[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans.
[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. AO-induced exon skipping producing functional truncated dystrophin exon has been demonstrated in animal models of DMD both in vitro and in vivo, and in DMD patient cells in vitro in culture, and in DMD muscle explants. More recently, AO-mediated exon skipping has been confirmed in DMD patients in Phase I clinical trials. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.
[Show abstract][Hide abstract] ABSTRACT: Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder.
We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250.
Two patients received 0.09 mg AVI-4658 in 900 microL (0.9%) saline and five patients received 0.9 mg AVI-4658 in 900 microL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44-79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26.4%), and the mean intensity was 17% (range 11-21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0.002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot.
Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD.
UK Department of Health.
The Lancet Neurology 09/2009; 8(10):918-28. DOI:10.1016/S1474-4422(09)70211-X · 21.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear:cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice.
[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) is a myodegenerative disorder caused primarily by mutations that create premature termination of dystrophin translation. The antisense oligonucleotide approach for skipping dystrophin exons allows restoration of the correct reading frame in the dystrophin transcript, thus producing a shorter protein. A similar approach in humans would result in the conversion of DMD to the milder Becker muscular dystrophy. It has been demonstrated previously that repeated intravascular injection of phosphorodiamidate morpholino oligomers (PMOs) in the mdx mouse induces more dystrophin expression than a single injection, but this approach is costly, and data demonstrating the safety of high doses of systemically injected PMO are unavailable. Furthermore, several publications have demonstrated the efficacy of peptide-conjugated PMOs, but the clinical applicability of such compounds is unclear at this stage. Here, we report that multiple intravascular injections of low doses of naked PMO show significantly more dystrophin-positive fibers in a variety of muscle groups, 8 weeks after administration compared with a single dose of the same total amount. After administration of a total of 200 mg of PMO per kilogram, histological features, such as the cross-sectional area, centronucleation index, and expression of the dystrophin-associated protein complex, showed significant improvement in mice treated by repeated injection. Furthermore, four administrations of just 5 mg/kg induced a significant amount of dystrophin expression. These results clearly demonstrate the key role of the optimization of dosing regimen for the systemic administration of PMO in patients, and support the clinical feasibility of this approach with naked PMO.
Human gene therapy 06/2009; 20(9):955-65. DOI:10.1089/hum.2008.157 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myostatin is a member of the transformating growth factor-beta (TGF-beta) superfamily of proteins and is produced almost exclusively in skeletal muscle tissue, where it is secreted and circulates as a serum protein. Myostatin acts as a negative regulator of muscle mass through the canonical SMAD2/3/4 signaling pathway. Naturally occurring myostatin mutants exhibit a 'double muscling' phenotype in which muscle mass is dramatically increased as a result of both hypertrophy and hyperplasia. Myostatin is naturally inhibited by its own propeptide; therefore, we assessed the impact of adeno-associated virus-8 (AAV8) myostatin propeptide vectors when systemically introduced in MF-1 mice. We noted a significant systemic increase in muscle mass in both slow and fast muscle phenotypes, with no evidence of hyperplasia; however, the nuclei-to- cytoplasm ratio in all myofiber types was significantly reduced. An increase in muscle mass in slow (soleus) muscle led to an increase in force output; however, an increase in fast (extensor digitorum longus [EDL]) muscle mass did not increase force output. These results suggest that the use of gene therapeutic regimens of myostatin inhibition for age-related or disease-related muscle loss may have muscle-specific effects.
Rejuvenation Research 05/2009; 12(2):85-94. DOI:10.1089/rej.2008.0815 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations of the human DMD gene. Antisense oligonucleotides (AOs) have previously been used to skip additional exons that border the deletions such that the reading frame is restored and internally truncated, but functional, dystrophin expressed. We have designed phosphorodiamidate morpholino oligomer (PMO) AOs to various exons of the human dystrophin gene. PMOs were designed to have their target sites overlapping areas of open RNA structure, as defined by hybridization-array analysis, and likely exonic splicing enhancer (ESE)/silencer sites on the target RNA. The ability of each PMO to produce exon skipping was tested in vitro in normal human skeletal muscle cells. Retrospective analysis of design parameters used and PMO variables revealed that active PMOs were longer, bound to their targets more strongly, had their target sites closer to the acceptor splice site of the exon, overlapped areas of open conformation (as defined by the hybridization or the RNA secondary structure prediction software), and could interfere with the binding of certain SR proteins. No other parameter appeared to show significant association to PMO-skipping efficacy. No design tool is strong enough in isolation; however, if used in conjunction with other significant parameters it can aid AO design.
[Show abstract][Hide abstract] ABSTRACT: To improve liver-directed retroviral-mediated gene transfer, we injected C57/BL10 mice intravenously with three adenoviral vectors encoding retroviral vector genome and structural components: AdGagPol expressing the respective structural genes of Moloney murine leukaemia virus, Ad10A1Env expressing the 10A1 envelope protein of 10A1-MuLV, and AdLEIN, encoding the LEIN retrovirus genome, expressing green fluorescence protein (eGFP) and the neomycin resistance gene.
The extent of eGFP expression was determined after 1 and 15 weeks by fluorescence microscopy and FACS analysis. Proviral integration was determined by a novel PCR-based technique.
Hepatocytes infected with all three Ad vectors generated LEIN retrovirus after one week and in situ transduction of neighbouring cells resulted in stable proviral integration associated with eGFP expression ranging from 4.3% to 20.5% in different liver cell populations 15 weeks post-infection.
Hybrid adeno-retroviral vectors can be efficiently used to improve the efficiency of retroviral-mediated gene transfer to the liver.
In vivo (Athens, Greece) 01/2009; 23(6):885-93. · 1.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.
Human gene therapy 10/2008; 19(10):1000-8. DOI:10.1089/hum.2008.063 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.