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MAR-Mediated Dystrophin Expression in Mesoangioblasts for Duchenne Muscular Dystrophy Cell Therapy

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A cornerstone of autologous cell therapy for Duchenne muscular dystrophy is the engineering of suitable cells to express dystrophin in a stable fashion upon differentiation to muscle fibers. Most viral transduction methods are typically restricted to the expression of truncated recombinant dystrophin derivatives and by the risk of insertional mutagenesis, while non-viral vectors often suffer from inefficient transfer, expression and/or silencing. Here we addressed such limitations by using plasmid vectors containing nuclear matrix attachment regions (MAR). Using in vitro transfection and intra muscular transplantation in nude and immunosuppressed mdx mice, we show that clones of mesoangioblast skeletal muscle progenitors can be generated to mediate stable expression from MAR-containing vectors, while maintaining their ability to differentiate in vitro and in vivo and to express dystrophin after transplantation in dystrophic mouse muscles. We conclude that the incorporation of MARs into plasmid vectors may improve non-viral plasmid-based cell therapy feasibility.
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Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
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ISSN: 2168-9547
Molecular Biology
van Zwieten et al., Mol Biol 2015, 4:3
http://dx.doi.org/10.4172/2168-9547.1000134
Research Article Open Access
MAR-Mediated Dystrophin Expression in Mesoangioblasts for Duchenne
Muscular Dystrophy Cell Therapy
Ruthger W van Zwieten1, Stefano Majocchi1, Pavithra Iyer1, Yves Dussere1, Stefania Puttini1, Francesco Saverio Tedesco2, Giulio Cossu3
and Nicolas Mermod1*
1Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL and Institute of Biotechnology, University of Lausanne, 1015 Lausanne, Switzerland
2Department of Cell and Developmental Biology, University College London, WC1E 6DE London, UK
3Institute of Inammation and Repair, University of Manchester M13 9PL Manchester, UK
Abstract
A cornerstone of autologous cell therapy for Duchenne muscular dystrophy is the engineering of suitable cells
to express dystrophin in a stable fashion upon differentiation to muscle bers. Most viral transduction methods are
typically restricted to the expression of truncated recombinant dystrophin derivatives and by the risk of insertional
mutagenesis, while non-viral vectors often suffer from inefcient transfer, expression and/or silencing. Here we
addressed such limitations by using plasmid vectors containing nuclear matrix attachment regions (MAR). Using in
vitro transfection and intra muscular transplantation in nude and immunosuppressed mdx mice, we show that clones
of mesoangioblast skeletal muscle progenitors can be generated to mediate stable expression from MAR-containing
vectors, while maintaining their ability to differentiate in vitro and in vivo and to express dystrophin after transplantation
in dystrophic mouse muscles. We conclude that the incorporation of MARs into plasmid vectors may improve non-viral
plasmid-based cell therapy feasibility.
*Corresponding author: Nicolas Mermod, Laboratory for Molecular Biotechnology,
Station 6, EPFL, 1015 Lausanne, Switzerland, Tel: +41 21 693 6151; E-mail:
nicolas.mermod@unil.ch
Received September 09, 2015; Accepted September 16, 2015; Published
September 23, 2015
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015)
MAR-Mediated Dystrophin Expression in Mesoangioblasts for Duchenne Muscular
Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Copyright: © 2015 van Zwieten RW, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.
Keywords: Matrix attachment regions (MAR); Dystrophin;
Insertional mutagenesis; Duchenne muscular dystrophy
Introduction
Duchenne muscular dystrophy is an X-linked progressive muscular
wasting disease that aects skeletal and cardiac muscles, and for which
there is currently no cure. It is caused by mutations in the dystrophin
gene, resulting in the lack or reduction of the protein. is decit leads
to a disruption of the dystroglycan complex and destabilization of the
sarcolemma, resulting in progressive muscle wasting [1]. Promising
experimental approaches that aim to restore the dystrophin complex at
the sarcolemmal membrane include i) exon skipping by pharmacological
strategies, ii) systemic gene therapy and iii) cell therapy [2,3]. Gene
therapy approaches aim to engineer vectors that eciently transduce
myobers with a dystrophin expression cassette, whereas cell therapy
approaches aim to deliver the dystrophin transgene to the myober
by stem/progenitor cells, while preferably replenishing the satellite
cell pool with genetically corrected or complemented autologous
cells. In this study, we assessed a novel approach to express full-length
dystrophin in a cell therapy setting.
Besides myoblasts, multiple myogenic stem/progenitor cells were
described for potential use in cell therapy. Unlike myoblasts that
require high-density injection and are limited to supercial muscles [4],
mesoangioblasts can cross the blood vessel wall and home into damaged
muscle aer intra-arterial delivery [5], thus providing an approach
towards systemic cell therapy [6-9]. Mesoangioblasts were isolated
from mice, dogs and humans, and they maintain their dierentiation
potential upon in vitro culture. Although aneuploidy and transformation
may be observed upon prolonged culture of murine cells, human and
canine mesoangioblasts maintained a normal karyotype and did not
escape eventual senescence in long-term cultures [10,11].
e therapeutic potential of wild-type mesoangioblasts
injected intra-arterially was shown in α-sarcoglycan null mice
and dystrophic dogs, leading to dramatic improvement of muscle
morphology and function [9,10,12]. In contrast, the therapeutic
ecacy of transplantations with viral vector-transduced dystrophic
mesoangioblasts was modest. is may result in part from the
transcriptional silencing of the transgene by epigenetic eects [13].
In addition, limitations of typical viral vector cargo size necessitated
the development of shorter, Becker-like dystrophins, which may have
reduced therapeutic eects [14]. Despite signicant advances in viral
vector engineering, safety concerns remain regarding genotoxic eects
and potential malignant cell transformation, because of the tropism of
some viral vectors for cellular genes [15]. Use of non-viral vectors or
gene correction may be promising alternative approaches. However,
their eciency remains generally low, and gene correction is also
limited by the breadth of mutations that aect dystrophic patients [16].
erefore, expression of truncated dystrophin derivatives from viral
vectors is still mostly used in experimental cell therapy approaches.
Strategies that allow the introduction of a functional copy of the full-
length dystrophin coding sequence into autologous cells might thus be
benecial. Current eorts are thus increasingly focused on the isolation
of single clones and on the characterization of the genomic integration
locus to reduce the risk of adverse eects. Here, we investigated a
novel non-viral transgene introduction approach as an alternative to
viral vector transduction, assessing whether the stable transfection of
nuclear matrix attachment regions (MAR)-containing plasmids may
allow the engineering of primary muscle precursor cells. A recent
genome-mining eort yielded potent human MARs (hMARs) that
enhance and stabilize transgene expression in cultured cells as well as in
murine muscles [17]. hMAR elements were shown to promote plasmid
integration in the host cell genome by homologous recombination-
related mechanisms, to increase transgene transcription and to oppose
epigenetic silencing eects in cultured rodent and human cells [17-21].
In this study we evaluated plasmid vectors containing distinct MAR
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 2 of 8
elements to promote transgene expression in mesoangioblasts, in an
attempt to improve non-viral engineering of muscle progenitors for
cell therapy. We show that MARs increase stable transfection eciency
up to 10-fold. Clones generated with MAR-containing vectors retained
their ability to dierentiate in vitro and in vivo while sustaining
transgene expression. is indicates that transfection-based cell therapy
approaches may be improved with MAR element-containing non-viral
vectors.
Materials and Methods
Cell culture
MDX mesoangioblasts cultures were established and cultured in
Dulbecco’s modied eagle medium (DMEM, Gibco) containing 20%
FBS as described previously, and provided by G Cossu, Milan [22].
C2C12 mouse myoblasts were maintained in DMEM (Gibco) plus 10%
FBS. All culturing was done in a humidied 37°C/5% CO2 incubator.
Dierentiation was induced by the co-culture of C2C12 cells and
mesoangioblasts at a 1:4 ratio in DMEM 20% for 24h, aer which the
culture medium was changed to DMEM plus 2% horse serum. Myotube
formation was conrmed by immunouorescence for α-actinin using
a mouse polyclonal antibody (Sigma). eGFP expression levels of
dierentiated and undierentiated mesoangioblasts were recorded by
uorescence microscopy.
DNA constructs and transfection
e construction of MAR-containing eGFP expression vectors was
as previously described [17]. pMDA (full length mouse dystrophin
cDNA driven by the muscle creatine kinase promoter) was kindly
provided by JS Chamberlain [23]. Plasmids were amplied in DH5α
bacteria and puried using a plasmid maxiprep kit (Genomed).
Transfections were done with Lipofectamine 2000 (Invitrogen),
Fugene 6 (Roche), Fugene HD (Roche), while electroporations were
performed using the Nucleofector (Amaxa) or the Neon electroporator
(Invitrogen) following the manufacturers’ instructions. For equimolar
transfections of various eGFP-expressing constructs, pUC18 was added
to maintain an equal amount of total DNA. Cells were plated 27 h
before transfection to allow an appropriate timing of the transfection
with the cell cycle, as adapted from [17]. At time of transfection cell
conuency was 80%, and 4.5 µl of Fugene HD was added together with
2 µg of plasmid DNA. For stable clone isolation, the cells were placed
aer 48 h post transfection in DMEM supplemented with 20% FBS
and 2.5 µl/ml puromycin dihydrochloride (Sigma). Aer 20-30 days,
eGFP expressing clones were isolated by mechanical dislodging of the
colony with a sterile pipet tip. Transfection ecacy was measured by
uorescence acquisition for eGFP using a uorescence-activated cell
sorter (FACSCalibur, BD biosciences). 100,000 events were counted
per given cell population. Statistical analyses were performed with the
Student’s t test. e described lentivirally transduced mesoangioblast
population was generated as described previously [6,9].
Plasmid rescue for genomic integration locus DNA
sequencing
We extracted total genomic DNA from cells with the Blood and
Tissue kit (QIAGEN), following the manufacturer’s instructions. Aer
digestion of 2 µg of genomic DNA with a unique restriction site cutter,
BamHI (NEB), we took 1/10 of digested DNA and ligated with 15 µl
T4DNA ligase (NEB) in 500 µl at 16°C over-night. e ligation was
dialysed against water, precipitated and resuspended in 4 µl, of which
1 µl was used to transform 20 µl of electrocompetent DH10B cells
(Invitrogen).
In vivo transplantation assay
5 x 105 cells were suspended in 30 µl phosphate buered saline
(PBS) and injected intramuscularly in the Tibialis anterior of 5
wk old CD-1 nude (Charles river), C57Bl6 or mdx5cv mice using a
29G ‘Myjector’ syringe (Terumo), while the contralateral muscle
was injected with PBS alone. 3 mice were transplanted for each cell
clone. During the procedures, the mice were anaesthetized by intra-
peritoneal injection with xylazine/ketamine. Mice were sacriced by
cervical dislocation at day 9, day 40 or 3 months post injection, and
muscles were collected and frozen in liquid nitrogen cooled isopentane
(Sigma). All precautions were taken to reduce animal suering, and the
procedures were approved by the Service de la consommation et des
Aaires vétérinaires of the Canton of Vaud (Lausanne, Switzerland).
Immunouorescence for eGFP was performed using a rabbit polyclonal
antibody (Invitrogen) on paraformaldehyde 4% xed sections.
Dystrophin staining was performed on non-xed 10 µm TA sections
using mouse monoclonal antibodies NCL dys1 and NCL dys2 from
Novocastra (Leica).
Fluorescence in situ hybridization
Mesangioblast clones were exposed for 2 hours to colcemid
(Invitrogen) to block cell division in metaphase. Aer harvesting,
cells were exposed to a hypotonic shock with 37.5 mM KCl for 20
minutes, xed with 25% acetic acid and 75% methanol and spread
onto superfrost microscope slides. Hybridization probes were prepared
using a nick translation DNA labeling system (Enzo Life Sciences) and
Orange 552 dUTP (Enzo Life Sciences) according to the manufacturer’s
instructions. e probe targeting eGFP was generated from the eGFP
expressing vector devoid of MAR whereas the dystrophin probe was
derived from the pMDA vector. Precipitated probes were resuspended
in hybridization buer (2x SSC, 50% formamide, 10% Dextran Sulfate),
denaturated for 10 min at 75°C, cooled down on ice and nally pre-
warmed at 37°C. Before applying the probes, slides were washed in PBS,
denaturated in denaturation buer (2x SSC, 70% formamide at 75°C),
dehydrated through ethanol series performed at room temperature
(70%, 85%, 100%) and air-dried. Hybridization occurred overnight at
37°C. Slides were rst washed for 90 seconds with 0.4x SSC and 0.3%
NP-40 at 72°C followed by a 1 minute wash in 2x SSC and 0.1% NP-40
at room temperature. Metaphases were counterstained with Vectashield
Mounting Medium with DAPI (Vector Labs) and observed using a
100X oil immersion objective on an Axio Vert Inverted microscope
(Carl Zeiss).
Imaging
Microphotographs of eGFP autouorescence, DAPI and secondary
antibodies conjugated with Alexa uor 546 were made with an Observer
A.1 equipped with an Axiocam (Zeiss) using the Axiovision soware.
Results
Optimizing transfection of primary murine mesoangioblasts
A panel of human and animal MAR elements was tested for their
eect on the establishment of stable cell clones and for transgene
expression level and stability using murine mesoangioblasts (Table 1).
e MAR elements were inserted upstream of the SV40 promoter and of
the eGFP coding sequence. As primary progenitor cells are oen dicult
to transfect, we rst tested several transfection reagents and assessed
the transient eGFP uorescence levels in primary mesoangioblasts that
were obtained from the commonly used mdx-5Cv mouse model for
Duchenne muscular dystrophy. Transient transfection with Fugene HD
was most ecient, with 14.5% and 9.3% of eGFP positive cells with the
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 3 of 8
MAR-devoid and hMAR X-29-containing plasmids, respectively, while
other reagents yielded lower transfection ecacies, as measured by
FACS for GFP autouorescence 48 h aer transfection. As transfections
were carried out with equimolar amounts of plasmid, the lower eGFP
obtained from the hMAR X-29 MAR construct likely resulted from
the larger plasmid size and thereby reduced transfection ecacy.
Electroporation also proved to be a very ecient transient transfection
method, yielding up to 20% eGFP expressing cells aer 48 h (Figure 1).
Transfection of primary murine mesoangioblasts by various
approaches
Transfection was done according to manufacturers instructions
using optimized amounts of DNA. e percentage of eGFP positive cells
was recorded 48h aer transfection by FACS cytouorometry (A) or
uorescence microscopy (representative elds shown, panel B). Scale bars
indicate 200 µm. FACS proles of eGFP expression obtained from the
hMAR X-29-containing vector of from a control construct devoid of any
insert (no MAR), as determined 48 h aer transfection with lipofectamine
2000 (C) or Fugene HD (D). Cells were transfected with equimolar
amounts of either the no MAR eGFP or the hMAR X-29 eGFP plasmid.
Eect of MARs on stable transfection ecacy of primary
murine mesoangioblasts
e MAR-eGFP or control vectors were then co-transfected with
a plasmid bearing the puromycin-resistance gene and puromycin was
added to the transfected cell pools for up to 30 days. is selection
period allowed for the formation of colonies of antibiotic resistant cells
of sucient size to be picked manually and individually. Despite the
low initial number of eGFP positive cells at 48 h, the vector containing
hMAR X-29 showed a statistically signicant 10-fold increase of stable
eGFP-expressing clones as compared to the MAR devoid construct,
and up to a ~ 60x increase when compared to the vector whose MAR
element was substituted by a genomic fragment of comparable size
but without MAR activity (Figure 2A). Overall, 1.8 x 105 transfected
cells yielded on average 17 eGFP-expressing clones when using hMAR
X-29. While electroporation yielded the highest ecacy of transient
eGFP expression, the subsequent selection of cells mediating sustained
transgene expression under antibiotic selection yielded no stable clones
from the two electroporation devices tested (Figure 1A). erefore,
subsequent transfections performed to generate stable clones relied on
the Fugene HD reagent.
With hMAR X-29, 82 ± 17 % of the obtained clones expressed
eGFP, which was signicantly higher than the 26 ± 14 % of expressing
clones obtained with the MAR-devoid construct (p<0.05, student’s
t-test), and the 11 ± 19% recovered for the construct containing
a non-MAR control genomic fragment (p<0.01; See S1 Table for a
summary of all clones). hMAR 1-68 also signicantly increased stable
transfection ecacy by ~ 6x as compared to the no MAR control
(p<0.05). Other human MARs, namely hMAR 1-6 and 1-42 also
showed a positive trend for stable transfection ecacy. e chicken
lysozyme (cLys) MAR was the only tested element that did not show a
detectable eect (Figure 2A).
MAR name Species of origin MAR size (bp)
Control genomic DNA Human Chr. 1 2275
hMAR 1-6 Human Chr. 1 4618
hMAR 1-42 Human Chr. 1 4660
hMAR 1-68 Human Chr. 1 3643
hMAR X-29 Human Chr. X 3343
cLys MAR Chicken Chr. 1 2827
Mouse MAR S4 Mouse Chr. 1 5457
These MAR elements were previously described in references [6,9].
Table 1: Overview of MAR elements used in this study.
Untransfected
No MAR
hMAR X-29
Untransfected
No MAR
hMAR X-29
Cell number
100 1 2 3 4
0
100
200
300
400
500
10 10 10 10
eGFP
Cell number
100 1 2 3 4
0
100
200
300
400
500
10 10 10 10
eGFP
0
5
10
15
20
25
eGFP positive cells (%)
Figure 1: Transfection of primary murine mesoangioblasts by various approaches. Transfection was done according to manufacturer’s instructions using optimized
amounts of DNA. The percentage of eGFP positive cells was recorded 48 h after transfection by FACS cytouorometry (A) or uorescence microscopy (representative
elds shown, panel B). Scale bars indicate 200 µm. FACS proles of eGFP expression obtained from the hMAR X-29-containing vector of from a control construct devoid
of any insert (no MAR), as determined 48 h after transfection with lipofectamine 2000 (C) or Fugene HD (D) Cells were transfected with equimolar amounts of either the
no MAR eGFP or the hMAR X-29 eGFP plasmid.
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 4 of 8
Overall, the maximum level of eGFP uorescence of individual
colonies was higher in presence of hMAR X-29 than for clones generated
without a MAR (Figure 2B and Table S1). Analysis of the mean
uorescence of the 4 highest expressing clones generated with hMAR
X-29 or without a MAR yielded 39 and 10.5 relative uorescence units
(RFU), respectively. Clones displaying the highest eGFP uorescence
were isolated from the transfections performed with or without the
hMAR X-29, and they were subsequently expanded without antibiotic
selection pressure. eGFP expression was rapidly lost in the isolated
clone generated using the vector containing the control genomic DNA,
whereas it was stable for the hMAR X-29-containing clone (Figure 2C
and 2D). While selection allowed the maintenance of eGFP expression
in the control MAR-devoid cells aer 30 days of culture, the selection
pressure had no noticeable eect on transgene expression in the hMAR
X-29-containing clone, indicating a lack of detectable silencing over
this time-period in presence of the MAR.
In vitro and in vivo dierentiation of clonal primary murine
mesoangioblasts
We next tested whether transfected mesoangioblasts retained
their myogenic dierentiation potential. us, we co-cultured the
mesoangioblasts with the C2C12 murine myoblast cell line under
myogenic dierentiation conditions, to induce the in vitro co-
dierentiation and fusion of these cells into myotubes. Most clones
generated with the MAR yielded eGFP-expressing myotubes in such
assay, indicating that the mdx mesoangioblast cells had maintained their
ability to fuse with dierentiating myotubes, and that dierentiation was
not accompanied by the silencing of the transgene (S1 Table and Figure
S1). In contrast, eGFP expression was low prior to dierentiation in the
cells generated without the MAR, and it became nearly undetectable
aer myotube dierentiation (Figure S2 and data not shown).
Overall, we were unable to obtain a clone devoid of the MAR that
would express sucient eGFP levels for detection in subsequent in vivo
dierentiation assays with this approach. us, we adapted an iterative
transfection procedure phased on the cell division cycle, as described
previously for CHO cells, in which the cells were transfected a second
time 27 h aer the rst transfection [19]. One of the resulting clones
had an expression level close to those of hMAR X-29-containing clones
(Figure 3A and Table S1).
When comparing the eGFP expression levels of stably transfected
clones to a lentiviral vector-transduced polyclonal population by
cytouorometry, the maximum eGFP uorescence levels were quite
similar between the hMAR-containing clone and the transduced
population (Figure 3A).
When the clone transfected twice with the MAR-devoid construct
was assessed in the dierentiation assay, eGFP expression could be
detected. However expression was low in comparison to that elicited
by the hMAR-containing clone, indicating an expression stabilizing
eect from the hMAR (Figure 3B). Fusion of mesoangioblasts
generated with the hMAR allowed signicant levels of transgene
expression in myotubes, despite the fact the latter are formed by an
excess of non-expressing C2C12 cells. Proper myogenic dierentiation
of the clonal mesoangioblast populations was conrmed by α-actinin
immunouorescent staining, indicating that the fusion of transfected
mesoangioblasts had not impaired myotube formation. e
transfection and selection protocol had virtually no negative eect on
the dierentiation potential of the mesoangioblasts, as 29 out of 30
tested eGFP-expressing clones gave rise to eGFP-positive myotubes
(Table S1).
*
**
*
Control DNA
hMAR X-29
Phase eGFP
Day 0, hMAR X-29
Day 23, no puromycin
Day 30, puromycin
Day 0, control DNA
Day 4, no puromycin
Day 30, puromycin
Cell number
10
0 1 2 3 4
0
100
200
300
400
500
10 10 10 10
eGFP (RLU)
Cell number
10
0 1 2 3 4
0
100
200
300
400
500
10 10 10 10
eGFP (RLU)
0
5
10
15
20
25
none 2.3kb
Contr. 1-6 1-42 1-68 X-29 cLys
Number of clones per transfection
Figure 2: Effect of MARs on mesoangioblast stable transfection efcacy.
(A) Mesoangioblasts were transfected with equimolar amounts of eGFP plasmid with or without a MAR insert, as indicated, together with the pSVpuro antibiotic
resistance plasmid, and selection with 2.5 µg/ml of puromycin was initiated 48 h later. Resistant eGFP positive colonies were quantied after 30 days of selection.
Statistical signicance was determined from at least three independent experiments by unpaired student’s t-test (*: p<0.05, **: p<0.01). (B) Primary colony morphology of
clones without and with the MAR (clones noMAR 2.3Kb.A and hMAR X-29.J, respectively, as described in the supplementary Table S1). hMAR X29.J has an increased
RFU of 910% as compared to the noMAR 2.3Kb.A control. These clones generated without (C) or with (D) the MAR displaying the highest expression were picked
after 20-30 days of antibiotic selection, and they were further cultured with or without antibiotic selection. eGFP expression levels were assessed by cytouorometry at
different time-points, where day 0 refers to the day of antibiotic removal.
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 5 of 8
Transgene integration site determination in transfected
stable mesoangioblast clones
We next wished to determine the number of transgene integration
sites by uorescent in situ hybridization (FISH). Aneuploidy was found
in metaphase spreads of parental cells and of the transfected clones,
as expected from the long-term culture of the mouse mesoangioblasts.
Similar chromosome numbers were found in the clonal populations
and in the parental cells prior to transfection, indicating that the
transfection procedure did not cause chromosomal abnormalities
per se (Figure S2A). A single integration site was found in all tested
clones, although the genomic integration locus varied from clone to
clone (Figure 4). While a unique integration locus is advantageous in
terms of safety when compared to vectors yielding multiple integration
sites like retroviral vectors, we nevertheless wished to determine if the
genomic integration site may be identied in individual clones, so as to
assess the potential insertional mutagenesis of cellular genes. us, we
next attempted to characterize the genomic integration site of clones
generated with MAR-eGFP plasmids.
Genome-integrated vectors were released by digesting total
genomic DNA with a restriction enzyme that cleaves the plasmid once
and fragments were circularized by ligation. Plasmids were rescued by
bacterial transformation and sequence determination of the genomic
anking DNA region of the plasmid rescued from the hX29.J clone
showed transgene integration into an intronic region of solute carrier
family 12 member 8 gene, on chromosome 16. As this and other related
genes have not been linked to oncogenesis, we predicted that the
hX29.J clone would not give rise to tumors in vivo. Although we were
unsuccessful in determining the transgene integration sites for other
clones due to the low intrinsic success rate of the protocol, no safety
issues related to mutagenesis arose as no tumorigenesis was observed
during subsequent mouse experiments.
Following the validation of the eGFP-expressing clones for in vitro
dierentiation and for the lack of potentially oncogenic mutagenic
event elicited by plasmid genomic integration, we investigated the
in vivo dierentiation potential of the hMAR X-29.J and noMAR.B
clones. 5 x 105 cells were injected intra-muscularly into the Tibialis
anterior (TA) of 5-week old CD-1 nude mice. Cells from the lentiviral
vector-transduced population were similarly injected as a control. Both
transfected clones dierentiated into eGFP-positive myobers aer
transplantation (Figure 3C). In agreement with in vitro dierentiation
results, the hMAR X-29.J transplanted TA resulted in bers displaying
higher eGFP levels than the muscle transplanted with the MAR-devoid
clone (Figure 3D).
Transfection and dierentiation of primary murine
mesoangioblasts with dystrophin
We then proceeded to generate dystrophin-expressing clones from
A Control
hMAR X-29
GFP
GFP
α-actinin
α-actinin
Phase
Phase
B
C
D
E
GFP GFP, WGA GFP, WGA, DAPI
No MAR control
Transduced
hMAR X-29
Cell number
100
0
1000
2000
3000
4000
101 102 103 104
eGFP (RLU)
Figure 3: In vitro and in vivo differentiation of clonal eGFP-expressing mesoangioblasts.
(A) Comparison of the eGFP expression levels from clones obtained from an iterative transfection of transfected clones of a MAR-devoid eGFP expression vector
(clone noMAR.B), from a single transfection of the hMAR X-29-containing vector (clone X-29.J), or from a polyclonal population of mesoangioblasts transduced with
an eGFP-expressing lentiviral vector. The cytouorometric proles for eGFP uorescence were monitored for each population. (B) Differentiation of the noMAR.B
and hMAR X-29.J cell clones after a co-culture with C2C12 cells for 7 days in differentiation medium. eGFP uorescence is shown in green, while the α-actinin
immunouorescence performed to determine myogenic differentiation and DAPI nuclear staining are displayed in red or blue, respectively. Scale bars indicate 50
µm. (C-E) In vivo transplantation of eGFP-expressing mesoangioblasts clones with hMAR X-29 (clone X29.J, panel C) or without a MAR (noMAR.B, panel D), and of
a lentiviral vector-transduced mesoangioblast polyclonal population without a MAR. A single intramuscular injection with 5 x 105 cells was done in the TA of 5 wk old
CD-1 nude mice, and muscles were isolated for immunostaining at day 9 after injection. Brightness and contrast were increased for better visibility of the muscle bers
delineation for the red channel (WGA) of the enlargement panels (C, D and E).
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 6 of 8
the mdx mesoangioblasts by co-transfecting the puromycin selection
gene with the full-length murine dystrophin cDNA placed under the
control of the muscle creatine kinase promoter. Various MAR-eGFP
expression plasmids were co-transfected at equimolar amounts to
supplement the dystrophin sequence with the MAR elements in trans,
as previous evidence indicated that distinct plasmids co-integrate at the
same genomic locus upon co-transfection [17]. No clone was obtained
without a MAR, despite good initial transfection ecacies, and we
did not obtain eGFP-expressing clones with hMAR X-29 either. is
could be attributed to the fact that in presence of hMAR X-29, the
muscle specic MCK promoter displayed a leaky expression in non-
dierentiated cells (Figure S3), which leads to cellular toxicity eects
due to ectopic dystrophin expression. However, relatively less potent
MARs such as the cLys and h1-6 MAR generated 1 and 3 clones,
respectively. Transfection with a mouse MAR termed S4 yielded three
additional clones. Consistently with the results from the earlier eGFP-
expressing clones, all dystrophin vector-transfected clones yielded a
single transgene integration locus (Figure S2B and S2C). In addition,
all these clones were found to co-dierentiate with C2C12 cells to yield
eGFP-expressing myotubes in vitro (Figure 5A-5C). ese ve clones
were transplanted in the TA muscles of 5-week old mdx mice, which
were immunosuppressed with FK506 until sacrice to prevent rejection
A B
C D
Figure 4: Clones derived from plasmid transfection have single genomic integration sites.
Representative FISH images of clones used for in vivo transplantation studies. Arrows indicate the transfected plasmid integration sites. Inlays are enlargements of
transgene-bearing chromosomes. (A) eGFP clone X29.J. (B) eGFP clone noMAR.B. (C) eGFP clone S4.B. (D) eGFP clone 1-6.E.
α
α
α
0
2
4
6
8
10
12
number of dystrophin
expressing fibers
Figure 5: Differentiation and in vivo dystrophin expression from transfected mesoangioblast stable cell clones.
In vitro differentiation assays were performed on all selected clones, and the eGFP expression, α-actinin staining and an overlay are shown at day 7 after a co-culture
with C2C12 cells in the co-differentiation assay conditions. A representative clone for each of the expression construct is shown, namely clone h1-6dys.E (panel A),
cLysdys.D (panel B) and mS4dys.C (panel C). Scale bars represent 100 µm. (D) Dystrophin staining on cross-section of TA from a 5 wk old C57Bl6 mouse (positive
control, panel D) and cross-section of a TA from a 5 wk old mdx mouse 1 month after a single intra-muscular injection with 5 x 105 cells of the S4dys.D clone (panel E).
Mdx mice were immunosuppressed by daily injections of FK506 before sacrice, and dystrophin-positive bers were counted in distal and proximal transversal sections
of each TA muscle. The scale bar represents 50 µm. (F) Dystrophin positive bers were counted for the negative control (n=6) and after transplantation of cells from the
h1-6dys.E (n=12), mS4dys.C (n=6), mS4dys.D (n=12), mS4dys.A (n=4), or cLysdys.D (n=10) clones.
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 7 of 8
of the allogeneic cells. e number of dystrophin positive bers was
then counted 1 month aer transplantation in immunostaining studies
of proximal and distal cross-sections of the TA of all mice. Overall, the
dystrophin expression levels of injected mdx TA muscles were lower
than those of wild-type myobers, but clusters of dystrophin expressing
bers could be observed in muscles transplanted with clones S4dys.D
and 1-6dys.E along with centrally located nuclei indicative of muscle
ber regeneration (Figure 5D and 5E). No such cluster of dystrophin-
expressing cells was detected from the transplantation of the other
three clones or from the muscles of non-transplanted animals (Figure
5F). Overall, we concluded that some of the transfected clones were
capable of dystrophin expression aer transplantation into the muscle
of dystrophic mdx mice.
Discussion
Non-viral cell-based therapy has oen been limited by gene
transfer and maintenance, and thus by the lack of cell populations
mediating stable and consistent expression. Here we showed that stable
clones could be obtained from the transfection of adult stem cells
propagated in vitro. is involved the development of methods allowing
antibiotic selection, the mechanical isolation of single clones, and
the subsequent expansion in tissue culture in conditions that prevent
anoikis, an oen-noted problem when cultivating isolated primary
cells. Although mesoangioblasts signicantly slowed cell cycling
when cultured at low density, and a minority of cells showed signs of
spontaneous dierentiation, i.e. a attened morphology of multiple
nucleated syncytia, most cells did not exit the cell cycle and expanded
to form clonal populations expressing the transgene at homogeneous
levels. Conversely, another limitation of muscle progenitor cells is
a loss of dierentiation potential resulting from contact inhibition,
when allowing the stable clones to form colonies. However, clones
grown according to this protocol were consistently able to form
eGFP-expressing myotubes in vitro, and the inclusion of MARs in the
expression vector had no negative eect on dierentiation.
We showed that several MAR elements of human origin enhanced
stable transfection eciency signicantly. is eect of MARs cannot
be related to a simple increase of the transfer of the DNA, as transient
transfection of MAR bearing plasmids consistently yielded fewer
uorescent cells than ones devoid of MAR, even when transfecting
equimolar amounts of plasmid. us, the eect of the MARs is rather to
increase transgene genomic integration, as required to establish clones
displaying stable transgene propagation and expression upon cell
division. is eect of the MARs can be readily explained by the recent
nding that MARs promote transgene genomic integration in cell lines
by a homologous recombination-related mechanism [19]. A similar
increase of the genomic integration of MAR-containing plasmids has
also been observed in vivo upon plasmid electroporation in murine
muscles [24]. us a more frequent establishment of mesoangioblasts
cell clones is well explained by the fact that primary cells are known
to have limiting recombination activity when compared to established
cell lines [24], and that MAR would thus be required to increase
integration by such a recombination mechanism. In this respect, it will
be interesting to characterize transgene integration sequences at a large
scale, to determine if MARs may promote more frequent occurrence of
integration at specic types of genomic loci or of chromatin structure.
Nonetheless, and despite the higher frequency, clone establishment
remained relatively rare events, yielding up to 10-20 clones per 105
cells for the most ecient MAR-containing vectors. erefore, isolated
primary mesoangioblast populations would have to be expanded
to close to a million cells in a clinical setting to yield 10-100 clones
that could be stored and characterized before transplantation. Given
the genomic stability of human mesoangioblasts and the maintenance
of their dierentiation potential upon culturing in vitro, this might
be a feasible goal provided that senescence may be avoided during
culture. is might be achieved for instance by the currently developed
reversible immortalization of these human cells [25].
In addition to the eect on transgene integration, the human
MARs were found to have a positive eect on transgene expression
levels and stability, the most potent in this respect being hMAR X-29.
In presence of hMARs, clones that expressed eGFP in mononucleated
mesoangioblasts were in most cases found to maintain eGFP expression
aer dierentiation, even in the absence of antibiotic selection pressure.
Although a clone could be isolated form a MAR-devoid plasmid with
a comparable expression level as those obtained from mesoangioblasts
transfected with a MAR, such clones usually had lower expression or
lost any detectable eGFP uorescence upon dierentiation to myotubes.
is likely results from the known adoption of less permissive chromatin
structures such as heterochomatin over large portions of the genome
upon the dierentiation of stem cells [26]. e ability of MARs to
maintain a transcriptionally permissive chromatin structure, even when
the chromatin structure is restricted upon myogenic dierentiation,
may thus explain their favorable eect on transgene expression upon
myotube formation. In addition to the favorable eect of MARs on
transgene expression, all clones had a single genomic integration site
as is oen the case for transfected cell lines, and this integration site
can be mapped to increase the safety of potential transfection-based
clinical protocols. is feature may be advantageous in terms of safety
when compared to viral or transposable vectors that oen integrate at
multiple and variable number of loci.
Results from in vivo transplantation experiments indicated that
transfected mesoangioblast clones may lead to eGFP positive myobers
upon intramuscular injection as early as 9 days aer administration.
In accordance with their in vitro dierentiation properties, the hMAR-
containing clone yielded clearly detectable eGFP expression in vivo,
whereas the clone without a MAR displayed low eGFP levels. Based
on these ndings we concluded that human MARs like X-29 could
fulll some of the requirements of autologous muscle cell therapy.
While we were able to obtain stable mesoangioblast clones that were
co-transfected with a plasmid encoding full-length dystrophin driven
by the muscle creatine kinase (MCK) promoter and some of the
MAR-bearing eGFP plasmids, transfections with the most potent
hMAR X-29-containing plasmid consistently yielded no clones. e
rationale for using a muscle-specic promoter to express dystrophin
stems from previous observations that the build-up of dystrophin
protein expression in undierentiated mesoangioblasts leads to toxicity
eects, possibly because of the lack of concomitant expression of other
components of the glycoprotein complex that dystrophin interacts with
[27,28]. us, a likely explanation for the lack of stable clones expressing
dystrophin in presence of the most potent hMAR X-29 is that the co-
integration of the MAR and the dystrophin construct caused leakiness
of the MCK promoter, and ectopic expression of dystrophin prior to
dierentiation. is interpretation is indeed supported by data showing
GFP expression from the MCK promoter in presence of hMAR X-29
in undierentiated mesoangioblast cells, which indicated an increased
leakiness of the muscle promoter in presence of the most potent MAR.
Nevertheless, dystrophin cDNA-containing clones were obtained
using other MAR elements like the mouse S4, the human 1-6 and the
chicken lysozyme MAR, and clones obtained with these elements were
capable of dierentiating in vitro and in vivo. Dystrophin expressing
bers were found from the injection of the clonal mesoangioblasts. A
minority of the bers had dystrophin expression, as expected from the
Volume 4 • Issue 3 • 1000134
Mol Biol
ISSN: 2168-9547 MBL, an open access journal
Citation: van Zwieten RW, Majocchi S, Iyer P, Dussere Y, Puttini S, et al. (2015) MAR-Mediated Dystrophin Expression in Mesoangioblasts for
Duchenne Muscular Dystrophy Cell Therapy. Mol Biol 4: 134. doi:10.4172/2168-9547.1000134
Page 8 of 8
previously reported relatively lower ecacy of a single intramuscular
injection relative to several consecutive injections (ref. [25] and
unpublished data from G.C. and collaborators), and also because
potentiating protocols like the transduction with MyoD vectors or pre-
injection exposure to growth factors were not used [8]. Nevertheless,
our observations provide a rst description of full-length dystrophin
expression from the stable plasmid transfection of muscle precursor
cells, followed by their incorporation in muscle bers in vivo. It thereby
facilitates a new strategy to pursue a possible treatment of muscular
dystrophies using genetically-corrected cells.
Overall, the presented approach may be limited to the cells that
maintain a normal genomic structure and dierentiation potential
when cultured in vitro, and to cloning procedures that allow the
selection of transgene expressing cells. Overall, we propose that the
ability to characterize the genomic structure of clonal populations aer
limited expansion may decrease the likelihood of adverse eects and
may thus open a feasible path towards cell-based therapies involving
gene transfer.
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Gene expression often cycles between active and inactive states in eukaryotes, yielding variable or noisy gene expression in the short-term, while slow epigenetic changes may lead to silencing or variegated expression. Understanding how cells control these effects will be of paramount importance to construct biological systems with predictable behaviours. Here we find that a human matrix attachment region (MAR) genetic element controls the stability and heritability of gene expression in cell populations. Mathematical modeling indicated that the MAR controls the probability of long-term transitions between active and inactive expression, thus reducing silencing effects and increasing the reactivation of silent genes. Single-cell short-terms assays revealed persistent expression and reduced expression noise in MAR-driven genes, while stochastic burst of expression occurred without this genetic element. The MAR thus confers a more deterministic behavior to an otherwise stochastic process, providing a means towards more reliable expression of engineered genetic systems.
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Matrix attachment regions (MARs) are DNA sequences that may be involved in anchoring DNA/chromatin to the nuclear matrix and they have been described in both mammalian and plant species. MARs possess a number of features that facilitate the opening and maintenance of euchromatin. When incorporated into viral or non-viral vectors MARs can increase transgene expression and limit position-effects. They have been used extensively to improve transgene expression and recombinant protein production and promising studies on the potential use of MAR elements for mammalian gene therapy have appeared. These illustrate how MARs may be used to mediate sustained or higher levels of expression of therapeutic genes and/or to reduce the viral vector multiplicity of infection required to achieve consistent expression. More recently, the discovery of potent MAR elements and the development of improved vectors for transgene delivery, notably non-viral episomal vectors, has strengthened interest in their use to mediate expression of therapeutic transgenes. This article will describe the progress made in this field, and it will discuss future directions and issues to be addressed.