HSV-1 amplicon vectors are a highly efficient gene delivery  system for skeletal muscle myoblasts and myotubes

Wang, Y.; Fraefel, Cornel; Protasi, Feliciano; Moore, R. A.; Fessenden, James D.; Pessah, Isaac N.; DiFrancesco, A.; Breakefield, Xandra O.; Allen, Paul D.

doi:10.1152/ajpcell.2000.278.3.c619

Cited by 60 publications

(56 citation statements)

References 23 publications

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“…The mutant E4032A RyR1 cDNA was introduced into 1B5 dyspedic myotubes by using the p-HSV amplicon system (21). 1B5 myotubes efficiently express E4032A RyR1 (which is the expected size by Western blot analysis; Fig.…”

Section: Resultsmentioning

confidence: 99%

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

Fessenden

Chen

Wang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca 2؉ release from endoplasmic reticulum͞sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high-and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null͞dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200 -500 M ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 s) gating events and insensitivity to Ca 2؉ . Addition of ryanodine restores Ca 2؉ -dependent channel activity exhibiting full, 3͞4, 1͞2, and 1͞4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

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Section: Resultsmentioning

confidence: 99%

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

Fessenden

Chen

Wang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Several genetically modified viruses, such as retrovirus, herpes simplex virus, Epstein-Barr virus, adenovirus (AdV) and adeno-associated virus (AAV), have all been tested for gene delivery into muscle. [2][3][4][5] Of these, AdV and AAV have been found to be most efficient so far for transducing muscle fibres. [6][7][8] AAV in particular is able to transfect non-proliferating muscle fibres and is considered to be non-pathogenic, as it is not found to be associated with any disease.…”

Section: Introductionmentioning

confidence: 99%

Non-viral gene delivery in skeletal muscle: a protein factory

Lu¹,

Bou‐Gharios²,

Partridge³

2003

Gene Ther

170

109

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Ever since the publication of the first reports in 1990 using skeletal muscle as a direct target for expressing foreign transgenes, an avalanche of papers has identified a variety of proteins that can be synthesized and correctly processed by skeletal muscle. The impetus to the development of such applications is not only amelioration of muscle diseases, but also a range of therapeutic applications, from immunization to delivery of therapeutic proteins, such as clotting factors and hormones. Although the most efficient way of introducing transgenes into muscle fibres has been by a variety of recombinant viral vectors, there are potential benefits in the use of non-viral vectors. In this review we assess the recent advances in construction and delivery of naked plasmid DNA to skeletal muscle and highlight the options available for further improvements to raise efficiency to therapeutic levels.

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“…Amplified fragments from WT RyR1 encoding aa 1681-2217 (Ch-17), 1924-2446 (Ch-21) and 1681-3770 (Ch-4) were inserted, in frame, into the endogenous restriction site(s) of HSV-RyR3 plasmid as described previously [20]. All chimeric constructs were cloned into the HSV-1 amplicon vector and packaged using a helper virus-free packaging system [22]. …”

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confidence: 99%

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Voss

Allen

Pessah

et al. 2008

Biochemical and Biophysical Research Communications

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We studied cation regulation of wild type ryanodine receptor type 1 ( WT RyR1), type 3 ( WT RyR3) and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [ 3 H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca 2+ titrations with WT RyR3 and three chimeras show biphasic activation that is allosterically coupled to attenuated inhibition relative to WT RyR1. Chimeras show biphasic Mg 2+ inhibition profiles at 3 and 10μM Ca 2+ , no observable inhibition at 20μM Ca 2+ and monophasic inhibition at 100μM Ca 2+ . Ca 2+ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca 2+ transients with inhibition kinetics that are significantly slower than those expressing WT RyR1 or WT RyR3. Four new aspects of RyR regulation are evident: 1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, 2) Ca 2+ facilitates removal of the inherent Mg 2+ block, 3) WT RyR3 exhibits reduced cooperativity between H activation sites when compared to WT RyR1, and 4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca transients.Keywords ryanodine receptors; calcium-induced calcium release; channel regulation Three isoforms of wild type ryanodine receptors ( WT RyR1, 2 and 3) are expressed in specialized regions of endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells where they function as Ca 2+ Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and Methods Chimeric RyR1/RyR3 constructsSpecific primers were designed for PCR amplification of the selected fragments using WT RyR1 as a template. Amplified fragments from WT RyR1 encoding aa 1681-2217 (Ch-17), 1924-2446 (Ch-21) and 1681-3770 (Ch-4) were inserted, in frame, into the endogenous restriction site(s) of HSV-RyR3 plasmid as described previously [20]. All chimeric constructs were cloned into the HSV-1 amplicon vector and packaged using a helper virus-free packaging system [22]. Cell culture, infection and membrane preparation1B5 myoblasts were cultured and differentiated into myotubes as described previously [20] and [23]. Plates with differentiated myotubes were infected with virion containing wild type and RyR1/RyR3 chimeric cDNA for 2 h and membrane extracts were prepared 36 h after infection. Myotubes were homogenized and membrane fractions obtained by differential centrifugation as described previously [24].[ cold buffer, placed into 5 ml scintillation cocktail (ScintiVerse; Fisher Scientific) and radioactivity counted. Equations for binding analysisCurve fitting was ...

show abstract

HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes

Cited by 60 publications

References 23 publications

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

Non-viral gene delivery in skeletal muscle: a protein factory

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

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