Developments in gene therapy for muscular dystrophy

Developments in Gene Therapy for Muscular DystrophyDENNIS HARTIGAN-O’CONNOR1,3 AND JEFFREY S. CHAMBERLAIN1,2,3,*1Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-06182Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-06183Center for Gene Therapy, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618

KEY WORDS dystrophin; Duchenne; limb-girdle; vector; adenovirus; gutted; gutless; helper-dependent; AAV; lentivirus

ABSTRACT Gene therapy for muscular dystrophy (MD) presents significant challenges,including the large amount of muscle tissue in the body, the large size of many genes defective indifferent muscular dystrophies, and the possibility of a host immune response against thetherapeutic gene. Overcoming these challenges requires the development and delivery of suitablegene transfer vectors. Encouraging progress has been made in modifying adenovirus (Ad) vectors toreduce immune response and increase capacity. Recently developed gutted Ad vectors can deliverfull-length dystrophin cDNA expression vectors to muscle tissue. Using muscle-specific promoters todrive dystrophin expression, a strong immune response has not been observed in mdx mice.Adeno-associated virus (AAV) vectors can deliver small genes to muscle without provocation of asignificant immune response, which should allow long-term expression of several MD genes. AAVvectors have also been used to deliver sarcoglycan genes to entire muscle groups. These advancesand others reviewed here suggest that barriers to gene therapy for MD are surmountable. Microsc.Res. Tech. 48:223–238, 2000. r 2000 Wiley-Liss, Inc.

INTRODUCTIONThe muscular dystrophies (MDs) are a common set of

genetic disorders. Since none of the underlying biochemi-cal defects were described until 10 years ago, treatmentfor these disorders has been mostly palliative. In theabsence of a biological explanation for muscle weak-ness, little progress toward identifying rational treat-ments could be expected. This situation changed withthe identification of dystrophin in 1987 (Koenig et al.,1987), which cleared the way for biochemical studies ofthe defect underlying DMD and offered hope that otherdystrophies might be similarly understood.

The genetics of many types of MD have now beendelineated in considerable detail. In addition to muta-tions of dystrophin and the dystrophin-associated glyco-proteins (Bonnemann et al., 1996; Ozawa et al., 1995),mutations in genes including calpain (Richard et al.,1995), caveolin-3 (Minetti et al., 1998), laminin-alpha2(Helbling-Leclerc et al., 1995), fukutin (Kobayashi etal., 1998), and emerin (Bione et al., 1994) have beenshown to cause forms of MD (Fig. 1). Each of thesemolecules must play a functionally important role inmuscle and so offers an opportunity for understandingthe biology of muscle and the pathogenesis of dystro-phy.

The potential of gene therapy for treatment of geneticdisease was understood long before the development ofpositional techniques for identification of disease genes(Freese, 1972). When the dystrophin gene was identi-fied, many scientists hoped that gene therapy for DMDwould follow quickly. However, the difficulties of usinggene transfer to treat DMD soon became clear. Themajor obstacles included the large size of the dystro-phin gene (Table 1), the large mass of post-mitoticmuscle cells in the body, and the tendency of theimmune system to reject novel antigens. It is now clear

that efficient, long-term transfer of dystrophin requiredsignificant new vector technologies beyond those avail-able in 1987.

Today there is room for cautious optimism that DMDand other muscular dystrophies will be treated withgene therapy. Scientists have achieved a new under-standing of the requirements for, and obstacles to,successful gene transfer. There is greater respect forboth the capabilities and the limitations of availableviral vectors. By choosing the best vector for use in aparticular setting, significant success has already beenachieved.

MUSCULAR DYSTROPHY AS A CANDIDATEFOR GENE THERAPY

The earliest clinical gene therapy protocols targetedcells of the hematopoietic system (Culver et al., 1991;Rosenberg et al., 1990). There were at least two advan-tages to this choice. First, gene transduction could beperformed ex vivo, which allowed for in vitro selectionof genetically altered cells. Second, there are diseases ofthe hematopoietic system in which corrected cells shouldhave a selective growth advantage over uncorrectedcells, allowing for successful therapy even when aminority of target cells are transduced. In principle, itcan be argued that the muscular dystrophies shouldshare these advantages. Dystrophic myoblasts can beisolated and manipulated ex vivo. Transplantation ofcorrected myoblasts into dystrophic muscle could re-store normal muscle function. Since myoblasts canproliferate considerably before fusing into myofibers,

*Correspondence to: Jeffrey S. Chamberlain, Program in Cellular and Molecu-lar Biology, University of Michigan Medical School, Ann Arbor, Michigan48109-0618. E-mail: [email protected].

Received 20 October 1999; accepted in revised form 21 October 1999

MICROSCOPY RESEARCH AND TECHNIQUE 48:223–238 (2000)

r 2000 WILEY-LISS, INC.

an individual myoblast or myogenic stem cell couldcorrect a large volume of muscle tissue (Engel andFranzini-Armstrong, 1994). Also, corrected muscle fi-bers would likely display a strong selective advantageover dystrophic fibers, which have a limited half-life(Morgan et al., 1993). Unfortunately, the replicativecapacity of myoblasts from dystrophic humans is low,which greatly limits the possibilities for culture, correc-tion, and reinfusion of these cells (Blau et al., 1983).Heterologous myoblast transfer, which could bring nor-mal dystrophin genes into the body, has met with littlesuccess (Mendell et al., 1995). This latter scenario hasbeen limited by immunologic rejection of donor myo-blasts and by the loss of myogenic potential followinglarge-scale culturing in vitro (Gussoni et al., 1996;Karpati et al., 1993). Nonetheless, recent advances inidentifying myogenic stem cells suggest that ex vivostrategies might one day be developed for the musculardystrophies (Ferrari et al., 1998; Gussoni et al., 1999).

At the present time, direct gene transfer to muscle isa more promising approach for gene therapy of MDs, sothis review is focused on transduction of muscle tissuein vivo. Muscle tissue has several advantageous charac-teristics for gene therapy. First, the bulk of skeletalmuscle is easily accessible for experimental manipula-tion. In addition, muscle tissue is efficiently transducedby commonly used viral vectors, including adenovirusand AAV (see Fig. 4; Kessler et al., 1996; Xiao et al.,1996). Myofibers have long lifespans in vivo, whichshould facilitate long-term gene transfer. Finally, fewalternative treatments are available for the musculardystrophies, which justifies the development of poten-tially expensive gene replacement therapies. On theother hand, gene therapy of muscle presents at leastone daunting challenge: muscle tissue comprises over40% of body mass. Most muscular dystrophies—withthe possible exception of merosin-deficient CMD (Vil-quin et al., 1996)—are caused by cell-autonomous de-fects, which argues that the majority of muscle cells willhave to be treated individually (Phelps et al., 1995;Rafael et al., 1994). Currently most gene delivery tomuscle is accomplished by intramuscular injection ofvector particles, an impractical approach for treatmentof all muscles in the body. In the future, vasculardelivery systems will have to be developed to make genereplacement practical (see Fig. 5; Greelish et al., 1999).

The various forms of MD are not equally well suitedto the development of gene replacement therapy. Du-chenne muscular dystrophy has received the mostattention because it is the most common type of dystro-phy and its pathogenesis was the first to be elucidatedat the molecular level (Fig. 1). Replacement of a singleisoform of dystrophin in striated muscle is sufficient forelimination of the major symptoms of the disease, animportant consideration given the widespread expres-sion of dystrophin isoforms (Cox et al., 1993). Further-more, low-level dystrophin expression in a simple major-ity of muscle fibers has been shown to suffice forelimination of symptoms (Phelps et al., 1995). One ofthe major difficulties with the development of gene

TABLE 1. DNA sequences used for gene therapyof muscular dystrophy

Sequence Sizea (approximate kb)

Conventional Ad 8–10BMD minigene 6.2DH2-R19 minigene 6.0Laminin-a2 9.3

Gutted Ad 27–36Dystrophin 11.2Utrophin 10.3

AAV 4.8a-sarcoglycan 1.2emerin 0.8

Lentivirus 8.9CMV 0.7MCK (full length) 6.5MCK (truncated) 3.3MCK (synthetic) 0.6Human skeletal a-actin 2.2

aFor viral vector systems, listed in bold, the approximate capacity for foreignDNA is given. For potentially therapeutic genes, the length of the coding region ofthe cDNA is given. The last five items in the table refer to frequently usedpromoters.

Fig. 1. The dystrophin-glycoprotein complex (DGC). The DGCforms a structural link between the actin cytroskeleton and laminin-2in the extracellular matrix. Integral components of this link includedystrophin, the dystroglycans, the sarcoglycans (SGs), sarcospan (SP),and various proteins associated with the C-terminus of dystrophin,including syntrophin (SYN), dystrobrevin (Db), and MAST. Many

forms of muscular dystrophy (MD) are caused by mutations in genescoding for DGC proteins. Shown are congenital MD, one form of whichis caused by mutations in a laminin subunit; limb-girdle MD, forms ofwhich are caused by mutations of each of the SGs; Duchenne MD,caused by mutations in the dystrophin gene; and Bethlem myopathy,caused by mutations in subunits of collagen type VI.

224 D. HARTIGAN-O’CONNOR AND J.S. CHAMBERLAIN

replacement therapy for DMD has proven to be thelarge size of the dystrophin gene, which has requireddevelopment of entirely new vector systems for efficientdelivery (see Figs. 4, 6; Table 1).

The sarcoglycanopathies, as targets for developmentof gene therapy, share some of the advantages of DMDand are caused by defects in relatively small genes (Fig.1; see Fig. 7; Table 1). Since alpha- and gamma-sarcoglycan are expressed primarily or exclusively instriated muscle, gene transfer to striated muscle shouldcorrect most symptoms of limb-girdle muscular dystro-phy (LGMD) 2D and 2C (Bonnemann et al., 1995;Coral-Vazquez et al., 1999; Jung et al., 1996; Lim et al.,1995; McNally et al., 1994; Nigro et al., 1996; Noguchiet al., 1995; Roberds et al., 1993, 1994; Straub et al.,1999). Beta- and delta-sarcoglycan, however, are ex-pressed in both smooth and striated muscle, so com-plete correction of LGMD2E and 2F may require morewidespread gene expression (Coral-Vazquez et al., 1999;Straub et al., 1999). Though the levels of gene transferrequired for functional correction have not been system-atically investigated, it is likely that correction of amajority of fibers would be sufficient, given the fact thatthe sarcoglycanopathies and DMD cause dystrophythrough an effect on the same protein complex (Fig. 1;Straub and Campbell, 1997). Perhaps most impor-tantly, the coding region of each sarcoglycan gene is lessthan 2 kb, enabling delivery using an adeno-associatedvirus (AAV) vector (Fig. 4; Table 1; Lim and Campbell,1998). AAV has been shown to deliver neoantigens tomuscle without elicitation of an immune response-thebasic goal of all gene replacement therapies (Kessler etal., 1996; Xiao et al., 1996). The major drawback tosarcoglycanopathies as models for gene therapy of MDis their relatively low incidence, which is probably lessthan 5% of that of DMD (Ljunggren et al., 1995).

Other muscular dystrophies have received limitedattention as candidates for gene therapy. Merosin-deficient CMD (MCMD), for example, appears to becaused by a defect that is not entirely cell-autonomous,which could allow phenotypic correction despite trans-duction of a lower proportion of fibers (Fig. 1; Vilquin etal., 1996). Correction of MCMD has been investigatedin transgenic mouse models, where it was shown thatexpression of laminin-alpha2 from a muscle promotercorrected the muscle phenotype but failed to correct arelatively minor neurological phenotype (Kuang et al.,1998). Unfortunately, the laminin-alpha2 mRNA isabout 9.5 kb (Kuang et al., 1998), too long for deliveryusing AAV or conventional Ad vectors (Table 1). Emery-Dreifuss muscular dystrophy (EDMD) is caused bydeficiency of emerin, a widely expressed protein of theinner nuclear membrane and intercalated disks (Bioneet al., 1994; Bonne et al., 1999; Cartegni et al., 1997;Manilal et al., 1996; Nagano et al., 1996). Since themost lethal features of EDMD are caused by absence ofthe protein in heart tissue, rather than skeletal muscletissue, this disease offers the opportunity for effectiveintervention through treatment of a relatively smallmass of tissue (Emery, 1987). In addition, the emerincoding region is less than 1 kb, which allows for itsdelivery in an AAV vector (Table 1). To date, develop-ment of gene therapy for EDMD has been hampered bythe lack of an animal model.

THERAPEUTIC GENES FOR DELIVERYTO DYSTROPHIC MUSCLE

For a recessive genetic disease or a dominant diseasecaused by haploinsufficiency, delivery of the diseasegene itself is an obvious therapeutic choice. Unfortu-nately, gene replacement will not be feasible in allcases. First, some muscular dystrophies are domi-nantly inherited and not amenable to gene replace-ment. Second, gene replacement could lead to an im-mune response against the therapeutic protein inpatients who do not express any significant proteinfrom their disease locus. Finally, the size of the intactdisease gene may be too large for delivery with the viralvector of choice (Table 1). To overcome these difficulties,it may be necessary to deliver a minigene, a homologousgene, or a modulatory gene instead of the disease gene.

The use of a minigene for therapy of DMD wasoriginally suggested by mutation analysis in patientswith mild forms of Becker muscular dystrophy (BMD;Love et al., 1990). A number of these patients had largedeletions in their dystrophin genes, which nonethelessled to the accumulation of small proteins with substan-tial functional capacity (Arahata et al., 1991; Bulman etal., 1991; Monaco et al., 1988). Considerable attentionhas been focused on a patient with a deletion of 46% ofthe dystrophin coding sequence but very mild BMD(BMD minigene in Table 1; England et al., 1990). Thisallele functions well in transgenic animals (Figs. 2, 3;Phelps et al., 1995). Similar minigenes have beendelivered to muscles using both adenoviral and retrovi-ral vectors and some efficacy was observed in young orimmunosuppressed animals (Deconinck et al., 1996;Dunckley et al., 1993; Ragot et al., 1994; Vincent et al.,1993). The obvious problem with the use of suchminigenes is that the means of their identification,study of patients with BMD, ensures that they are lessthan completely effective (Figs. 2, 3). It is, therefore,hoped that creation of recombinant dystrophin mini-genes in the laboratory might yield more effectiveminigenes. For example, we have observed that modifi-cation of some naturally occurring BMD minigenes torestore an integral number of spectrin-like repeatsresulted in a smaller molecule with superior effective-ness (DH2-R19 in Table 1; Figs. 2, 3; Chamberlain et al.,unpublished data). Several other promising alleles,shown to restore the dystrophin-associated glycopro-tein complex and small enough to be delivered usingAAV, have unfortunately failed to provide functionalcorrection in transgenic mice (Cox et al., 1994, Chamber-lain et al., unpublished data).

Delivery of a homologous gene may also prove usefulin some circumstances. This approach could eliminateconcerns about an immune response triggered by deliv-ery of a formerly missing protein. The best example isdelivery of utrophin for therapy of DMD. Utrophin is aprotein closely related to dystrophin, but which isnormally localized to the neuromuscular and myotendi-nous junctions (Helliwell et al., 1992, 1994). Expressionof utrophin uniformly on the sarcolemma using trans-genic animal technologies prevented development ofdystrophic pathology in mdx mice (Tinsley et al., 1998).Utrophin expression on the sarcolemma could in theorybe achieved either by delivery of utrophin in a viralvector or by delivery of factors that stimulate transcrip-

225DEVELOPMENTS IN GENE THERAPY FOR MD

Fig. 2. Histology of wild-type, mdx, and transgenic mdx muscle.Top-left panel, marked DH2-R19, shows muscle histology in an mdxmouse made transgenic for a synthetic dystrophin minigene lackingan integral number of spectrin-like repeats. The histology shown isnormal, as can be seen by comparison with the wild-type panel atlower-right: variation in fiber size is minimal and myonuclei arelocated at the periphery of their fibers. The top-right panel, markedD17–48, shows muscle histology in an mdx mouse made transgenic for

a minigene isolated from a patient with mild Becker musculardystrophy. The histology of this muscle is close to normal, but anoccasional central nucleus is observed, indicating low-level muscleregeneration. The bottom-left panel shows typical muscle histology inmdx mice. Extreme variation in fiber size, frequent centrally nucle-ated fibers, and some fibrosis are observed. Prominent central nucle-ation and fiber size variation indicate ongoing muscle regeneration.


tion of the endogenous gene (Gramolini et al., 1999;Khurana et al., 1999). This approach might be advanta-geous because the utrophin coding region is almost aslarge (10.3 kb) as that of dystrophin (Table 1; Tinsley etal., 1992). The possibility of using homologous geneproducts for therapy has also been raised in other formsof muscular dystrophy, though there is no evidence yetthat this would work. For example, M-laminin ormerosin appears later in muscle development than doesan earlier isoform called A-laminin, which differs frommerosin only in the identity of its heavy chain (Fig. 1;Leivo and Engvall, 1988; Sanes et al., 1990; Xu et al.,1994). If the A-laminin heavy chain could be deliveredto muscle or expression of the endogenous locus couldbe forced, perhaps this approach would be sufficient toalleviate dystrophy. Similarly, epsilon-sarcoglycan is awidely expressed gene with similarity to alpha-sarcogly-can, and it has been suggested that high-level expres-sion of epsilon-sarcoglycan might allow for restorationof the sarcoglycan complex in LGMD type 2D (Ettingeret al., 1997).

Another approach to treating the muscular dystro-phies would be to deliver a modulatory gene togetherwith, or instead of, a replacement for the mutant gene.The MDs are characterized by constant cycles of musclefiber degeneration and regeneration that eventuallyfail (Fig. 2; Blau et al., 1983; Emery, 1993; McArdle etal., 1995). If these cycles could be slowed, or if regenera-tion could be made more robust, it is reasonable toassume that the dystrophic process would be delayed oralleviated. Genes that might achieve these goals in-clude trophic factors that stimulate muscle hypertro-phy, such as IGF-1 (Barton-Davis et al., 1998); regula-tory molecules that can stimulate formation of newmyogenic precursors, such as MyoD (Lattanzi et al.,1998; Megeney et al., 1996); or inhibitors of necrosis orapoptosis, such as calpastatin or Bcl family members(Tidball et al., 1995).

REQUIREMENTS FOR GENE THERAPYOF MUSCULAR DYSTROPHY

Most muscular dystrophies are caused by absence ofa protein in muscle cells. Although some dystrophieshave less obvious effects on the central nervous system,dysfunction of heart or skeletal muscle leads to death.

For DMD and MCMD, it has been proven that genereplacement in striated muscle alone can alleviate themajor features of the disease (Cox et al., 1993; Kuang etal., 1998). For these reasons, gene delivery to musclecells is the most likely route to an effective treatment.Achievement of this goal will require (1) a suitablevector that can be mass-produced cheaply and (2) anefficient means for delivery of the vector to the surfaceof muscle fibers. Vector delivery to the muscle surface isusually accomplished on a small scale by multipleinjections throughout the muscle; however, a systemicvascular delivery system would be vastly superior if onecould be developed. Dystrophin-positive fibers appearto have a survival advantage over negative fibers,which might allow for use of a less efficient deliverysystem in the long term, especially if this selectiveadvantage also applies to the other forms of dystrophy(Morgan et al., 1993).

A vector that can achieve long-term persistence willbe required. Since many patients with MD are diag-nosed early in life, and the goal of an ideal therapy isextension of lifespan into the normal range, the idealtherapy would retain its effectiveness for about 70years. This goal could be achieved by multiple treat-ments throughout life, but each individual treatmentshould retain effectiveness for at least several years.Experience has shown that long-term effectivenessdepends on avoidance of the host immune response, soan appropriate vector must evade the immune systemor be administered together with immune suppression.

A suitable vector must also be capable of carrying anddelivering the therapeutic gene(s). In some cases thedisease gene will be large, but even small genes mayneed to be regulated using large, tissue-specific regula-tory elements or might need to be delivered togetherwith a modulatory gene. Several approaches can betaken to facilitate delivery of long stretches of DNA.First, development of new viruses as gene deliveryvectors should be pursued. Development of a herpessimplex virus (type 1) system, for example, is encourag-ing in this regard (Huard et al., 1995; Marconi et al.,1999). Second, conventional vector systems could beadapted so as to increase their capacity for foreignDNA, as has been recently achieved with the develop-ment of gutted Ad vectors (Fig. 4; see Fig. 6; Fisher et

Fig. 3. Functional correction of dystrophyin transgenic mdx mice. The specific force, orforce per unit cross-sectional area, producedby dystrophic muscle (mdx) is reduced com-pared to that produced by healthy muscle(C57). Transgenic mdx mice bearing a Beckermuscular dystrophy (BMD) dystrophin mini-gene, called D17–48, display partial correc-tion of this parameter. Transgenic mice bear-ing a synthetic minigene with an integralnumber of spectrin-like repeats, called DH2-R19, are fully corrected.


al., 1996; Kochanek et al., 1996; Kumar-Singh andChamberlain, 1996). Use of these vectors eliminatesthe host immune response against viral proteins andmay reduce the response against the therapeutic pro-tein (Schiedner et al., 1998). These vectors have re-cently allowed our laboratory and others to deliverdystrophin without elicitation of a major immune re-sponse (Fig. 4; see Fig. 6).

VIRAL VECTORS FOR MUSCLEGENE THERAPY

Adenoviral Vector TechnologyAdenoviral replication is usually divided into two

stages, early and late, which are divided by the onset ofDNA replication. Only a subset of viral genes, called E1,E2, E3, and E4, are expressed during the early phase(Fig. 4). These genes prepare the host cell for viralreplication by stimulating production of necessary pre-cursors and helping to prevent a host immune re-sponse. During the late phase genes for structuralcomponents of the Ad virion are expressed. Obviously,these structural proteins must produced at high levels;perhaps because of this, they are also the principaltargets of the host immune response (Jooss et al.,1998a). Expression of the late genes requires DNAreplication, though the mechanism behind late geneactivation is not entirely understood (Thomas andMathews, 1980). As a result, expression of late genes

and production of immunogenic late proteins occursonly if the early phase progresses through the onset ofviral DNA replication.

Conventional Ad vectors are created by the replace-ment of viral early genes with an exogenous expressioncassette (Fig. 4). The vectors can be propagated inpackaging cell lines that express the deleted earlygenes, but are essentially replication-defective in non-complementing cell lines and in vivo. First-generationvectors lack E1, which is involved in transactivation ofother viral genes, so immunogenic viral proteins areexpressed at a greatly reduced level (Gaynor and Berk,1983; Nevins, 1981; Yang et al., 1996b).

Adenoviral vectors have some advantageous proper-ties for gene therapy. The serotypes used in creation ofgene delivery vectors usually cause only mild or subclini-cal disease in the wild (Brandt et al., 1969; Schmitz etal., 1983). As a result, inadvertently generated, replica-tion-competent virus is unlikely to harm an immune-competent patient. Adenoviruses can be readily grownto very high titer. Since Ad virions remain tightlyassociated with lysed cells, within whose nucleus theyare packed into crystalline arrays, concentrated stocksare easily prepared through low-speed centrifugationand collection of lysed cells. Adenoviruses efficientlyinfect most human cell types, including immaturemuscle cells (see Fig. 7), though most lymphocytes arerelatively resistant. Finally, the popularity of Ad vec-

Fig. 4. Vectors for gene therapy. The genome of Wild-type Ad maybe divided into early and late regions. Early regions, designated bygradient shading, are replaced by exogenous DNA in conventional Advectors. 1st generation vectors contain deletions in the E1 and E3regions. 2nd generation vectors contain deletions in additional earlyregions-E2B in this example. Ad5bdys is an example of a gutted Advector, in which virtually the entire genome can be replaced byexogenous DNA. AAV vectors also contain deletions of nearly the

entire viral genome, except for the viral ITRs, which are required forreplication and packaging of the vector. The ITRs are only 145 bp insize and are not shown in this diagram. Lentiviral vectors contain aninitial short stretch of viral RNA required for export and packaging offull-length transcripts in the packaging line. The remainder of thegenome may be replaced with exogenous sequences, except for a smallregion near the polyadenylation signal that is required for reversetranscription.


tors has led to development of simple means for theirmanipulation (Chartier et al., 1996; reviewed in Gra-ham and Prevec, 1991).

Unfortunately, Ad vectors have drawbacks that limittheir usefulness for gene replacement therapy of muscu-lar dystrophy. The cloning capacity of first-generationvectors is only about 8 kb, which is the amount of spacemade available by deletion of E1 and E3 (Fig. 4; Table 1;Bett et al., 1993). Obviously, first-generation Ad vectorscannot deliver full-length dystrophin, since the codingregion of dystrophin alone is 11 kb. These vectors can,however, be used to deliver shortened forms of dystro-phin that are generated in the laboratory. Ad vectorscan also be used to deliver smaller genes involved inother forms of muscular dystrophy (see Fig. 7).

A more serious difficulty with first-generation Advectors is leaky expression of immunogenic viral pro-teins in vivo. Despite deletion of E1, viral gene expres-sion and even limited replication can occur in non-complementing cells over a longer time scale. Expressionof viral proteins leads to a host immune response andelimination of gene expression from transduced tissues(Dai et al., 1995; Dong et al., 1996; Gaynor and Berk,1983; Nevins, 1981; Van Ginkel et al., 1995; Yang et al.,1994, 1996b; Yang and Wilson, 1995; Zsengeller et al.,1995). The resulting inflammatory process can evenlead to muscle damage and exacerbation of weakness.Immunosuppressive drugs can partially overcome thisproblem (Lochmuller et al., 1996), but immunosuppres-sion has its own risks and so development of animproved vector would be preferable.

Second Generation Ad VectorsSeveral groups created new Ad vectors lacking addi-

tional early genes in an effort to address problems withfirst-generation vectors (Fig. 4; Amalfitano et al., 1998;Armentano et al., 1995; Gorziglia et al., 1996; Schaacket al., 1995; Wang et al., 1995; Weinberg and Ketner,1983). The deletions target E2A, E2B, and E4, whichare the remaining early regions of the Ad genome. Foreach deletion a corresponding packaging cell line mustbe generated, which expresses the missing proteins intrans. These second-generation vectors provide addi-tional cloning capacity and should further attenuatethe virus. Further inhibition of viral replication isdesirable for two reasons. First, these highly modifiedsecond-generation vectors are less likely to generatereplication-competent virus during large-scale, clinical-grade vector preparation. Second, since expression ofviral late genes requires replication, complete inhibi-tion of Ad genome replication should abolish late geneexpression, which would eliminate the host immuneresponse against late proteins (Amalfitano et al., 1998).

E2A, E2B, and E4 deletions introduced into Advectors have provided about 1.4 kb, 1 kb, and 1.9 kb ofadditional cloning capacity, respectively. Individually,none of the deletions yields a dramatic increase incloning capacity; nonetheless, combining two or three ofthese additional deletions might allow delivery of full-length dystrophin via an Ad vector. However, sincemany second-generation vectors grow to lower viraltiters than first-generation vectors or wild-type Ad(Zhou et al., 1996), combining all these deletions into asingle vector may prove difficult.

Second-generation vectors have been shown to elicita reduced immune response and thereby allow pro-longed transgene expression (Gao et al., 1996; Hu et al.,1999; O’Neal et al., 1998; Wang et al., 1997). Forexample, an E1-, E2B-, and E3-defective vector de-signed in our laboratory demonstrated prolonged trans-gene expression and persistence of vector genomes,along with reduced toxicity, in the liver (Hu et al.,1999). Similar experiments to test the properties ofE2B-defective vectors in muscle are underway. Second-generation vectors therefore offer significant promise insome settings, but further investigation of their advan-tages for treatment of MD are required.

Gutted Adenoviral VectorsGutted, or helper-dependent, Ad vectors may over-

come many drawbacks associated with conventional Adtechnology (Fig. 4). These deleted genomes were thefirst Ad vectors to be developed (Solnick, 1981; Thum-mel et al., 1981), but interest in their use has expandedrecently with several demonstrations of their advan-tages for gene transfer. Growth and purification ofthese viruses was extremely laborious until the recentdevelopment of new techniques (Hardy et al., 1997;Hartigan-O’Connor et al., 1999; Parks et al., 1996).Further improvements will still be required beforeroutine, large-scale growth of clinical-grade guttedvirus is feasible.

Gutted vectors contain cis-acting DNA sequencesthat direct adenoviral replication and packaging but donot contain viral coding sequences (Fig. 4; Fisher et al.,1996; Kochanek et al., 1996; Kumar-Singh and Cham-berlain, 1996). Theoretically, the vectors can accommo-date up to about 37 kb of exogenous DNA, though 28–30kb is more typical (Fig. 4; Table 1). Since gutted vectorsdo not contain any viral genes, expression of viralproteins is not possible. Gutted vectors are defectiveviruses produced by replication in the presence of ahelper virus, which provides all necessary viral pro-teins in trans. Like other defective viruses, guttedviruses are normally prepared as a mixture with helpervirus.

The starting material for production of all guttedviruses is plasmid DNA (Fisher et al., 1996; Kochaneket al., 1996; Kumar-Singh and Chamberlain, 1996).These plasmids contain the viral origins of replication(ITRs), the packaging signal (psi), and therapeutic DNAto be carried by the gutted virus. Co-transfection of thisplasmid and helper viral DNA into a packaging cell lineleads to replication of the helper virus and concomitantreplication of the gutted virus, as directed by the viralITRs contained in the starting plasmid. Robust helpervirus replication causes lysis of the transfected cells.The resulting lysate contains a large number of helpervirions and a relatively small number of gutted virions.To increase the number and proportion of gutted virionsin the lysate, the initial mixture must be seriallypassaged. During serial passage, for unknown reasons,gutted virus is amplified more quickly than helper virusand eventually substantial enrichment occurs. Par-ticles containing gutted viral genomes, rather thanhelper genomes, must then be purified on the basis oftheir lower density (Fisher et al., 1996; Kochanek et al.,1996; Kumar-Singh and Chamberlain, 1996).


A gutted vector can accommodate not only the full-length dystrophin cDNA but also expression cassettescoding for marker or modulatory proteins. The guttedvector Ad5bdys, for example, contains the full-lengthdystrophin cDNA driven by a 3.3 kb muscle creatinekinase promoter as well as a lacZ gene controlled by theCMV promoter for monitoring vector production invitro and gene delivery in vivo (Fig. 4; Kumar-Singhand Chamberlain, 1996). We have developed othervectors that contain dystrophin, either murine or hu-man, and combinations of other regulatory, reportergene, and phenotypic modulatory sequences (see Fig.6). The very large cloning capacity of gutted vectorsopens a new world of therapeutic possibilities that werepreviously not possible.

Ad5bdys delivers full-length dystrophin (and b-galac-tosidase) to the skeletal muscles of mdx mice with anefficiency comparable to that of first-generation viruses(Kumar-Singh and Chamberlain, 1996; Hauser et al.,unpublished data). Gene expression persists for over 1year in immune-deficient SCID/mdx mice, which indi-cates that the complete absence of viral gene expressiondoes not destabilize gutted vector genomes in vivo. Inimmune-competent mdx mice, by contrast, expressionof dystrophin persists for less than 1 month, suggestingthat dystrophin expression may be eliminated by animmune response. Removal of all viral coding se-quences from Ad5bdys was therefore insufficient, byitself, to eliminate a host immune response against allantigens. A newer version of this gutted vector in whichb-galactosidase is expressed from an inducible pro-moter leads to dystrophin expression for at least sev-eral months in immune-competent mice (Hauser et al.,unpublished data). Gutted vectors should be designedto provide targeted expression of therapeutic proteinsin the affected tissue only, a principle that was notfollowed in construction of Ad5bdys. As is discussedfurther below, limiting gene expression to the targettissue may allow avoidance of the immune responseand long-term gene expression.

Adeno-Associated Virus (AAV) VectorsAAV particles were first identified by electron micro-

scopic examination of human and simian Ad prepara-tions (Atchison et al., 1965; Melnick et al., 1965). Theparticles were soon recognized as defective viruses thatnormally replicate only in the presence of Ad (Hogganet al., 1966). It is now known that several other virusesand a variety of genotoxic treatments can also providehelper functions (Schlehofer et al., 1986; Yakinoglu etal., 1988; Yakobson et al., 1987, 1989). In fact, no helpervirus genes are directly involved in replication of AAVDNA; instead, the helper virus seems to maximizesynthesis of cellular proteins involved in AAV replica-tion (Muzyczka, 1992).

AAV is a 4,680-bp parvovirus with a fascinating andunusual life cycle. In the absence of helper virus, AAVinfection is nonproductive and no progeny AAV par-ticles are produced. Instead, the ssDNA genome may beconverted to a double-stranded form and become cova-lently associated with cellular DNA at a specific locuson chromosome 19 (Kotin et al., 1990). If a helper virussubsequently infects the cell, the AAV genome is excisedand begins to undergo lytic growth. The growth of AAVreduces production of helper virus particles and lysis of

the cell eventually produces more AAV particles thanAd particles (Atchison et al., 1965).

Gene delivery vectors based on AAV are prepared byreplacement of all viral coding sequences with therapeu-tic DNA (Fig. 4; Muzyczka, 1992). The total amount ofexogenous DNA that can be carried by AAV vectors iscurrently less than 5 kb, which unfortunately elimi-nates many muscular dystrophy genes from consider-ation (Table 1). The only remaining viral sequences arethe AAV ITRs, which direct replication and packagingof the vector construct. Both AAV proteins and helpervirus proteins must be provided in trans, an arrange-ment that will be recognized as similar to that of guttedadenovirus vectors. Originally, AAV vector stocks weretherefore produced as a mixture of vector particles andhelper virus particles (Hermonat and Muzyczka, 1984).Within the last 2 years, however, helper-virus-freepackaging systems have been developed based on abetter understanding of the requirements for efficientAAV replication (Matsushita et al., 1998; Salvetti et al.,1998; Xiao et al., 1998). Using these systems, pure AAVvectors can be produced at titers nearly equivalent tothose of wild-type Ad.

AAV was originally developed as a gene therapyvector based on the hope that genomic integrationwould allow long-term expression of transgenes. AAVhas, in fact, been a successful vector because in sometissues it provides very long-term expression of trans-genes in the absence of an immune response. Forexample, Xiao et al. (1996) found that expression ofb-galactosidase from an AAV vector was stable for morethan 8 months in the muscle of adult, immune-competent animals. Though humoral responses againstthe AAV particles were observed, no cellular immuneresponse was apparent. This result is astonishing giventhe robust cellular response observed after transduc-tion of the b-galactosidase gene using Ad vectors.

It has turned out that lack of a cellular immuneresponse, not genomic integration, is the most impor-tant factor in maintenance of gene expression in musclecells. Jooss et al. (1998b) showed that administration ofan Ad-lacZ vector can elicit a cellular immune responsethat destroys muscle fibers previously transduced byAAV-lacZ, which could otherwise survive and continueto express b-galactosidase. These authors also demon-strated that AAV-lacZ vectors fail to transduce antigen-presenting cells (APCs) whereas Ad vectors efficientlytransduce such cells. These data suggest that AAVvectors achieve immune evasion and persistent geneexpression through avoidance of antigen presentationby professional APCs such as dendritic cells. Further-more, it seems that most of the AAV genomes in muscletissue are present in the form of large circular multi-mers (Duan et al., 1998). Formation of such multimersmay play a role in the persistence of vector DNA, but Adgenomes can also survive inside the nucleus for longperiods, so physical persistence of episomal DNA can beachieved through several mechanisms.

The striking success of AAV vectors in long-termtransduction of muscle tissue has given rise to hopethat muscular dystrophies caused by defects in smallgenes may soon be treated using this vector. Some of theexciting results achieved so far are described below.Just as importantly, our insight into the mechanismsused by AAV to achieve persistent gene expression may


be applicable to other vector systems that have not yetachieved comparable success.

Lentiviral VectorsMurine retroviral vectors were used in some of the

earliest efforts at gene replacement for DMD (Dunckleyet al., 1992, 1993). As murine retroviral vectors areunable to stably infect non-dividing cells, post-mitoticmuscle fibers were not targeted in these studies. In-stead, investigators attempted to transduce proliferat-ing myoblasts in vitro or in vivo. Although transductionof myoblasts with retroviral vectors is effective anddoes lead to gene transfer after myoblast transplanta-tion, the significant technical hurdles to myoblast trans-plantation remain. Transduction of myoblasts in vivomight be expected to be effective, since muscle regenera-tion is such a prominent feature of dystrophic pathology(Fig. 2). Unfortunately, direct injection of a murineretrovirus carrying minidystrophin resulted in transduc-tion of only a few percent of fibers at the site of injection(Dunckley et al., 1993). Injection of bupivicaine, whichinduces muscle necrosis and regeneration, doubled thenumber of transduced fibers. More recently, implanta-tion of retroviral-producer cells resulted in transduc-tion of greater than 10% of fibers (Fassati et al., 1997).Such an approach is, however, limited by problemsassociated with immunological rejection of the pro-ducer cells. Finally, the 7.8 kb cloning capacity ofretroviral vectors prevents delivery of full-length dystro-phin cDNA clones. As a result, the use of the murineretroviruses for gene replacement therapy in musclewill require significant technical innovation before itbecomes a promising approach.

Recently, however, new retroviral vectors have beenintroduced that can integrate into the genome of post-mitotic cells (Carroll et al., 1994; Naldini et al., 1996;Poeschla et al., 1998). These vectors are based onhuman or feline lentiviruses, which infect nondividingcells as part of their normal life cycle (Lewis et al., 1992;Weinberg et al., 1991). Like the older murine vectors,these vectors are produced by expression of a package-able vector construct in a cell line that expresses viralproteins (Fig. 4). Vector particles bud from the surfaceof such cells continuously and can be harvested fromthe supernatant. Because the lentivirus life cycle isnonlytic and because the enveloped vector particles arenot as stable as naked Ad or AAV particles, the vectortiters obtained are relatively low (about 109 per ml)even after concentration. The size of the vector con-struct is limited to about 10 kb by constraints onretroviral packaging, which again prohibits transfer ofthe full-length dystrophin cDNA (Table 1).

Lentiviral vectors efficiently transduce post-mitoticneurons, hepatocytes, and muscle fibers (Kafri et al.,1997; Naldini et al., 1996). Injection of a lentiviralvector expressing GFP into muscle tissue resulted intransduction of about half the muscle fibers at the siteof injection (Kafri et al., 1997). Gene expression wasmaintained for at least 2 months without elicitation of amajor immune response. It should be noted, however,that the number of particles injected was several ordersof magnitude lower than the number of Ad or AAVparticles that are usually injected. Elicitation of asignificant cellular immune response may require at-tainment of a certain gene expression threshold, which

may not have been achieved in this case (Tripathy et al.,1996). Alternatively, perhaps the VSV-G envelope pro-tein, with which the vector was pseudotyped, does notallow efficient infection of dendritic cells. Further devel-opment of this technology might someday allow foreffective treatment of muscular dystrophy.

DELIVERY OF VIRAL VECTORSTO MUSCLE TISSUE

A single injection into muscle tissue typically resultsin transduction of cells within 0.5–1 cm of the injectionsite. This limited vector diffusion means that satura-tion of a single muscle group would require multipleinjections. Saturation of many large human muscles,including the heart and diaphragm, would requirehundreds of injections and is probably impractical on alarge scale. Therefore, if viral vectors can be provensuitable for stable transduction of muscle on a smallscale, a means for vector delivery to a large mass ofmuscle tissue needs to be developed.

Arterial delivery holds promise as a means to over-coming this difficulty (Fig. 5; Greelish et al., 1999;Welling et al., 1996). Unfortunately, transport of viralvectors across normal vascular endothelium is poor,even when high hydrostatic pressure is applied (Greel-ish et al., 1999; Jejurikar et al., 1997). Greelish et al.(1999) found that arterial instillation of Ad-lacZ underelevated hydrostatic pressure led to efficient transduc-tion of vasculature but resulted in limited muscleinfection (Fig. 5A–C). After vasodilation with papaver-ine and endothelial permeabilization with histamine,however, widespread transduction of entire musclegroups was achieved (Fig. 5D, G, H). Transduction wasstrictly limited to muscle groups served by the perfusedarteries, confirming that gene delivery was accom-plished through arterial delivery (Fig. 5E, F). Thetreatment was effective in both hindlimb and heartusing either Ad or AAV vectors (Fig. 5I). If arterialdelivery can be performed safely and routinely, thentransduction of the majority of human muscle tissue forgene replacement may be feasible.

Effective gene delivery depends not only on transportof vector to target cells, but also on subsequent infec-tion. Tropism of Ad vectors for muscle fibers has beenobserved to decline with muscle maturation, duringwhich process myofibers down-regulate expression ofcellular receptors for adenovirus (Acsadi et al., 1994a;Acsadi et al., 1994b; Nalbantoglu et al., 1999). As aresult, infection of immature or regenerating muscle byAd is more efficient than infection of mature muscle.AAV, in contrast, seems to infect mature muscle asefficiently as immature muscle (Snyder et al., 1997). Itis unknown whether the infectability of dystrophichuman muscle will present a serious impediment togene therapy of MD with Ad vectors, as little is knownabout Ad tropism for human muscle. Some success hasbeen attained in modifying the tropism of adenovirus toimprove infection of macrophage, endothelial, smoothmuscle, fibroblast, and T cells (Douglas et al., 1996;Stein et al., 1999; Wickham et al., 1997); however,engineering of an adenovirus with dramatically in-creased tropism for skeletal muscle has not yet beenachieved. Combining a vascular delivery method with atropism-modified Ad vector could result in efficient gene


transfer to human muscle using a systemic deliverymethod.

GENE REPLACEMENT FOR DMDTransgenic mice have proven the feasibility of gene

replacement in muscle as a means for treatment ofDMD (Figs. 2, 3; Corrado et al., 1996; Cox et al., 1993;Phelps et al., 1995; Rafael et al., 1994; Wells et al.,1995). Several investigators have created mdx miceexpressing a variety of levels of full-length or truncated

dystrophins in muscle tissue. More recently, similarstudies have been performed using utrophin vectors(Tinsley et al., 1996, 1998). These animals have pro-vided detailed information on the level of gene expres-sion needed to prevent occurrence of dystrophy, thefunctional capacity of different therapeutic molecules,and the percentage of muscle fibers that must expressthe transgene to prevent disease progression. Aftergeneration of transgenic mice and confirmation that aparticular molecule is effective in preventing dystrophy,transduction using viral vectors can be attempted.Obviously, it has been more difficult to demonstratefunctional correction of dystrophy using viral vectorsthan to demonstrate prevention of dystrophy withtransgenes that are uniformly expressed in all muscles.

Since most viral vectors are unable to carry a full-length dystrophin, many studies have focused on dystro-phin minigene vectors. Transgenic mdx mice have beencreated that express a truncated dystrophin minigeneof 6.3 kb in length (Fig. 2; Phelps et al., 1995). Thismolecule, missing about half of the coding region, wasbased on the mutation discovered in a mildly affectedBecker muscular dystrophy patient with a deletion ofexons 17–48 (BMD minigene in Table 1). Few signs ofdystrophy were observed in mice expressing this mini-dystrophin, though low-level muscle regeneration wasdetected on careful histological analysis (Phelps et al.,1995). Muscles from these animals also display slightdeficits in their ability to generate force and resistinjury (Fig. 3). These data show that this minidystro-phin does not completely prevent dystrophy; however,the observed lack of fibrosis and almost normal forcedevelopment indicate that smaller proteins could beuseful in a clinical setting. In fact, a recombinantadenovirus expressing this minidystrophin was shownto prevent muscle pathology if injected within one weekof birth (Deconinck et al., 1996; Vincent et al., 1993).Older mdx mice and dystrophic humans represent moredifficult challenges due to a functional immune system,advanced pathology, and down-regulation of adenovi-rus receptors. Despite these difficulties, the same viruswas found to be reasonably effective when injected intoolder mdx mice that had been immunosuppressed withFK506 (Lochmuller et al., 1996; Yang et al., 1998).

These data offer hope that more effective minidystro-phin molecules might be designed in the laboratoryusing knowledge of dystrophin structure and function.The BMD deletion described above results in the loss ofa non-integral number of spectrin-like repeats formingthe rod domain of dystrophin. Since this truncationlikely disrupts the structure of the dystrophin roddomain, a variety of modified truncations have beenengineered in attempts to develop a highly functionaldystrophin encoded by a cDNA less than 6 kb in size(Phelps et al., 1995; Rafael et al., 1994; Yuasa et al.,1998; Hauser and Chamberlain, unpublished data).Transgenic mdx mice expressing some of these modifiedalleles have recently been observed to be more effectivethan the exon 17–48 truncation in preventing musculardystrophy in mdx mice (Figs. 2, 3). Experiments toassess the efficacy of delivery of these modified cDNAsto dystrophic muscle using Ad and retroviral vectorsare currently in progress.

As described earlier, sarcolemmal expression of utro-phin, a dystrophin-related protein normally localized to

Fig. 5. Gene transfer across the endothelial barrier: histamineand papaverine increase permeability to viral particles. A–C: Patternof gene transfer in the absence of inflammatory mediators demon-strates interference of microvascular barrier with adenovirus trans-port. A: b-galactosidase activity in whole-mount-stained leg of adultrat 4 days after arterial infusion with AdCMVlacZ. X-gal shows thatvirus uptake is limited to microvasculature; muscle fibers do not stain.B,C: Staining is seen in most capillaries but absent in muscle fibers ofthe tibialis anterior. D–I: Efficient gene transfer to adult skeletalmuscle fibers after forced exudation with histamine and papaverine.D: Entire hindlimb from rat dissected before whole-mount-staining toexpose multiple cross-sections; universal fiber uptake is visible onmost cross-sections. E,F: Gross (E) and light microscopic (F) appear-ance of marker gene distribution in the quadriceps shows detail onadjacent rectus femoris and vastus medialis. A tourniquet at the levelof the common femoral artery occluded blood supply to the rectusfemoris, preventing virus delivery and infection. G: Semimembrano-sus and adductor brevis with adjacent saphenous artery. H: Nomarskimicrograph of tibialis anterior shows unstained wall of arteriole (rightcenter) against a backdrop of uniformly stained muscle fibers. I:Heterotopically transplanted heart after isolated perfusion with hista-mine and papaverine analogous to that used for isolated limb.Reprinted from Greelish J P, Su L T, Lankford E B, Burkman J M,Chen H, et al. 1999. Stable restoration of the sarcoglycan complex indystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector. Nature Med 5:439–443, with permission of thepublisher.


neuromuscular and myotendinous junctions, preventsdystrophy in transgenic mdx mice (Tinsley et al., 1998).This observation is important because it is possible thatDMD patients with null alleles could mount an immuneresponse against dystrophin, which might be perceivedas a neoantigen. Even mdx mice, which express a lowlevel of dystrophin in revertant myofibers, have beenshown to mount a CTL response against dystrophinexpressed by transplanted C57/BL10 myoblasts (Oht-suka et al., 1998). In contrast, no strong immuneresponse has been noted in experiments using guttedvectors to deliver dystrophin to mdx mice, so concernsabout immune response in human patients may proveunfounded. Considerable work has been done with asynthetic utrophin minigene analogous to the commonexon 17–48 deleted minidystrophin (Deconinck et al.,1997; Tinsley et al., 1996). In transgenic mdx mice, thisallele provides significant improvement in dystrophicpathology based on histological and functional criteria;in fact, the improvement in phenotype is roughlysimilar to that obtained with the 6.3 kb minidystrophin(Phelps et al., 1995).

Undoubtedly the most direct and efficacious route togene therapy of DMD would be delivery of full-lengthdystrophin itself-true gene replacement (Fig. 6). Expres-sion of even low levels of dystrophin in transgenic mdxmice eliminated the major symptoms of dystrophy.Dystrophin expression at levels between 20 and 5,000%of wild-type prevented all signs of dystrophy withoutdeleterious side effects (Cox et al., 1993; Phelps et al.,1995). Mouse and human dystrophin molecules werefound to be equally effective in these studies (Phelps etal., 1995). Animals with expression levels below 20% ofwild-type level displayed intermediate signs of dystro-phy, indicating that even low-level dystrophin expres-sion may have therapeutic benefit. Some lines of trans-genic mice produced dystrophin in a variable pattern(Phelps et al., 1995; Rafael et al., 1994). These non-uniformly expressing lines provide data on the percent-age of fibers that must be transduced to alleviatesymptoms. Comparison of the dystrophic pathology ofseveral such lines indicated that at least 50% of musclefibers must accumulate moderate amounts of dystro-phin to prevent a severe dystrophy.

Fig. 6. Delivery of full-length dystrophin using a gutted adenoviralvector. Ghumdys is a gutted adenoviral vector containing the full-length human dystrophin cDNA driven by a muscle creatine kinase(MCK) promoter. The vector contains no reporter gene. Top: Expres-sion of dystrophin in mdx mouse muscle 5 days after injection of thevector. Note cytoplasmic accumulation of dystrophin, indicating high-level expression. Bottom left: Marked DYS, shows immunofluores-

cence staining for dystrophin. Arrowheads have been placed withintwo weakly positive fibers. Bottom right: Marked Merge, showsaccumulation of Evans blue dye in dystrophin-negative fibers. Evansblue is a dye that permeates fiber with sarcolemmal disruption,indicating severe injury. Note that the weakly dystrophin-positivefibers do not take up dye, despite their proximity to damaged fibers.


The major difficulty with delivery of full-length dys-trophin is the fact that most gene delivery vectorscannot accommodate the large size of the gene (Table1). Until recently, full-length dystrophin could only bedelivered to muscle using myoblast transplantation ornaked DNA injection. However, as discussed above,recent improvements in helper-dependent or ‘‘gutted’’Ad vectors have allowed delivery of full-length dystro-phin (Fig. 6; Kochanek et al., 1996; Kumar-Singh andChamberlain, 1996). We found that a gutted vectorcontaining both dystrophin and b-galactosidase expres-sion cassettes, Ad5bdys (Fig. 4; Kumar-Singh andChamberlain, 1996), efficiently delivered full-lengthdystrophin to skeletal muscle, but expression persistedfor less than 1 month. The presence of a lymphocyticinfiltrate surrounding dystrophin- and b-galactosidase-positive fibers suggested that an immune response wasresponsible for loss of expression.

Since Ad5bdys does not contain any viral codingsequences, it seemed likely that the immune responsewas directed against either dystrophin or b-galactosi-dase, the two transgenes carried by the vector (Fig. 4).b-galactosidase, which plays no therapeutic role in thevector, is known to elicit a potent T-cell-mediatedresponse when delivered to immune-competent miceusing conventional Ad vectors (Tripathy et al., 1996;Yang et al., 1996a). To reduce expression of b-galactosi-dase, we created a new form of the gutted vector,GEbdys, in which the inducible ecdysone promoterdrives lacZ expression. Use of this promoter greatlylimits expression in mouse and human tissues, whichdo not express the ecdysone receptor (EcdR), but allowsfor titering of gutted vector preparations in modified293 cells expressing EcdR. Since provocation of a strongCTL response is thought to require gene expression inantigen-presenting cells, attenuation of gene expres-sion by use of such a promoter should reduce theimmune response. In fact, lymphocytic infiltration ofmdx muscles injected with GEbdys was dramaticallyreduced, indicating successful reduction of the CTLresponse despite robust dystrophin expression in myofi-bers. Muscles injected with GEbdys expressed dystro-phin for at least 4 months post-injection, which was thelongest time point tested (Salvatori et al., unpublisheddata).

These data suggest that gutted vectors will allowdelivery and long-term expression of full-length dystro-phin, although avoidance of immune response remainsa critical issue.Additional studies addressing the persis-tence and functional capacity of gutted vectors lackingall reporter genes have yielded highly encouragingresults, suggesting that phase I clinical trials of guttedvectors should proceed (Fig. 6).

GENE REPLACEMENT FORSARCOGLYCANOPATHIES

The dystrophin-glycoprotein complex found in skel-etal muscle includes four related proteins named sarco-glycans (Fig. 1; Campbell and Kahl, 1989; Ervasti et al.,1990; Yoshida and Ozawa, 1990). Sarcoglycan defi-ciency is now known to cause four forms of autosomalrecessive LGMD-one for each sarcoglycan molecule(Bonnemann et al., 1995; Jung et al., 1996; Lim et al.,1995; Noguchi et al., 1995; Roberds et al., 1994).

Collectively these forms of muscular dystrophy arecalled sarcoglycanopathies.

A naturally occurring hamster mutant, the BIO 14.6cardiomyopathic hamster, is available for delta-sarcogly-can (delta-SG) deficiency, so gene replacement studieshave focused on this gene (Fig. 7; Nigro et al., 1997;Sakamoto et al., 1997). It is likely that a gene replace-ment therapy effective in amelioration of delta-SGdeficiency could be directly adapted to the other sarco-glycanopathies. Holt et al. were the first to explore thefeasibility of gene replacement in the BIO 14.6 hamster(Fig. 7; Holt et al., 1998). Due to the small size of thesarcoglycan genes, these authors were able to deliverdelta-SG in a conventional E1, E3-deleted adenovirus(Fig. 7A, Table 1). They found that injection of delta-SGAd, but not control alpha-SG Ad, restored all foursarcoglycans to the sarcolemma (Fig. 7B). Injection ofyoung (3-week-old) hamsters with the Ad vector re-sulted in expression for at least several months, whichalso occurs after injection of Ad into young, but notmature, mice. Delta-SG Ad injection also prevented thedevelopment of morphological abnormalities, as as-sessed by the percentage of muscle fibers with centralnucleation, and maintained sarcolemmal integrity.These results are very similar to those achieved inyoung mdx mice after delivery of full-length dystrophin

Fig. 7. Recombinant d-SG adenovirus mediates high efficiencygene transfer into BIO 14.6 hamster muscle. A: Age-matched quadri-ceps from F1B, BIO 14.6, and BIO 14.6 injected with 109 particles ofa-SG adenovirus or d-SG adenovirus (tissue harvested 7 days post-injection) were analyzed by immunofluorescence using an antibodyspecific for d-SG. Bar 5 50 µm. B: d-SG adenovirus particles (109 ) wereinjected into the quadriceps femoris muscle of BIO 14.6 hamster.Seven days later, muscle cryosections were prepared and subjected toimmunofluorescence using antibodies specific for individual sarcogly-can proteins, as shown. Bar 5 50 µm. Reprinted from Holt K H, Lim LE, Straub V, Venzke D P, Duclos F, et al. 1998. Functional rescue of thesarcoglycan complex in the BIO 14.6 hamster using delta-sarcoglycangene transfer. Mol Cell 1:841–848, with permission of the publisher.


or dystrophin minigenes (Deconinck et al., 1996; Vin-cent et al., 1993).

Unlike the dystrophin gene, the sarcoglycan genesare all small enough to be delivered using AAV (Table1). Since AAV has proven capable of gene delivery toadult animals without elicitation of an immune re-sponse, it should be possible to inject older BIO 14.6hamsters and maintain expression for a long period.For example, Greelish et al. (1999) injected 9-week-oldhamsters and evaluated injected muscle 13 weeks aftergene transfer, when stabilization of sarcolemmal integ-rity was demonstrated through exclusion of procionorange dye. Such studies offer hope that sarcoglycandeficiency could be corrected in adult, immune-compe-tent humans. This hypothesis will be the focus of anumber of limited human clinical trials over the nextfew years (Mendell and Wilson, personal communica-tion).

CONCLUSIONThere has been encouraging progress towards estab-

lishing gene replacement as a viable therapy for muscu-lar dystrophy. Understanding the limitations of avail-able gene transfer vectors led to the development ofnew options including gutted Ad, AAV, and lentiviralvectors. Gutted Ad vectors have allowed delivery offull-length dystrophin to adult muscle for the first time.Even in immune-competent animals, gene expressioncan be maintained for at least several months. In thefuture, these vectors may provide the capacity neces-sary for tightly regulated expression of therapeuticgenes or delivery of multiple expression cassettes.Recent advances in the growth and purification of thesegutted vectors should allow evaluation of their useful-ness in disparate settings.AAV vectors have had encour-aging success in gene delivery to the muscles of immune-competent animals. Even proteins that are known to bevery immunogenic, such as b-galactosidase, can beexpressed for long periods. It is hoped that expression ofsmall muscular dystrophy disease genes using AAV willbe comparatively easy.

Many challenges remain before gene replacementtherapy for MD can be declared a success. Production ofviral vectors at very large scale and high purity re-mains problematic, so further improvements will berequired before delivery of full-length dystrophin tohumans is practical. Long-term functional correction,as opposed to prevention, of the dystrophic phenotypein mouse models has not yet been demonstrated. Theimmunologic consequences of delivering disease genesto patients with null mutations are still unknown.Finally, safe, consistent delivery of viral vectors to alarge mass of muscle tissue has not been shown to bepractical.

Stable gene transduction that provides functionalcorrection at the level of single cells has been demon-strated. In the future, we hope that viral vectors suchas gutted Ad, AAV, and others being developed willallow meaningful correction of muscular dystrophy atthe level of whole muscles and human patients. Makingthis leap in scale is the challenge of the years to come.

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Developments in gene therapy for muscular dystrophy