Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model

ARTICLEdoi:10.1016/j.ymthe.2004.09.016

Optimization of Regional Intraarterial NakedDNA-Mediated Transgene Delivery to Skeletal Muscles

in a Large Animal Model

Gawiyou Danialou,1 Alain S. Comtois,2 Stefan Matecki,1 Josephine Nalbantoglu,3

George Karpati,3 and Renald Gilbert4 Pascale Geoffroy5 Sandra Gilligan5

Jean-Francois Tanguay5 Basil J. Petrof1,*

1Meakins–Christie Laboratories and Respiratory Division, McGill University, Montreal, QC, Canada H3A 1A12Department of Kinanthropology, University of Quebec at Montreal, Montreal, QC, Canada H3C 3P8

3Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montreal, QC, Canada H3A 2B44Biotechnology Research Institute, National Research Council of Canada, Montreal, QC, Canada H4P 2R2

5Montreal Heart Institute, Montreal, QC, Canada H1T 1C8

*To whom correspondence and reprint requests should be addressed. Fax: (514) 843 1695. E-mail: [email protected].

Available online 28 October 2004

MOLECULA

Copyright C

1525-0016/$

Effective gene therapy for muscular dystrophy will likely require intravascular administration.Although plasmid DNA (pDNA) contained within a large volume and rapidly infused into a majorartery can achieve gene transfer within downstream muscles, this is associated with substantialmuscle edema. Here we hypothesized that excessive edema-related increases in intramuscularpressure (IM pressure) developed during intraarterial pDNA injections could hinder successful genedelivery. Accordingly, we monitored IM pressure during injection of pDNA carrying a LacZ transgeneinto the femoral artery of rats and pigs. Large variations in IM pressure were found betweendifferent muscles. There was a significant inverse relationship between IM pressure and thesubsequent level of gene transfer to muscle. Modification of the injection protocol to reduce IMpressure led to greatly increased pDNA-mediated gene expression and reduced muscle damage inpigs. Under the most optimized conditions, average transfection within eight different muscles ofthe pig hind limb amounted to 22% of all fibers, attaining a maximum of 60% in the gastrocnemiusmuscle. We conclude that IM pressure monitoring is a simple and useful procedure, which can beapplied in both small and large animals to help optimize pDNA-mediated gene transfer to skeletalmuscles by the intraarterial route.

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Key Words: nonviral vector, muscular dystrophy, intravascular administration, systemic delivery,intraarterial injection, plasmid DNA, pig muscles, dystrophin

INTRODUCTION

Gene therapy is one of the principal approaches beingconsidered for the treatment of muscular dystrophies andother inherited myopathies. One major target for such anapproach is Duchenne muscular dystrophy (DMD), adisease for which little treatment is currently availableother than purely supportive measures. At the presenttime, there is no consensus regarding the best vectorsystem for delivering therapeutic genes to skeletal musclein DMD. Different vectors have been tested in normal aswell as dystrophic muscles, and each vector system has itsown advantages and disadvantages. Naked plasmid DNA(pDNA) combines the advantages of low inherent toxicityand a large gene insert capacity and is also much more

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amenable to large-scale production than viral vectors [1].Indeed, since the seminal observation by Wolff andcolleagues [2] that exogenous gene expression can beachieved in vivo after direct intramuscular injection ofpDNA, there has been great interest in using pDNA totreat muscular dystrophies. A major limitation to the useof pDNA for this purpose has been the very low degree ofgene transfer achieved after intramuscular injection [2,3],although this can be greatly improved upon by employ-ing adjunctive measures such as electroporation [4] orsonoporation [5].

It is important to recognize that a highly desirable andeven essential attribute of any vector system beingconsidered for the treatment of DMD is an ability to be

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ARTICLE doi:10.1016/j.ymthe.2004.09.016

safely and effectively delivered through the bloodstream.Although relatively little study has been devoted to thisarea, it is clear that the development of an intravasculardelivery method would greatly improve the prospects foreffective clinical application of gene therapy for DMD.This is because the widespread nature of the diseasemakes direct intramuscular injections impractical, sincean exceedingly large number of injections would berequired to treat all of the involved muscles. In addition,it is well known that after direct intramuscular depositionof vector particles, transgene expression is inhomoge-neously distributed and often limited to a small areawithin the muscle [6]. Based on studies in transgenic mdxmice demonstrating that the evenness of distribution ofdystrophin expression within a muscle is a majordeterminant of physiological efficacy [7], this wouldconstitute a major problem. On the other hand, vectoradministration via the arterial system should permit amuch more widespread and even distribution of trans-gene expression, by virtue of the fact that skeletal muscletissue contains an extensive capillary network, which liesin close proximity to the muscle fibers.

In previous studies it was shown that when geneexpression vectors are delivered intraarterially underconditions of high injectate volume and increased hydro-static pressure, it is possible to achieve substantial genetransfer to distal hind limb muscles in various animalmodels, including dystrophic mice [8–12]. The elevatedinjectate volume and associated increase in intravascularhydrostatic pressure are presumed to drive vector particlesout of the microcirculation and into the tissue throughseveral mechanisms [10]. However, there is also anincrease in bulk fluid flow into the interstitial compart-ment of the muscle under these conditions, which causesprominent muscle swelling and edema both during andimmediately after the procedure [9,10,12].

In the present study, we hypothesized that theincreased intramuscular tissue fluid pressure (IM pres-sure) associated with muscle edema is a potentiallyimportant hindrance to the successful outcome of intra-arterial pDNA administration, in terms of both genetransfer efficiency and safety of the procedure. Becauseeach individual skeletal muscle behaves as an anatomi-cally closed compartment, large increases in muscleinterstitial fluid content lead to an increase in IMpressure, which will in turn cause intramuscular arterio-lar and venous compression [13,14]. Therefore, if themagnitude and rate of increase in IM pressure duringintraarterial pDNA injection are excessive, vector particledelivery and hence gene transfer will be reduced by acutecollapse of the capillary bed. Sustained increases in IMpressure after pDNA injection may also cause ischemicdamage to muscle fibers, a condition known clinically asa bcompartment syndromeQ [13,15]. Furthermore, whilethe use of histamine has been proposed as a method toincrease vascular permeability and thus facilitate vector

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egress out of the microcirculation [11], histamine couldaccelerate capillary closure by further increasing thedegree of intramuscular edema/pressure developed underthese conditions. Histamine also has the potential tocause systemic hemodynamic instability.

In the current study, our overall goal was to develop arational physiological basis for optimizing regionaldelivery of naked DNA to skeletal muscles via theintraarterial route. Our investigation was performed inrats as well as in a large animal (pig) model. The latterwas selected to approximate the body weight of a DMDchild, since information gained in larger mammals willbe essential for guiding the future application of thisapproach in dystrophy patients. Here we specificallyaddressed the following questions: (1) What is therelationship between IM pressure achieved during intra-arterial pDNA injection and the subsequent level of geneexpression within the same muscles? (2) What are theeffects of histamine on IM pressure development, bloodpressure, and gene transfer efficiency under these con-ditions? (3) Is there a relationship between the IMpressure achieved during and after the intraarterialinjection and the subsequent level of myofiber damage?Our data indicate that IM pressure monitoring is asimple and useful procedure, which can be applied insmall as well as large animals to optimize gene transfer toskeletal muscles during regional intraarterial delivery ofnaked pDNA.

RESULTS

Rat ExperimentsBecause we hypothesized that large increases in IMpressure during large volume intraarterial injectioncould hinder skeletal muscle fiber transfection, we firstsought to ascertain the relationship between IM pressuremeasurements and regional muscle transfection effi-ciency in the rat model. As shown in Fig. 1A, IMpressure values generated during intraarterial injection(volume 25 ml/kg into the femoral artery) were signifi-cantly higher in the tibialis anterior (TA) than in thegastrocnemius (GAST) muscle (P b 0.001 at all timepoints shown). There was a dramatic increase in IMpressure in the TA, which peaked at an average meanvalue of 200.2 F 17.7 mm Hg above baseline atapproximately 4 s after the onset of the injection. Inmarked contrast, the peak value of IM pressure in theGAST was only 15.6 F 5.3 mm Hg, and this occurred ata later time point (approximately 8 s) after the onset ofthe injection. Thereafter, the IM pressure fell slowlytoward its baseline value in both TA and GAST. Fig. 1Bshows the level of h-galactosidase (h-gal) transgeneexpression in the TA and GAST under these conditions.As can be seen, both the number of fibers stainingpositively for h-gal on muscle sections and the wholetissue levels of h-gal determined by chemiluminescence

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were approximately 10-fold greater in the GAST than inthe TA muscle (P b 0.001).

To optimize further the intraarterial pDNA deliverymethod, we next evaluated the effects of administeringhistamine directly into the femoral artery 5 min prior topDNA injection. Fig. 1C shows the negative correlationbetween IM pressure values and the number of trans-fected fibers, which was highly significant either with(P b 0.001) or without (P b 0.02) histamine. Histamineitself did not appear to have any impact upon IMpressure values attained during pDNA injection in eitherthe GAST or the TA. However, pretreatment withhistamine produced a significant change in the relation-ship between IM pressure and transfection efficiency, asindicated by the significantly different slopes (P =0.003) of the two regression lines shown in Fig. 1C.Hence the use of histamine was associated withsignificantly improved transfection efficiency onlyunder the low IM pressure conditions found in theGAST (1406 F 168 versus 772 F 194 h-gal-expressingfibers, P b 0.05) and not the high IM pressureconditions found in the TA (52 F 26 versus 75 F 29h-gal-expressing fibers).

Pig ExperimentsWe sought to determine whether the physiologicalprinciples established in the rat could also be successfullyapplied in a large animal model to achieve high-levelmuscle transfection after intraarterial injection of pDNA.A representative tracing of the physiological recordingsobtained in pigs during the intraarterial injection proce-dure is shown in Fig. 2B. Fig. 3 shows the values ofmaximal IM pressure for the TA and GAST, as well assystemic arterial blood pressure at the same time points,after rapid injection of different volumes into the pigfemoral artery. Note that the peak IM pressure values inthe TA exceeded systolic arterial blood pressure at allvolumes tested. In contrast, IM pressure values in theGAST exceeded systolic blood pressure only at the twohighest volumes tested (19 and 25 ml/kg) and did notdiffer from diastolic blood pressure at the two lowervolumes examined (12.5 and 16 ml/kg). As in the rats, theIM pressure values in TA and GAST of pigs showed agradual return toward baseline preinjection values, with

FIG. 1. (A) Time course of changes in IM pressure within the rat tibialis anterior

(TA) and gastrocnemius (GAST) during intraarterial injections. Values for IM

pressure were significantly higher in the TA at all time points shown after

injection. (B) Quantification of h-gal expression in rat hind limb muscles after

pDNA administration into the femoral artery. Both the total number of h-gal-

expressing fibers (open bars) and the amount of h-gal protein (filled bars)

were significantly greater in the GAST compared to the TA. (C) Relationship

between peak IM pressure and the number of h-gal-expressing fibers in rat TA

and GAST muscles. Note the significant negative correlation between IM

pressure and number of h-gal-expressing fibers, irrespective of the use of

histamine. Symbols represent the following: circles, GAST; squares, TA; open

symbols, without histamine; filled symbols, with histamine.

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FIG. 2. (A) Schematic illustration of experimental setup

in the pig (BP, arterial blood pressure; CVP, central

venous pressure). (B) Representative physiological

recordings of IM pressure and hemodynamic variables

during 25 ml/kg volume injection into the pig femoral

artery. Double-headed arrow indicates the onset of the

injection.


90% recovery at 218 F 28 and 396 F 31 s postinjectionfor the 12.5 and 25 ml/kg groups, respectively.

We next determined the efficiency of gene transfer topig hindlimb muscles using the two opposite ends of theinjection volume spectrum (12.5 and 25 ml/kg), butcontaining the same absolute amount (50 mg) of pDNA.Histamine infusion led to a transient reduction in meanarterial blood pressure (from 48.9 F 5.4 to 33.6 F 3.0 mmHg, P b 0.005), thus indicating that the tourniquet systemwas not completely effective in eliminating venous out-flow from the hind limb. However, an anti-histamine(diphenhydramine) and volume expander (dextran) wereadministered intravenously to counteract this effect, andmean arterial pressure demonstrated full recovery tobaseline values 5 min later. Eight distal hind limb

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muscles, evenly divided between the anterior and theposterior compartments of the leg, were analyzed. Fig. 4shows that for each of the muscles examined, trans-fection efficiency was higher when pDNA was deliveredusing the lower injection volume of 12.5 ml/kg. This wastrue in terms of both the percentage cross-sectional areaof transfected muscle fibers (Fig. 4A) and overall tissuelevels of h-gal as determined by chemiluminescence(Fig. 4B). When all of the muscles were analyzed together,the mean percentage of muscle cross-sectional areademonstrating h-gal positivity amounted to 22.1 F3.9% versus 3.0 F 0.9% ( P b 0.001) for injection volumesof 12.5 and 25 ml/kg, respectively.

Even at the lower injection volume of 12.5 ml/kg,transfection efficiency was generally higher in posterior



FIG. 3. Differential effects of intraarterial volume delivery on IM pressure and

arterial blood pressure during 12.5 (n = 2), 16 (n = 1), 19 (n = 1), and 25 (n =

2) ml/kg volume administration into the pig femoral artery.


than in anterior compartment muscles, although someoverlap was present. Interestingly, differences in trans-fection efficiency between the posterior and the anteriorcompartment muscles were most pronounced for theGAST and TA, respectively. Approximately 60% of thetotal muscle area was transfected in the GAST, whereasonly about 3% of the TA muscle area demonstratedpositive h-gal staining under the same conditions (seeFigs. 4A and 5). Fig. 6 shows that for most musclesexamined, there was also a proximal-to-distal gradient intransfection efficiency, with higher values being found inthe more proximal portions of the muscle. We did not



find any major differences in transfection either betweendeep and superficial components of the muscles orbetween the medial and the lateral gastrocnemius inthe pig.

Finally, we also evaluated two potential adverse effectsof intraarterial pDNA delivery: (1) unwanted transfectionof various organs and (2) muscle damage. With respect tothe former, we found no increase in h-gal expression inporcine lungs, heart, or liver with either the 12.5 or the25 ml/kg injections (data not shown). The animalsambulated normally on the day after the surgery.Histological analysis revealed that only 5 of 16 musclesanalyzed (n = 8 for each injection volume) demonstratedsmall areas of inflammatory cell infiltration at the time ofeuthanasia, with the mean value for the damagedmuscles (n = 5) representing 1.44 F 0.006% of musclecross-sectional area. There was no significant differencein the level of histological damage induced by the twoinjection volumes. However, serum assays for creatinekinase (CK) and lactate dehydrogenase (LDH) (Fig. 7)performed in a larger number of animals (n = 4 for eachinjection volume) revealed higher values of LDH in the25 ml/kg group at 24 h postinjection ( P = 0.01), with asimilar trend being found at the same time point for CK( P = 0.09). The CK and LDH values had returned tobaseline in both injection volume groups by 72 h post-injection. White blood cells, hematocrit, and plateletswere unaffected.

DISCUSSION

The present study is the first to address the potential roleof raised IM pressure as a factor that could inhibit pDNA-mediated gene transfer during intraarterial plasmiddelivery to skeletal muscles. Importantly, there was asignificant negative correlation between raised IM pres-sure and muscle fiber transfection. Moreover, modifica-

FIG. 4. Quantification of (A) total number of h-gal-

expressing fibers and (B) h-gal protein, after pDNA

administration into the pig femoral artery using an

injection volume of either 12.5 or 25 ml/kg. Four

muscles were sampled from the anterior compart-

ment (TA; EDL, extensor digitorum longus; PT,

peroneus tertius; PL, peroneus longus) as well as

the posterior compartment (GAST; SOL, soleus; TC,

tibialis caudalis; FDL, flexor digitorum longus) of the

pig hind limb. Data represent the average values

pooled from all regions (proximal, middle, and

distal) of each muscle.

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FIG. 5. Representative photomicrographs of

histochemical staining for h-gal in GAST,

SOL, EDL, and TA at the two different

injection volumes (12.5 and 25 ml/kg).

Magnification is identical for all photomicro-

graphs; scale bar, 1 mm.


tion of the injection protocol to reduce IM pressure led togreatly improved levels of gene transfer. Under the mostoptimized conditions tested in our study, approximately60% of muscle fibers within the pig GAST expressed theh-gal transgene. The other major findings of the presentstudy can be summarized as follows: (1) changes in IMpressure during intraarterial injections varied greatly inmagnitude as well as timing between the differentmuscles examined; (2) there was mild muscle damageassociated with the intraarterial injections, whichappeared to be exacerbated by higher IM pressureconditions; and (3) while intraarterial administration ofhistamine prior to pDNA delivery had no effect on IMpressure, it significantly increased transfection efficiency,but only under the low IM pressure conditions found inthe GAST.

Comparison with Previous StudiesFew studies have performed intraarterial delivery ofnaked DNA to skeletal muscles [8,9,12], and to ourknowledge there is only one other published report in alarge animal model [9]. In prior investigations, a widespectrum of injection volumes was employed, rangingfrom approximately 40 to 80 ml/kg in mice [12], 50 to 65ml/kg in rats [8,9], and 13 to 30 ml/kg in the lower limbsof monkeys [9]. The dosage of pDNA injected has

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generally ranged from approximately 1.5 to 3.0 mg/kgof body weight, although Liang et al. [12] administeredsubstantially higher levels (about 15 mg/kg). In general, ahigh degree of variability in transfection efficiency wasnoted among different muscles in these previous studies.In addition, substantially higher levels of transfection inthe posterior compared to the anterior compartmentmuscles of the lower hindlimb were observed [8,9].However, the reasons for these variations in transfectionefficiency were not determined. Our findings suggestthat different levels of IM pressure among differentmuscles could account for this large variability in trans-fection efficiency.

Based upon the information provided in their report,Zhang et al. [9] delivered 1.3–3.0 mg/kg pDNA andachieved a transfection level averaging 7.3% of myofiberswithin the lower limb muscles of monkeys. In thecurrent study, we administered 1.6–2.0 mg/kg pDNA topigs and achieved transfection levels averaging 22.1% ofall myofibers, but only under low IM pressure conditions.Our study is also the first to determine the effects ofintraarterial histamine and large volume of pDNAadministration on systemic hemodynamics in a largeanimal model. Interestingly, we found that despitefemoral vein clamping and application of a tourniquetabove the level of injection, a significant effect of



FIG. 6. Number of h-gal-expressing fibers quantified within proximal, middle,

and distal muscle regions under optimized conditions (12.5 ml/kg injection

volume). In most cases, there was higher transfection within the more

proximal regions of targeted muscles.


histamine upon systemic arterial blood pressure wasnonetheless observed in the pigs. This is consistent withthe fact that small increases in central venous pressurewere also found to occur during the intraarterial injec-tions. However, histamine effects were transient andeffectively counteracted by the use of an anti-histaminemedication and dextran, such that the fall in bloodpressure after histamine administration was well toler-ated by the animals.

FIG. 7. (A) Creatine kinase and (B) lactate dehy-

drogenase activities at 24, 48, 72, and 120 h after

injection of either 12.5 or 25 ml/kg volume into the

pig femoral artery. The mean data are expressed as

a percentage of baseline values obtained for each

animal immediately prior to the intraarterial injec-

tion. Data include pigs that received intraarterial

volume injections without pDNA, which did not

differ in their enzyme leakage responses.



Potential MechanismsDuring intraarterial injections as performed in the presentstudy, the associated changes in hydrostatic and colloidosmotic pressures favor fluid extravasation out of themicrovessels and into the skeletal muscle tissue [16–19]. Itis by virtue of this increased fluid flow into the muscleinterstitial compartment that convective transport ofpDNA is also able to occur. Hence, a certain degree ofmuscle edema is clearly necessary to achieve successfulskeletal muscle transfection using this approach. How-ever, optimizing transfection requires that one achieve theproper physiological balance between attaining sufficientfluid flow into the muscle on the one hand and avoidingthe point at which muscle edema begins to generateexcessive increases in IM pressure on the other hand. Thepoint at which the latter occurs will be a function of thecompliance characteristics of the muscle compartment,which is largely determined by its anatomic configurationand location [13,20]. Accordingly, if the muscle is largelyconstrained by surrounding structures with little room forvolume expansion, this will constitute a relatively low-compliance (i.e., stiff) compartment in which smallincreases in muscle volume lead to large rises in IMpressure. This likely explains the much larger and morerapid increase in IM pressure within the poorly transfectedTA, which is enveloped by immediately adjacent bone(tibia) and a thick fascial layer on its anterolateral surface.Indeed, the TA is the muscle most frequently involved inthe compartment syndrome in clinical practice [15]. Onthe other hand, the bidealQ target for intraarterial pDNAdelivery would appear to be a high-compliance (i.e.,nonstiff) muscle compartment, which will permit a highamount of fluid to be pumped into the muscle with arelatively small associated increase in IM pressure.

What is the mechanistic basis for the negative relation-ship between IM pressure levels and transfection effi-

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ciency under these conditions? One major possibility is adirect effect of increased IM pressure in reducing micro-circulatory flow to the muscle and hence pDNA delivery.Because both small-caliber arterioles and venules arecollapsible structures, they will inevitably undergo closureonce transmural pressure across the vessel wall (i.e.,extraluminal minus intraluminal pressure) reaches acritical level. This occurs even when large vessel distalpulses remain intact, since it is the microcirculation thatis predominantly affected [13]. It has been reported thatblood flow to the microcirculation ceases when the IMpressure rises to a level that approaches (within 20 mmHg) the diastolic blood pressure [14]. Although one wouldexpect the intraarterial injection to alter this relationshipby increasing intraluminal pressure, it is interesting tonote that peak IM pressure in the porcine GAST wasessentially equivalent to diastolic blood pressure at thelower (12.5 ml/kg) injection volume, and these conditionswere associated with high transfection efficiency. Incontrast, IM pressure values in the TA at both injectionvolumes as well as in the GAST at the higher injectionvolume (25 ml/kg) greatly exceeded diastolic bloodpressure, and this was also associated with extremely poorlevels of muscle fiber transfection.

Although we favor a reduction in pDNA delivery to themicrocirculation due to high IM pressure and consequentarteriolar/venular collapse as the most likely explanationfor our findings, other mechanisms are possible. Forexample, it is conceivable that excessive muscle edemainterferes with transfection by a dilutional effect onpDNA, which could decrease the chances for physicalcontact between pDNA particles and myofibers. On theother hand, it is equally plausible that movement of fluidwithin the muscle interstitium after intraarterial injec-tion would increase the probability of such pDNA–myofiber contacts occurring. To the extent that an activetransport mechanism has been proposed for pDNAuptake by muscle fibers [21], this could also be affectedin some as yet undetermined fashion by differentinjection volumes. Finally, although the muscle damageinduced by intraarterial injections was negligible at thelight microscopic level, serum assays for CK and LDHindicated significant muscle fiber membrane leakinesswithin the first 24 h postinjection. Because this was morepronounced after the 25 ml/kg injection volume, it ispossible that greater myofiber membrane damage underthese conditions prevented efficient transfection,although one might also posit the opposite effect sincesarcolemmal disruptions could theoretically favor pDNAentry into the cell [22]. Whatever the precise mechanismsinvolved, our data strongly suggest that IM pressureincreases produced by large volume/high pressure intra-arterial injection begin to hinder muscle transfectiongreatly at a certain point. This problem can be monitoredas well as successfully modified by the use of a simpletechnique to measure IM pressure levels.

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Clinical Implications and LimitationsBecause skeletal muscle has a rich capillary network thatlies in close contact with the surrounding muscle fibers,intraarterial delivery of pDNA is a theoretically attractivemethod for achieving effective therapeutic gene transferin patients suffering from DMD. The results of our studyprovide optimism that this may be feasible. It is note-worthy that dystrophin expression at approximately 20%of wild-type levels in transgenic mdx mice, if present in afairly even distribution throughout the muscle, is suffi-cient to prevent muscle weakness [7]. Accordingly, if thelevels of pDNA-mediated gene transfer achieved by intra-arterial injection under the optimized conditions estab-lished in this study can also be attained in human DMDpatients, this would be expected to have a clinicallyrelevant therapeutic effect on dystrophic muscle func-tion. Interestingly, it has recently been reported thatsignificant skeletal muscle transgene expression can alsobe attained after high-pressure intravenous injection ofpDNA, provided that proximal venous outflow from thelimb is occluded as in the present study [23]. The samefundamental physiological principles outlined in ourstudy should also be applicable during such intravenousinjections, although the quantitative relationshipbetween IM pressure and factors such as injectate volume,speed of injection, and so forth would likely differ. Inaddition, it is important to recognize that a particular setof injection conditions may be optimal for one musclegroup but not another. Therefore, the injection parame-ters that are ultimately selected for clinical applicationwill likely represent a compromise position, which isdesigned to optimize transfection efficiency in the mostfunctionally important muscles.

Finally, it should be noted that some caution iswarranted in extrapolating the findings of the presentstudy to DMD patients. First, local tissue factors indystrophic muscles could adversely affect pDNA-medi-ated gene transfer by the intraarterial (as well as theintravenous) route. For example, to the extent thatfibrosis eventually leads to increased stiffness of dystro-phic muscles, intraarterial injection conditions that aregeared to normal muscles could lead to excessively largeand rapid increases in IM pressure in DMD muscles.Therefore, monitoring of IM pressure in dystrophicmuscles is needed to determine the optimal volume andinfusion rate to be applied under these specific condi-tions. A second important factor to consider is thepotential adverse impact of this approach on cardiacfunction, since patients with DMD eventually develop asignificant cardiomyopathy [24]. Although the transienthypotension induced by histamine and subsequent smallincrease in central venous pressure associated with largevolume infusion into the femoral artery were welltolerated in normal pigs, this might not be so in thesetting of a preexistent cardiomyopathy. This problemcould presumably be managed by appropriate monitoring




of central hemodynamics, as was done in the presentstudy, and further minimized by instituting therapy at arelatively early point in the disease process prior to thedevelopment of major cardiac dysfunction. Last, althoughthe muscle damage incurred by our intraarterial genedelivery approach was transient and caused no significantmuscle fiber necrosis in normal animals, this may not bethe case in diseased muscles. This particular issue will alsoneed to be studied in greater detail in animal models ofDMD as well as other potential disease targets.

MATERIALS AND METHODS

Plasmid DNA preparation. pDNA was produced according to standard

methods and purified to remove contamination with endotoxin using the

Qiagen EndoFree plasmid preparation kit (Qiagen, Valencia, CA, USA).

The expression plasmid used in all experiments has been previously

described [22] and contained a LacZ cDNA under the control of a chicken

h-actin promoter/cytomegalovirus enhancer construct that is highly

active in skeletal muscle.

Animal procurement and preparation. All experimental protocols were

approved by the institutional animal care and ethics committee, in

accordance with the guidelines issued by the Canadian Council on

Animal Care. Surgical procedures were performed under sterile condi-

tions. Adult male Sprague–Dawley rats (Charles River Laboratories, St.

Constant, QC, Canada) weighing 300–350 g were entered into the study

at 8–10 weeks of age. All surgical procedures in rats were performed during

spontaneous breathing, after anesthesia by intraperitoneal injection of

sodium pentobarbital (65 mg/kg). Specific-pathogen-free, conditioned

pigs (Denis Vadnais, Inc., Drummondville, QC, Canada) weighing 25–30

kg were premedicated by intramuscular injection of tiletamine (5 mg/kg)

and zolazepam (5 mg/kg) together with atropine (0.8 mg). The pigs were

then intubated with a No. 6.0 endotracheal tube and mechanically

ventilated with a tidal volume and respiratory rate of 15 ml/kg and 18 per

minute, respectively. General anesthesia in pigs was maintained using an

inhalational mixture of isoflurane and oxygen. Rats and pigs received

postoperative analgesia for 24–48 h with buprenorphine as required.

Intramuscular tissue fluid pressure. In both rats and pigs, IM pressure

was measured in the anterior (TA) and posterior (GAST) compartments of

the hind limb using a previously validated slit catheter technique [13,14].

Briefly, a 22-gauge catheter (Cathlon IV; Jelco, Raritan, NJ, USA) was

introduced into the midportion of the TA and GAST in rats, such that the

cannula entered into the muscles at an angle of approximately 458. The

inner needle was removed, and a liquid-filled polyethylene tube (PE-50;

Becton–Dickinson, Parsippany, NJ, USA) was introduced through the

catheter and then flushed with isotonic saline. Prior to insertion, the PE

tubing was modified to incorporate four 5-mm-long slits at the tip of the

end placed within the muscle. The 22-gauge catheter was removed from

the muscles so that only the PE-50 tubing remained, and the latter was

then connected to a calibrated pressure transducer system (Transbridge,

Model TBM4-E; World Precision Instruments, FL, USA). Measurements of

IM pressure in the TA and GAST were made in the same way for pigs,

except that the introducing catheter (14-gauge) and polyethylene tubing

(PE-190) diameters were larger. The physiological signals were fed into a

paper chart recorder (Gould Instrument Systems, Valleyview, OH, USA),

and the integrity of the signals was verified before each experiment by

demonstrating an appropriate dynamic response to palpation over the

anterior and posterior hind-limb compartments.

Hemodynamic measurements. To allow for continuous monitoring of

arterial and central venous blood pressures in pigs, 8-Fr introducer

catheters (Cordis Corp., Miami, FL, USA) were inserted into the carotid

artery and internal jugular vein, respectively, via a percutaneous

approach. These were then connected via 12-Fr tubing to hemody-



namic pressure transducers (Transpac; Abbot Critical Care System,

Chicago, IL, USA).

Intraarterial pDNA injections. Under general anesthesia as outlined

above, the femoral artery was surgically isolated to allow intraarterial

administration of different fluid volumes and pharmacological agents as

well as pDNA. The overall approach was similar in rats and pigs, but with

the following variations in instrumentation and dosing in accordance

with the different body sizes of the two animal species:

(i) Rats: The femoral artery was cannulated with a 23-gauge

Butterfly needle, with the tip of the catheter directed distally toward

the foot. The venous circulation of the same hind limb was then

obstructed by placing a vascular clamp directly on the femoral vein.

Over a period of approximately 30 s, 2 mg (1 mg/ml solution in

normal saline) of papaverine (Sabex, Inc., Boucherville, QC, Canada)

was first infused into the femoral artery. This was followed 5 min

later by intraarterial administration of 500 Ag of pDNA diluted in 25

ml/kg normal saline, which was manually injected over a period of 5–

10 s as previously described [8]. In a subset of rats, 2 mg of histamine

(Sigma Chemical Co., St. Louis, MO, USA) mixed with 2 mg of

papaverine was given, followed 5 min later by pDNA administration

injected in the same manner as described above. The clamp on the

femoral vein was removed 20 min after pDNA administration, the

skin layer was closed, and animals were allowed to recover from

anesthesia.

(ii) Pigs: An 8-Fr pigtail catheter was introduced into the femoral

artery, and a tourniquet was placed high in the thigh at a location

proximal to the femoral arterial catheter. The tourniquet was held in a

stable position with the aid of surgical clamps and consisted of heavy wire

surrounded by soft rubber tubing to avoid trauma to the skin. After

venous outflow from the hind limb was obstructed by tightening the

tourniquet and applying a vascular clamp to the femoral vein, 10 mg of

papaverine was infused into the femoral artery over a period of

approximately 30 s, followed 5 min later by administration of 10 mg of

histamine mixed with 10 mg of papaverine. This was followed by rapid

intraarterial bolus injection of different volumes of normal saline (12.5,

16, 19, and 25 ml/kg of body weight) into the hind limb, delivered by a

peristaltic pump (Masterflex L/S Easyload; Cole-Parmer Instrument Co.,

Vernon Hills, CT, USA) at an infusion rate of 3400 ml/min. In a subset of

pigs (12.5 and 25 ml/kg groups), 50 mg of pDNA was diluted in the

infusate. The onset of the pDNA infusion was timed to correspond to the

maximal effect of histamine as judged by the trough in arterial blood

pressure, which generally occurred 20–40 s after histamine delivery. The

pigs were then treated with the anti-histamine diphenhydramine (50 mg;

Sabex, Inc., Boucherville, QC, Canada) and the volume expander Dextran

40 (500 ml; Baxter Corp., Toronto, ON, Canada), to counteract the

adverse effects of histamine administration on systemic arterial blood

pressure. The tourniquet was gradually released over the next 10 min, and

radiographic contrast dye was injected into the femoral artery to confirm

fluoroscopically the absence of arterial rupture or leakage into the

surrounding tissues. The femoral arterial catheter and venous clamp were

removed, and the fascia and overlying skin were repaired. The total

duration of anesthesia was approximately 2 h.

Detection of transgene expression by chemiluminescence. Animals were

euthanized 5 days after surgery, and the muscles, lung, heart, and liver

were collected. Quantitation of h-galactosidase, the LacZ gene product,

was performed on tissue homogenates using a commercially available kit

(Galacto-Light; Tropix, Inc., Bedford, MA, USA). Each organ sample was

blotted dry and weighed before being frozen in liquid nitrogen. It was

then pulverized into a powder, suspended in lysis solution, and

homogenized. The homogenate was assayed for h-gal activity by

chemiluminescence according to the manufacturer’s instructions. A

standard curve was carried out using commercially available h-gal protein

(Boehringer Mannheim, Laval, QC, Canada). Values for h-gal expression

were normalized to total protein within the sample.

Morphometric quantification of gene transfer efficiency and muscle

damage. Muscle, lung, heart, and liver sections (10 mm) were fixed in

1.5% glutaraldehyde in PBS for 3 min and then rinsed with PBS at room

265


temperature. Sections were stained overnight for h-gal activity by

incubation at 378C with 400 mg/ml X-gal, 1 mM MgCl2, 5 mM potassium

ferrocyanide, and 5 mM ferricyanide in PBS. To evaluate muscle damage,

muscle sections were also stained with hematoxylin and eosin. Micro-

scopically visualized sections were photographed using a digital camera

and the image was stored on computer.

Muscle fiber counts were performed from cross sections exhibiting

the maximal number of blue fibers, irrespective of the intensity of blue

staining. Similarly, the muscle sections with the greatest amount of

inflammatory cell infiltration were selected for the muscle damage

evaluation. For rats, total cross sections taken from the midbelly of the

muscles were analyzed. For pigs, muscles were first divided longitudi-

nally in their midline, so that both deep and superficial portions of the

muscle were included; these were then analyzed in their entirety, and

the results were combined. In pig gastrocnemius, the lateral and medial

portions of the muscles were analyzed separately in this manner and

combined. Analysis of the number of individual muscle fibers was

performed by manual tag using a commercial software package (Image-

Pro Plus, Media Cybernetics, Silver Springs, MD, USA). To quantify the

percentage area of h-gal-positive fibers and muscle damage in pigs, a

100-point grid was superimposed on the microscopic images and a

standard stereologic point-counting method was employed as previously

described [5].

Hematologic assays. White blood cells, hematocrit, platelets, and CK and

LDH activity were measured in pigs. Blood samples were taken from the

jugular vein at the time of surgery (prior to intraarterial injection) and

then again at 24, 48, 72, and 120 h postsurgery.

Statistical analysis. All data were analyzed with a statistics software

package (SigmaStat, SPSS, Inc., Chicago, IL, USA). Differences between

histamine treatment and nontreatment groups were determined using

Student’s t test for independent samples. Differences in blood pressure

before and after histamine infusion were determined using a paired t test.

Comparisons of the effects of different intraarterial volumes were

evaluated by one-way ANOVA, with post hoc application of the Tukey

procedure where appropriate. Correlation analysis was performed using

the Pearson product moment correlation method. Statistical significance

was defined as P b 0.05.

ACKNOWLEDGMENTS

This study was supported by the Muscular Dystrophy Association (USA), the

Canadian Institutes of Health Research, the Fonds de la Recherche en Sante du

Quebec, and the Association Francaise contre les Myopathies. This is NRC

Publication No. 37730.

RECEIVED FOR PUBLICATION JUNE 30, 2004; ACCEPTED SEPTEMBER 22,

2004.

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Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model