gene therapy is now a realistic prospect for the treatment of a variety of musculoskeletal disorders because of the emerging knowledge concerning proteins that govern the processes of growth and regeneration of tissues [ 1 , 2 ] .
research has identified numerous growth factors and other gene products with the ability to promote regeneration .
the most extensively studied are members of the transforming growth factor ( tgf ) superfamily , collectively known as bone morphogenetic proteins ( bmps ) . because gene therapy provides the gene rather than just a degradable protein , this technique may result in higher and more reproducible levels of protein production with the potential for long - term protein delivery .
this would allow either local protein production for tissue healing and so forth or potentiate long - term systemic delivery .
the most commonly used in the orthopaedic field have been viral vectors and ex vivo manipulation with considerable preclinical success [ 5 , 6 ] .
there are concerns regarding viral recombination / infectivity , immunogenicity , and possible carcinogenicity [ 79 ] .
they are expensive to produce and as vectors have biophysical and genetic limitations . also many gene therapy strategies prove ineffective , suffering from gene silencing .
non - viral therapy has not been extensively studied in orthopaedic research [ 11 , 12 ] . in theory ,
non - viral therapy lacks some of the drawbacks associated with viral systems . in this study ,
the long - term viability of murine skeletal muscle plasmid gene expression in vivo was assessed using live whole body imaging of luciferase expression , along with the potential for expression control using an inducible model .
finally an ex vivo model was developed in murine musculoskeletal tissue and conditions replicated to examine plasmid gene expression in human ex vivo mesenchymal tissue .
the commercially available plasmid pcmv - luc was purchased from promega ( wisconsin , usa ) .
david gould ( bone & joint research unit , barts and the london , queen mary 's school of medicine and dentistry , university of london , london , england ) .
b - alb c mice were obtained from harlan laboratories ( oxfordshire , england ) .
they were kept at a constant room temperature ( 22c ) with a natural day / night light cycle in a conventional animal colony .
female mice in good condition , without fungal or other infections , weighing 1622  g and of 68 weeks of age , were included in experiments .
all in vivo and ex vivo animal experiments were approved by the ethics committee of university college cork .
administration of 200  g xylazine and 2  mg ketamine . for liver transfection ,
a 1  cm subcostal incision was made over the liver and the peritoneum opened .
the right lobe of the exposed liver was administered plasmid by electroporation as described below .
the wound was closed in two layers  peritoneal and skin  using 4/0 prolene sutures ( promed ) .
all ex vivo human tissue experiments were approved by the clinical research ethics committee of the cork teaching hospitals .
informed consent was obtained from patients scheduled for lower limb amputation and the surgeon 's permission obtained . upon removal of sample
samples were cut to a size approximating the medial thigh muscle of balb - c mice ( 1.5  cm ) . for plasmid delivery by electroporation ,
a custom - designed applicator with 2 needles 4  mm apart was used , with both needles placed through the skin central to the tissue .
tissue was injected between electrode needles with plasmid dna in sterile injectable saline in an injection volume of 50  l .
plasmid concentration was determined using the nanodrop spectrophotometer ( nd-1000 spectrophotometer , labtech int , east sussex , uk ) .
the number of plasmid copies was determined using the following calculation : 
 ( 1)mass  of  plasmid  dna  ( g )  = no .  of  gene  copies  ( bp )  (plasmid  size1.09610(21 ) ) . 
 
plasmid sizes for pcmv luc and pgtrtl were 6.9  kbp and 7.5  kbp , corresponding to a mass of 6.05  10 g and 6.576  10 g , respectively .
after 80 seconds , square - wave pulses ( 1200  v / cm 100  sec  1 and 120  v / cm 20  msec , 8 pulses ) were administered in sequence using a custom - designed pulse generator ( cythorlab , aditus , lund , sweden ) . in all cases , a drop in impedance measurement was taken as evidence for successful tissue cell poration . replication incompetent recombinant adenovirus 5 particles encoding the luciferase gene under the transcriptional control of the cmv promoter were a kind gift from prof .
andrew baker , university of glasgow ; they were generated and titrated as described previously .
viral vector particles were administered by direct intramuscular injection in a volume of 50  l . 1  10  vp of replication incompetent recombinant adenovirus 5 particles was used per administration .
the entire medial thigh muscle tissue was harvested from sacrificed b - alb c mice ( 1.5  cm ) .
samples were stored as per the experimental protocol to assess survival in either rpmi ( roswell park memorial institute medium ) or dmem ( dulbecco / vogt modified eagle 's minimal essential medium ) at 37c for 24 hours .
the effect of electroporation on cell survival was assessed by administering the plasmid as described previously and assessing viability at 24 hours .
cell viability was assessed using the propidium - iodide - based nucleocounter kit as previously described .
samples were washed and processed through a fine mesh filter ( bd biosciences , oxford , uk ) , using a pestle and pbs ( phosphate buffered saline ) until a suspension was obtained .
the suspension was made up to a standard volume , and cell viability was assessed .
solutions were prepared by adding 200  g / ml of doxycycline ( sigma ) to distilled water containing 10% sucrose as previously described .
all drinking bottles were wrapped in aluminium foil and were renewed every 2 - 3 days .
a 20  g / ml solution was added to wells with pgtrtl ex vivo samples as the 200  g / ml resulted in excessive cell death ( data not shown ) . 
 in vivo luciferase activity from tissues
was analysed at set time points after transfection as follows : 80  l of 30  mg / ml firefly luciferin was injected intraperitoneally .
ten minutes after luciferin injection , live anaesthetised mice were imaged for 3  min at high sensitivity using an intensified ccd camera ( ivis imaging system , xenogen ) .
the exposure conditions were maintained at identical levels so that all measurements would be comparable .
all data analysis was carried out on the living image 2.5 software package ( ivis imaging system , xenogen ) .
luminescence levels were calculated using standardised regions of interest ( rois ) for all 3 anatomical areas .
actual levels were obtained by subtracting the corresponding roi of an untransfected mouse to account for background luminescence . for comparison between plasmids ,
flux was calculated per gene copy number . at each time point , either one - way a two - sampled t - test was used to compare mean luminescence within each experimental group .
luciferase - coding plasmid dna featuring the cmv ( pcmv - luc ) promoter ( figure 1 ) was delivered to liver or thigh muscle by electroporation .
ivis live whole body imaging was used at time points over 370 days to determine luciferase expression ( figure 2 ) .
expression driven by the cmv promoter in liver was initially high but reduced rapidly to background levels by day 14 .
a different temporal pattern of expression was observed in muscle tissue , with no gross reduction in expression over time observed .
total luminescence for the period was 1.33  10  p / sec / cm / sr / plasmid copy .
unlike liver , muscle therefore has the potential to act as a long - term conduit for gene expression .
luciferase - coding plasmid dna featuring the cmv ( pcmv - luc ) promoter or adenovirus 5 particles encoding the luciferase gene under the transcriptional control of the cmv promoter was delivered to murine thigh muscle as described .
ivis live whole body imaging was used at time points over 18 days to compare luciferase expression between the two constructs .
maximum expression was significantly higher using adenoviral delivery when compared to that of plasmid delivery .
expression following adenoviral delivery , however , decreased over the study period to background levels at 18 days ( figure 3(b ) ) .
luciferase expression from an inducible  tet - on  promoter was examined in muscle tissue using the pgtrtl plasmid , both in the presence and absence of induction by doxycycline ( figure 4 ) .
there was a low initial expression from reporter gene preinduction from the time of delivery .
however , following induction with doxycycline at day 8 , expression was increased by a factor of 11 ( p = 0.0001 ) at day 11 . following withdrawal of doxycycline at day 12 ,
expression was reinduced at day 105 and had increased by a factor of 9.2 by day 108 ( p = 0.001 ) and by a factor of 10.7 at day 116 .
expression remained constant at a maximal level between days 108 and 116 with no statistically significant change in magnitude observed ( p = 0.77 ) . following withdrawal of doxycycline at day 117 ,
there was no difference in maximal expression between the two induction periods ( p = 0.28 ) .
it can also be reinduced on a temporal basis to give reproducible levels of expression .
cell viability following incubation in dmem at 37c for 1 , 4 , and 24 hours was 85% , 82% , and 38% , respectively ( figure 5 ) .
this was significantly better than cell survival following incubation in rpmi ( 22% , p < 0.05 ) .
there was no difference in cell survival following plasmid electroporation when compared to untreated samples ( 34% and 38% , resp .
, p = 0.5 ) . to assess translation of the murine findings to a clinical setting
, samples of muscle , tendon , and bone with intact periosteal tissue were harvested from a human leg amputated for end - stage peripheral vascular disease .
4 samples were taken of each tissue type and transfected with pcmv - luc or pgtrtl as previously described .
these were incubated overnight in conditions described above and luminescence assessed at 24  h ( figure 6 ) .
luciferase gene expression was observed in all tissues electroporated with plasmid , indicating successful plasmid delivery and subsequent gene expression in this setting .
localised gene expression offers certain advantages in the setting of tissue healing . it may provide a more accurate system of protein delivery to the desired tissue , whilst lessening systemic side effects .
in addition , it has the potential for sustained protein delivery , both intracellularly and extracellularly without the need for repeated administration . however , inactivation of gene expression in specific cell types has important therapeutic implications , as does the prospect of uncontrolled protein production . here
, we have demonstrated that plasmid - based gene expression in murine muscle does not decrease with time .
long - term plasmid gene expression has been observed in muscle previously , although not using an in vivo luminescence system for a period greater than one year [ 15 , 16 ] . in vivo luminescence
it is also an ideal method of assessing an inducible agent as an  on - off  switch to control protein expression , by allowing repeated measurements in the same subjects over a long time period .
successful bone and tendon healing in animal models have been reported following adenoviral transfer of bmp 2 and bmp 14 [ 5 , 6 , 18 ] . here , we have compared luciferase expression between a plasmid construct and viral particles .
although maximum expression was significantly greater with adenovirus , this was rapidly silenced to background levels by day 18 .
the attraction of the plasmid system would therefore lie in its capacity for sustained levels of predictable expression over time .
electroporation - assisted intramuscular delivery of plasmid dna has been shown to efficiently transfect cells of myogenic origin with no evidence of transgene expression in infiltrating inflammatory cells .
this is broadly in keeping with our observation of long - term expression , with transfection of a nontransient cell population in muscle and the absence of a significant immune response .
silencing has previously been observed with adenovirus , with the mechanism postulated as methylation of the cmv promoter .
why this does not occur when the promoter is delivered using a plasmid construct , however , is unclear .
for instance , in the clinical use of recombinant bone morphogenetic protein ( rhbmp ) for augmentation of fracture healing and joint fusion , heterotopic ossification and other complications have been reported . in this instance , the protein is eventually cleared from the body . following gene delivery , however , it would be difficult to arrest protein production in the event of unwanted side effects .
it is , therefore , desirable to have a plasmid with an  on - off  switch .
we have demonstrated that this can be achieved using pgtrtl , a tet - on inducible promoter .
after plasmid delivery , gene expression initiated with the commencement of oral doxycycline administration and promptly ceased upon withdrawal .
this strategy has also proven to be successful in a preclinical arthritis study . in the clinical setting , should side effects occur , gene expression could be turned off simply by omitting the oral agent . in addition ,
growth factors may not be clinically beneficial until days after intervention , while the delivery of rhbmp at the time of surgery has been shown to impair tissue healing by inducing antagonist factors [ 18 , 22 ] . by using pgtrtl
, we have demonstrated that the timing and duration of gene expression can be tightly controlled in vivo .
this would decrease the chance of antagonist induction , whilst optimising the timescale of protein production .
this would allow us to repeatedly treat tissue with a known quantity of protein in recalcitrant situations .
as the system is orally administered , there would be no need for interference with the physical environment of tissue healing after the initial application .
one issue with tetracycline compounds is that they do have an effect on the tissue healing environment and as such may not be the ideal tool for the investigation of therapeutic plasmid use . in terms of principle , however , it demonstrates the potential applicability of an inducible plasmid - based system in a clinical setting .
finally , we have demonstrated , for the first time , successful plasmid - based gene delivery to human ex vivo muscle tissue .
levels of luciferase expression in ex vivo tendon and muscle were similar when examined at 24 hours .
although the exact reasons for the similar levels of expression are unclear , it may be related to a threshold effect or differential tissue survival in the ex vivo environment .
expression may differ in the human in vivo setting , particularly with regard to increased metabolic activity in muscle tissue .
nonetheless , we believe this to be an important step in the demonstration of the applicability of this technique in the in vivo setting , as expression capability can not be definitely extrapolated from the murine model to the human setting .
the expression of plasmid - based delivery in human ex vivo musculoskeletal tissue is encouraging on two levels ; firstly , it proves that the system can function in human tissue , and secondly , it raises the possibility of autograft or allograft transfection , which could lead to the acceleration of graft incorporation .
the luminescence imaging strategy utilised here provided a rapid , robust and reliable method for ex vivo assessment of transgene expression in patient samples .
the method of culturing tissue ex vivo has been demonstrated to provide high viability for up to 1 week post resection , while bioluminescence imaging provides a highly sensitive readout for real - time transgene expression in viable cells .
other reporter gene imaging techniques may also be applicable in this context ( fluorescence , pet etc . )
these results indicate , when administered using electroporation , that plasmid constructs result in long - term in vivo gene expression and that this expression can be reliably induced with oral agents . finally , for the first time , successful plasmid gene transfection with electroporation in human ex vivo mesenchymal tissue has been demonstrated .
 
 purpose . in vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing . 
 we aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues . 
 methods . using bioluminescence imaging , plasmid and adenoviral vector - based transgene expression in murine quadriceps in vivo was examined . 
 temporal control was assessed using a doxycycline - inducible system . 
 an ex vivo model was developed and optimised using murine tissue , and applied in ex vivo human tissue . 
 results . in vivo plasmid - based transgene expression 
 did not silence in murine muscle , unlike in liver . 
 although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid , expression reduced over time . 
 the inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline . 
 expression was re - induced to a similar level on a temporal basis . 
 luciferase expression was readily detected ex vivo in human muscle and tendon . 
 conclusions . 
 plasmid constructs resulted in long - term in vivo gene expression in skeletal muscle , in a controllable fashion utilising an inducible promoter in combination with oral agents . 
 successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time . 
 