to describe the characteristics and intensity of pain and QoL in FSHD type 1 patients registered with the UK FSHD Registry. The 416 patients registered were asked to complete questionnaires assessing persistent and recurrent localised pain, together with validated questionnaires (Short-Form McGill questionnaire and the Individualized Neuromuscular Quality of Life Questionnaire (INQoL)). The pain questionnaires were completed by 379 patients; persistent pain was reported by 50% and recurrent localised pain by 43%. Patients with localised pain described it as discomforting (16%) and for persistent pain it was described as discomforting in 19.7% and as distressing in 14.4% of cases. The shoulder was the most affected location for both persistent (40.4%) and localised pain (23.6%). The population with localised pain reported no change in muscle bulk or weakness after the painful episodes. INQoL was completed by 354 patients; the overall mean score was 50.87 where 100 is the greatest impact on QoL. Muscle weakness has the greatest impact (mean 66.32) followed by fatigue (mean 46.89). Activities and body image were the life domains most affected (mean 57.07 and 55.21). QoL is not significantly affected by current age, age of onset of disease or sex. Pain had a moderate impact on QoL (mean 37). INQoL score correlated positively with ambulation (p < 0.001). Score for ambulant patients was 41.6 and non-ambulant patients 61.5. In conclusion patients with FSHD have a high occurrence of pain that is most frequently persistent and has no correlation with ambulation status of the patient. It seems that pain has a moderate impact on QoL. The most affected domains are activities and body image.

The United Kingdom (UK) Facioscapulohumeral Dystrophy (FSHD) Patient Registry launched in May 2013. Funded by the Muscular Dystrophy Campaign and supported by the TREAT-NMD Alliance. This patient driven registry collects the internationally agreed core dataset, an outcome of an ENMC Workshop held in 2010 [1], through a novel online portal (http://www.fshd-registry.org/uk). Genetic details are added by a nominated neuromuscular specialist. In addition questionnaires about pain, quality of life and scapular fixation are included. In the 12 months between May 2013 and May 2014 over 400 people registered, 92% with a diagnosis of FSHD1. Similar proportions of patients registered from both sexes and 59% of patients were between 40 and 70 years old (mean 47.39). Muscle weakness was widely reported with periscapular shoulder weakness occurring most frequently (89%) followed by weakness of the hip girdle (73%), facial muscles (72%) and foot dorsiflexor (71%). The onset of facial weakness was reported significantly earlier than weakness in other areas with 66% experiencing facial weakness before 20 years old. Full time wheelchair use was reported in 18% of cases, 62% having lost ambulation between 31 and 60 years old (mean 41.61). Use of a wheelchair or other assistive device part time was reported in 44% of cases. A small proportion of patients report hearing loss (18%), retinal vascular disease (2%) and using ventilation (7%). Additional questionnaires on pain were completed by 350 patients during this time and the majority reporting at least some pain, most often described as tiring or aching. Persistent pain (experienced for at least 3 months in a year) was reported by 92% with 53% of people describing this pain as distressing, horrible or excruciating. The location of the pain is variable but most often reported in the shoulder. A broad spectrum of patients has registered providing a new insight into the FSHD population in the UK. The Registry aims to help facilitate and accelerate clinical research and trials, sharing a common dataset with a growing number of FSHD registries around the world will allow the registry to achieve this locally and internationally. The registry is well placed to inform future clinical research and help develop of standards of care.

•The manuscript elaborates on what is clinically meaningful and how to measure this in DMD.•We discuss methods of efficacy measurement – strength versus function.•We discuss animal models and biochemical outcome measures for DMD.•We review the strategy and design of clinical studies – extrapolating to other stages of the disease.

After the organization of nearly 200 workshops on neuromuscular diseases (NMD), and the publication in Neuromuscular Disorders of an equal amount of workshop reports, the European NeuroMuscular Centre (ENMC) recently celebrated its 20th anniversary as European consortium of patient organizations. A strategic meeting with patient organizations and other representatives of the neuromuscular community took place in June 2012 in Kørsor, Denmark, while a special celebration occurred earlier in October 2011 in Amsterdam, The Netherlands.

The Muscular Dystrophy Campaign, a London-based charitable organization, funds research on muscle function and muscle disease, including the study of muscle stem cells. Dr. Marita Pohlschmidt, the Muscular Dystrophy Campaign's director of research, describes its vision and goals.

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 small interfering RNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus vectors to express short-hairpin RNAs 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 upregulation of utrophin. This supports our hypothesis and suggests new pathophysiology of 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.

Duchenne Muscular Dystrophy (DMD) is one of a group of genetically heterogeneous muscular dystrophies that are characterized by progressive weakness and wasting of skeletal muscle. Loss of myofibres occurs in response to a deficiency of dystrophin, a protein which is believed to be responsible for myofibre maintenance and integrity. Dystrophin forms a link between the cytoskeleton and the membrane-spanning dystrophin-associated glycoprotein complex (DAPC), indicative of a structural role for dystrophin.

Anderson–Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by α-galactosidase A (AGA) deficiency [1.MacDermot K.D. Holmes A. Miners A.H. Anderson–Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males.J. Med. Genet. 2001; 38: 750-760Crossref PubMed Scopus (556) Google Scholar]. There is evidence that LSDs could be corrected by gene transfer and expression and enzyme secretion from a subset of cells or a specific tissue [2.Ohshima T. et al.Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 6423-6427Crossref PubMed Scopus (85) Google Scholar, 3.Ziegler R.J. et al.Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.Hum. Gene Ther. 1999; 10: 1667-1682Crossref PubMed Scopus (87) Google Scholar, 4.Xu F. et al.Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA.J. Gene. Med. 2004; 7: 171-178Crossref Scopus (30) Google Scholar]. Such gene therapy has several advantages over an alternative enzyme replacement approach [5.Ioannou Y.A. Zeidner K.M. Gordon R.E. Desnick R.J. Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice.Am. J. Hum. Genet. 2001; 68: 14-25Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6.Eng C.M. et al.Safety and efficacy of recombinant human alpha-galactosidase A—replacement therapy in Fabry's disease.N. Engl. J. Med. 2001; 345: 9-16Crossref PubMed Scopus (1352) Google Scholar], as it offers long-term therapeutic effect, eliminates risks associated with repeated parenteral administrations, and is less expensive. We describe here a nonviral AGA gene delivery and expression/secretion from skeletal muscle in AGA knockout mice (animal model of Fabry disease) [7.Ohshima T. et al.α-Galactosidase A deficient mice: a model of Fabry disease.Proc. Natl. Acad. Sci. USA. 1997; 94: 2540-2544Crossref PubMed Scopus (282) Google Scholar]. We studied the combination of plasmid vectors with the SP1017 Pluronic block copolymer, a nonionic carrier capable of increasing the level and duration of expression of genes injected into muscle [8.Lemieux P. et al.A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle.Gene Ther. 2000; 7: 986-991Crossref PubMed Scopus (197) Google Scholar]. Single intramuscular administration of naked plasmid DNA encoding AGA resulted in enzyme secretion into the bloodstream and enzyme reuptake by distant organs. However, when plasmids were used in combination with SP1017 to enhance gene expression, a highly specific dependence on the promoter type emerged. SP1017 increased only the CMV-driven expression of AGA and had no effect on the muscle-specific muscle creatine kinase (MCK) promoter-controlled AGA expression. There was a drastic and rapid decrease in AGA activity when plasmid that contains AGA under the transcriptional control of the CMV promoter was used. It was caused by immune responses against AGA recognized as a neoantigen. The decline in AGA activity could be delayed by putting AGA under the control of the MCK promoter or prevented by transient immunosuppression, but combination with SP1017 did not alter the specific anti-AGA responses. This promoter-specific effect of Pluronics on gene expression has implications for the future design and applications of polymeric adjuvants. The targeting vectors used in this study contained human α-galactosidase A cDNA under the control of one of two promoters combined with the rat myosin light-chain 1/3 enhancer [9.Donoghue M. Ernst H. Wentworth B. Nadal-Ginard B. Rosenthal N. A muscle-specific enhancer is located at the 3′ end of the myosin light-chain 1/3 gene locus.Genes Dev. 1988; 2: 1779-1790Crossref PubMed Scopus (124) Google Scholar]: the ubiquitously active human CMV immediate-early promoter [10.Hennighausen L. Fleckenstein B. Nuclear factor 1 interacts with five DNA elements in the promoter region of the human cytomegalovirus major immediate early gene.EMBO J. 1986; 5: 1367-1371PubMed Google Scholar] or the mouse MCK promoter [11.Larochelle N. et al.Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.Gene Ther. 1997; 4: 465-472Crossref PubMed Scopus (43) Google Scholar] selectively active in muscle cells. After we injected the CMV-driven AGA construct (designated pX61) into tibialis anterior (TA) muscles of Fabry mice, AGA activity in injected muscles measured 1 week postinjection was significant (Fig. 1A ). Moreover, in animals with the highest muscle activity, we could detect AGA in their sera (7.2 ± 0.8 nmol/h/ml serum, n = 4) and livers (4.0 ± 1.8 nmol/h/mg protein, n = 8, compared to 0.8 ± 0.4 nmol/h/mg protein in uninjected mice, n = 2). This liver activity was equivalent to ∼30% of the values found in wild-type livers (13.1 ± 6.4 nmol/h/mg protein, n = 2) and therefore within levels shown previously to be therapeutic [2.Ohshima T. et al.Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 6423-6427Crossref PubMed Scopus (85) Google Scholar, 3.Ziegler R.J. et al.Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.Hum. Gene Ther. 1999; 10: 1667-1682Crossref PubMed Scopus (87) Google Scholar, 12.Abe A. et al.Reduction of globotriaosylceramide in Fabry disease mice by substrate deprivation.J. Clin. Invest. 2000; 105: 1563-1571Crossref PubMed Scopus (143) Google Scholar, 13.Takahashi H. et al.Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer.Proc. Natl. Acad. Sci. USA. 2002; 99: 13777-13782Crossref PubMed Scopus (82) Google Scholar, 14.Park J. et al.Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer.Proc. Natl. Acad. Sci. USA. 2003; 100: 3450-3454Crossref PubMed Scopus (73) Google Scholar]. This confirmed that the fully functional AGA enzyme had been expressed and secreted from the skeletal muscle into the bloodstream and subsequently been taken up and accumulated by liver cells. At the later time points AGA activity in injected muscles decreased by approximately 90% per week and at 2–3 weeks after plasmid injection AGA was largely undetectable (Fig. 1A). Escalated dose of plasmid did not produce increased expression at any time beyond 1 week postinjection (data not shown). The most likely explanation for this decline in extraneously expressed enzyme levels was the destruction of the AGA-expressing cells by immune responses. The knockout Fabry mice completely lack AGA; therefore, the expressed enzyme could act as a neoantigen in these animals. Indeed, the analysis of the anti-human AGA antibody levels in the sera of pX61-injected mice at different time points showed that the treatment generated strong antibody responses against AGA within 2 weeks after injection (Fig. 2). Moreover, immunohistochemical analysis of muscles 2 weeks postinjection showed massive infiltrates of cytotoxic CD8+ T lymphocytes (CTLs) (Fig. 3B ) and macrophages (Fig. 3H). These findings show that in addition to the humoral response, a cellular reaction against AGA was present. The maximum of the humoral and cell-mediated responses coincided with the decline in AGA activity. The cellular effector mechanism was quickly moderated; no significant infiltrates were found at 5 weeks postinjection (Figs. 3D and 3J), while the levels of anti-AGA antibodies remained elevated significantly longer (Fig. 2). A short course of immunosuppression with cyclophosphamide (CY) prevented the decline in AGA activity in injected muscles at 3 weeks postinjection (Fig. 1B), further confirming that immune responses were responsible for this effect.Fig. 3Analysis of CD8+ lymphocyte and macrophage infiltrates following injection of plasmid vectors into Fabry mouse muscles. Plasmid vectors (A, C, E, G, I, K) pX61MCK and (B, D, F, H, J, L) pX61 were injected into TA muscles of 4-week-old Fabry mice and muscle sections were taken 2 weeks postinjection. (A–F) Immunolocalization of CD8+ T cells. Red fluorescence signal corresponds to CD8+ expression on cytotoxic T lymphocytes infiltrating injected muscle; nuclear counterstaining (YOPRO-1) is shown in green. (I–N) Immunolocalization of macrophages. Purple staining denotes macrophage-specific staining; nuclear counterstaining (methyl green) is in blue-green. (G, H, O, and P) Serum-only negative controls. Note the significant number of CD8+ cells and macrophages infiltrating muscle fibers injected with pX61 at 2 weeks postinjection compared to no staining in muscle injected with pX61MCK at any time point or with pX61 at 5 weeks postinjection. For detection of CD8+ T cells rat anti-mouse CD8 antibody (Serotec; YTS 169.4) was used (1/500 in 10% goat serum) with chicken anti-rat IgG conjugated with Alexa Fluor 647 (1/200 in 2% goat serum) secondary detection and YOPRO-1 (both from Molecular Probes) cell nucleus counterstaining. Samples were analyzed using an LSM 510 confocal microscope (Zeiss), with excitation at 650 and 488 nm and emission at 668 and 510 nm for Alexa and YOPRO-1, respectively. For detection of macrophages, rat anti-mouse F4/80 biotinylated antibody (Serotec; MCA 497B) was used (1/100 in 10% goat serum) with immunohistochemical visualization (VECTAstain and VIP kits; Vector Laboratories). In both cases negative controls were incubated with 10% goat serum only.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The observed rapid decrease in AGA levels could be explained by the widespread expression pattern triggered by the CMV promoter used in pX61 vector. Targeting of pDNA and direct transgene expression in professional APCs is known to increase antigen presentation [15.Sbai H. Schneider J. Hill A.V. Whalen R.G. Role of transfection in the priming of cytotoxic T-cells by DNA-mediated immunization.Vaccine. 2002; 20: 3137-3147Crossref PubMed Scopus (23) Google Scholar]. It appears that the ubiquitously active CMV promoter triggered AGA transgene expression in antigen-presenting cells (APC) located in the area surrounding the injection site. APCs, both dendritic cells (CD11c+) and macrophages (CD11b+), are abundant in mouse muscle between 2 and 7 weeks of age [16.Hartigan-O'Connor D. Kirk C.J. Crawford R. Mule J.J. Chamberlain J.S. Immune evasion by muscle-specific gene expression in dystrophic muscle.Mol. Ther. 2001; 4: 525-533Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar] (plasmid injection was performed at 4 weeks). In addition, recruitment of CTLs was more likely to be initiated when expression of AGA was targeted to APCs [17.Kutzler M.A. Weiner D.B. Developing DNA vaccines that call to dendritic cells.J. Clin. Invest. 2004; 114: 1241-1244Crossref PubMed Scopus (98) Google Scholar]. In an attempt to prolong the expression of AGA, we modified the pX61 vector by replacing the CMV promoter with a short version (1.35 kb) of the MCK promoter (vector designated pX61MCK) to restrict the expression to muscle cells [11.Larochelle N. et al.Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.Gene Ther. 1997; 4: 465-472Crossref PubMed Scopus (43) Google Scholar]. Indeed, we found that at 2–3 weeks postinjection the levels of AGA were still significantly high in pX61MCK-injected muscles and there was no significant decrease in enzyme activity after this critical (2–3 weeks) period (Fig. 1A). From 2 to 5 weeks postinjection, the AGA activity detected in muscles injected with pX61MCK was always significantly higher than for pX61 vector (Fig. 1A) (P ≤ 0.006 for 3 and 5 weeks). When we observed a decrease in AGA activity it was significantly later than with pX61 (between 3 and 5 weeks postinjection) (Fig. 1A). It was also associated with the start of the humoral response against AGA; however, the titer of anti-AGA antibodies was significantly lower and the onset of production delayed (P = 0.005) (Fig. 2). Moreover, in a clear contrast to the massive infiltration observed 2 weeks after injection of pX61 (see above) immunohistochemical analysis showed no significant infiltrates of CTLs (Figs. 3A and 3C) or macrophages (Figs. 3G and 3I) at any time postinjection of pX61MCK plasmid within the period studied here. It turned out that the MCK-driven expression of AGA, in keeping with the MCK inability to target antigen expression to APCs, resulted in delayed induction of immune responses against this protein. In addition, it was limited to the humoral response only, the type that is far less destructive for therapeutic applications. It has been shown previously that antibodies raised against the transgene have often not caused any significant problems in terms of safety or stability of expression [18.Schiffmann R. et al.Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease.Proc. Natl. Acad. Sci. USA. 2000; 97: 365-370Crossref PubMed Scopus (327) Google Scholar]. Although our finding of immunization even with the MCK-driven transgene is contrary to some other data [19.Bojak A. Hammer D. Wolf H. Wagner R. Muscle specific versus ubiquitous expression of Gag based HIV-1 DNA vaccines: a comparative analysis.Vaccine. 2002; 20: 1975-1979Crossref PubMed Scopus (20) Google Scholar], it is compatible with the known mechanisms of antigen recognition by the immune system. AGA secreted from skeletal muscle could be taken up, processed, and presented by professional APCs and ultimately trigger the effector phase of the response [20.Villadangos J.A. Ploegh H.L. Proteolysis in MHC class II antigen presentation: who's in charge?.Immunity. 2000; 12: 233-239Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar]. Our data suggested that plasmid vectors could be effective in targeting the AGA gene into skeletal muscle. However, two problems became apparent: first, expressed AGA was immunogenic in the knockout mice. Nevertheless, in a clinical setting, immunogenicity of AGA may pose a lesser problem as a significant proportion of patients have residual enzymatic activity or synthesize nonfunctional enzyme, which would decrease the risk of immunization [5.Ioannou Y.A. Zeidner K.M. Gordon R.E. Desnick R.J. Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice.Am. J. Hum. Genet. 2001; 68: 14-25Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6.Eng C.M. et al.Safety and efficacy of recombinant human alpha-galactosidase A—replacement therapy in Fabry's disease.N. Engl. J. Med. 2001; 345: 9-16Crossref PubMed Scopus (1352) Google Scholar, 18.Schiffmann R. et al.Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease.Proc. Natl. Acad. Sci. USA. 2000; 97: 365-370Crossref PubMed Scopus (327) Google Scholar, 21.Yasuda M. Shabbeer J. Osawa M. Desnick R.J. Fabry disease: novel alpha-galactosidase A 3′-terminal mutations result in multiple transcripts due to aberrant 3′-end formation.Am. J. Hum. Genet. 2003; 73: 162-173Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 22.Okumiya T. Ishii S. Kase R. Kamei S. Sakuraba H. Suzuki Y. Alpha-galactosidase gene mutations in Fabry disease: heterogeneous expressions of mutant enzyme proteins.Hum. Genet. 1995; 95: 557-561Crossref PubMed Scopus (57) Google Scholar]. The second problem (to some extent linked with the previous one) was the insufficiently high level and short duration of AGA expression. Pluronic block copolymer (SP1017) has been shown to be a particularly good enhancer for naked pDNA injected into muscle [8.Lemieux P. et al.A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle.Gene Ther. 2000; 7: 986-991Crossref PubMed Scopus (197) Google Scholar]. Therefore we formulated plasmid constructs pX61 and pX61MCK with SP1017 and injected them into TA muscles of Fabry mice, as before. The AGA activity measured 1 week postinjection of pX61 was significantly greater (five fold; P = 0.004) when pDNA was combined with SP1017 (Fig. 1C). Addition of SP1017 did not prevent the drop in AGA activity (P = 0.000) observed 3 weeks after injection of this construct (Fig. 1C). Analyses of anti-AGA antibody levels and muscle infiltrates (data not shown) confirmed that, like for the naked plasmid, immune responses were responsible for this decrease. Therefore SP1017 can significantly increase gene expression following intramuscular injection; however, it has no effect on the time of onset or the intensity of resulting immune reactions against the transgene acting as neoantigen. Pluronic block copolymer does not appear to alter the targeting and expression patterns of CMV-driven plasmids. Surprisingly, in stark contrast to the strong enhancing effect of SP1017 on pX61, there was no increase in the levels of AGA expression at any time postinjection when we used the MCK-driven construct (Fig. 1C). This was not caused by increased immune reactions following gene targeting with SP1017: like with the naked pX61MCK, the antibody responses in SP1017/pX61MCK-injected animals were very low and no significant cellular infiltrates in injected muscle were found (data not shown). This raises an interesting question regarding the mechanisms by which Pluronic SP1017 enhances gene expression. SP1017 does not condense pDNA (data not shown) and it is not even clear if it forms complexes with DNA. It has been shown before that SP1017 significantly augmented pDNA distribution throughout the injected muscle and may increase its bioavailability [23.Alakhov V. Klinski E. Lemieux P. Pietrzynski G. Kabanov A.V. Block copolymeric biotransport carriers as versatile vehicles for drug delivery.Expert Opin. Biol. Ther. 2001; 1: 583-602Crossref PubMed Scopus (59) Google Scholar]. It was also suggested that this polymer might increase cellular uptake of plasmids, increase membrane permeability, and promote escape from endosomes [8.Lemieux P. et al.A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle.Gene Ther. 2000; 7: 986-991Crossref PubMed Scopus (197) Google Scholar]. It emerges from our data that the effects of Pluronics depend strongly on the type of promoter used; only plasmids driven by the ubiquitous CMV promoter were responsive to SP1017 enhancement, while the plasmid driven by the muscle-specific promoter was not. This suggests that SP1017 may act as an exogenous, synthetic response modifier/enhancer of CMV promoter-driven expression. As suggested by some earlier data [24.Kabanov A.V. Batrakova E.V. Sriadibhatla S. Yang Z. Kelly D.L. Alakhov V. Polymer genomics: shifting the gene and drug delivery paradigms.J. Controlled Release. 2005; 101: 257-259Crossref Scopus (94) Google Scholar] it appears that NFκB binding sites (present in the CMV promoter) could be involved in trans-activation of the transcription by SP1017. While, in the light of stronger immunogenicity of transgenes expressed under the CMV promoter, this finding could be exploited for the development of DNA vaccines, it is not useful in applications requiring long-term transgene expression. It is of importance to investigate whether other muscle-specific promoters responsive to amphiphilic polymer enhancement could be identified or engineered. It is also interesting to study the mechanism of such a specific enhancement by SP1017, as it could find other applications in regulation of gene expression. Finally, our data indicate that any analyses of polymeric vectors/enhancers need to take into consideration the possibility of similar promoter-dependent effects. Concluding, Pluronic block copolymer SP1017 enhancement of plasmid-derived gene expression shows a striking promoter specificity. Identification of an appropriate combination of muscle-specific regulatory elements and polymeric enhancers could lead to strong and stable expression and secretion of functional AGA from skeletal muscle. Compared to viral approaches, effective plasmid gene targeting into muscle could be safer, simpler to produce, and less expensive to administer. We confirmed that the AGA transgene could have different immunostimulatory potentials depending on the vector, promoter, and route of administration used. Muscle-specific regulatory elements did not eliminate the risk of immunization entirely, but did allow for longer expression. Restriction of the transcription of a therapeutic gene to a particular cell type is also of importance for safety reasons, as this allows a better control of the expression and limits the toxicity. This work was supported in part by grants from the Fabry Support Group, UK; the Wellcome Trust; and the Deutsche Forschungsgemeinschaft and the Duchenne Parents Project of Germany (Action Benni & Co.) to H.L. H.L. is a member of the German Network on Muscular Dystrophies (01GM0302) funded by the German Ministry of Education and Research (Bonn, Germany). M.D.L. receives a Ph.D. studentship from the University of Portsmouth. The authors thank Ashok Kulkarni, National Institute of Dental and Craniofacial Research, National Institutes of Health (Bethesda, MD, USA) for a colony of Fabry mice; Jeanette Beveridge for technical assistance; and Chun-Fu Lien for helpful suggestions and C. Alexander for critical comments on the manuscript.