Human insulin production and amelioration of diabetes in mice by electrotransfer-enhanced plasmid DNA gene transfer to the skeletal muscle

Martinenghi, Sabina; Angelis, Maria Gabriella Cusella De; Biressi, Stefano; Amadio, Stefano; Bifari, Francesco; Roncarolo, Maria‐Grazia; Bordignon, Claudio; Falqui, Luca

doi:10.1038/sj.gt.3301804

Cited by 36 publications

(25 citation statements)

References 44 publications

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“…Tibialis anterior and quadriceps muscles were electroporated as previously described with minor modifications (Protocol 2, Martinenghi et al 2002). Briefly, mice were anesthetized by avertin injection, and expression vectors were injected with a 0.5-m insulin syringe through a 27-gauge needle into quadriceps and tibialis anterior muscles of 9-d CD1 male mice (20 µg of each plasmid in a costant volume of 20 µL of PBS) and into tibialis anterior muscles of 2-mo-old CD1 female mice (50 µg of each plasmid in 50 µL of PBS).…”

Section: In Vivo Electroporationmentioning

confidence: 99%

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle

Berghella¹,

Angelis²,

Buysscher³

et al. 2008

Genes Dev.

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Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate.[Keywords: CsdA; MSY-3; myogenin; Y-box; innervation; muscle maturation] Supplemental material is available at http://www.genesdev.org.

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Section: In Vivo Electroporationmentioning

confidence: 99%

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle

Berghella¹,

Angelis²,

Buysscher³

et al. 2008

Genes Dev.

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“…We and others have demonstrated constitutive secretion of fully processed human insulin for up to 8 weeks following intramuscular injection of a preproinsulin plasmid mutated at PC2 and PC3 cleavage sites to form tetrabasic consensus recognition sites for the ubiquitous trans-Golgi network protease, furin (Martinenghi et al 2002. Glucoselowering without dangerous hypoglycaemia has been confirmed in normal and diabetic rodents.…”

Section: Introductionmentioning

confidence: 91%

Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle

Wilson

Scougall²,

Ratanamart³

et al. 2005

Journal of Molecular Endocrinology

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Long-term secretion of insulin by host muscle following transduction with an insulin gene construct offers the potential of gene therapy for diabetes without immunosuppression. Clinical implementation will be dependent on proof of principle in human tissue and a system for safely regulating basal insulin levels. Liposomal co-transfection with a tetracycline-responsive wild type human preproinsulin (pTRE-hppI1) or mutant construct (pTRE-hppI4), in which PC2 and PC3 cleavage sites were altered to form tetrabasic consensus sites for furin, together with pTet-off (coding for a transactivating protein) was evaluated in the C2C12 mouse myoblast cell line and human myoblasts following establishment in primary culture. In the absence of tetracycline, (pro)insulin secretion in C2C12 and human myoblasts transfected with tetracycline-responsive hppI1 and hppI4 constructs was comparable to that following transfection with equivalent constructs under the control of a constitutively active cytomegaloviral promoter. Percentage processing to mature insulin was ,5% in C2C12 and human myoblasts transfected with pTet-off/pTRE-hppI1 but .90% in C2C12 cells and 45-60% in human myoblasts on transfection with pTet-off/pTRE-hppI4. Incremental dose-responsive suppression of proinsulin secretion was demonstrated in C2C12 and human myoblasts expressing pTet-off/pTREhppI1 following incubation with tetracycline (0-100 µg/ml) for up to 72 h. Reversibility was confirmed following tetracycline withdrawal. Dose-responsive tetracycline-inducible repression of mature insulin secretion was confirmed in C2C12 cells following transfection with pTet-off/pTRE-hppI4. Regulation of human proinsulin biosynthesis and secretion has been attained in vivo following plasmid-mediated gene transfer to rat skeletal muscle and oral tetracycline administration. In conclusion, processing to mature insulin has been confirmed following plasmid-mediated gene transfer to human muscle in addition to in vitro-and in vivo-regulated human proinsulin secretion employing the safe and well-tolerated antibiotic, tetracycline.

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“…and electroporated in the mouse streptozotocin (STZ)-induced diabetes model resulted in animal survival for the course of the 10-week experiment, while diabetic control mice died after 3 weeks. 10 The vaccination approach has been effective in the generation of selfantibodies against plasmid-encoded chemokine antigens (including MCP-1, MIP-1a, and RANTES) with therapeutic effect in experimental adjuvant arthritis; 11 in similar studies in an experimental autoimmune encephalomyelitis (EAE) model, the encoded antigen was the chemokine IP-10. 12 In NZB/NZW F1 lupus-prone mice, vaccination with plasmid DNA encoding MHC class I epitopes of the heavy chain variable region of anti-DNA antibodies induced cytotoxic T lymphocytes (CTL) that killed autoreactive B cells.…”

Section: Naked Dnamentioning

confidence: 99%

“…180 Other in vivo studies have also examined systems in muscle. 10,22 Transcriptionally regulated systems are characterized by a significant time lag (approximately 6 h) between induction of gene expression and protein synthesis. In comparison, secretion of endogenous insulin from b islet cells occurs within approximately 20 min after glucose uptake.…”

Section: Targeting Immune Cellsmentioning

confidence: 99%

Vectors for the treatment of autoimmune disease

Gould

Favorov

2003

Gene Ther

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Human insulin production and amelioration of diabetes in mice by electrotransfer-enhanced plasmid DNA gene transfer to the skeletal muscle

Cited by 36 publications

References 44 publications

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle

Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle

Vectors for the treatment of autoimmune disease

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