Human Molecular Genetics, 2002, Vol. 11, No. 15 1719-1730
© 2002 Oxford University Press
Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle
1Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway – University of London, Egham, Surrey TW20 0EX, UK, 2Gene Targeting Unit, Department of Neuromuscular Diseases, Imperial College School of Medicine, Charing Cross Hospital, St Dunstan's Road, London W6 8RP, UK, 3Laboratoire de Thérapie Génique, CHU Hotel Dieu, Bâtiment Jean Monnet, 44000 Nantes, France, 4Department of Molecular Genetics, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187, Japan and 5Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, Unité de Virologie Humaine, INSERM U412, Ecole Normale Supérieure de Lyon, 69364 Lyon, France
Received March 25, 2002; Accepted May 15, 2002
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag–pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of ß-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in ß-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdµDyIN), we were able to partially correct the mdx dystrophic phenotype. AdµDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%±6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdµDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Duchenne muscular dystrophy (DMD) is a fatal X-chromosome-linked muscle wasting disease affecting human juvenile males and caused by mutations in the 2.7 Mb dystrophin gene. It currently affects 1 in 3500 newborn males, one-third of these cases being spontaneous (1). The dystrophin protein is a large (427 kDa) membrane-associated protein that is thought to maintain myofibre structural integrity by connecting the extracellular matrix through the dystrophin-associated glycoprotein (DAG) complex to the cytoskeleton, although it is now apparent that dystrophin and the associated complex may also play a role in mediating intracellular signals (2–4). Dystrophin cDNA exceeds 12 kb and is therefore too large for helper-independent adenoviral and retroviral vectors used in current clinical trials. In order to circumvent this problem, a number of truncated mini-dystrophin (6.3 kb) (5) and micro-dystrophin (3.5–4.5 kb) (6,7) genes have been developed based on mutations observed in patients suffering from mild forms of muscular dystrophies. The ultimate goal of somatic DMD genetic therapy is to introduce a functional cDNA-based dystrophin gene into deficient muscle cells of a patient so that it is expressed for life.
In preclinical studies, the adenovirus has been the vector of choice to deliver dystrophin constructs to the muscle of mdx mice, the mouse model of DMD (8–15). However, adenovirally mediated expression of dystrophin to levels required to ameliorate the dystrophic phenotype is transient in nature. There are two reasons for this. Firstly, adenovirus is highly immunogenic, eliciting a CD8+ T-cell-mediated immune response to adenoviral protein and transgene sequences (16–18). Secondly, adenovirus is an episomal vector, and, as such, transduced DNA is not integrated into the host cell genome, and is subsequently lost during muscle cell turnover in dystrophic tissue (19). Therefore, identification of an alternative vector system resulting in stable integration of the dystrophin gene is required for an effective therapy of DMD.
Retroviral and, potentially, adeno-associated viral (AAV) vectors retain the ability to integrate their DNA into the host genome, and have also been used in mdx mice (20–24). While use of the AAV vector in muscle holds promise due to its ability to evade the immune response, resulting in long-term transgene expression, its use to treat DMD is restricted because of the limit on packageable DNA. Alternatively, retroviral vectors are large enough to accommodate current dystrophin mini-genes that are widely accepted to provide therapeutic benefit (20,23). However, although retroviral vector technology is improving at the bench, increasing production of these vectors to a pharmaceutical scale remains difficult. Moreover, complement-mediated particle lysis of retroviral vector particles often results in a low efficiency of in vivo gene delivery (25), although this can now be overcome by pseudotyping (26,27). Owing to the low efficiency of transduction, most retroviral vector gene therapy protocols adopt an ex vivo approach, where cells are genetically modified by retroviral vector in vitro, selected and expanded, before being implanted back into the patient (28–30). This procedure is inefficient, resulting in the transduction of only a small population of target cells and hampered by an immune response to factors required for culturing the cells in vitro. Alternatively, it is possible to improve retroviral transduction in vivo by implanting retroviral producer cells into the target tissue (22,23,31). However, this approach can generate severe inflammatory response and may result in the formation of tumours derived from retroviral producer cells (22).
In an attempt to circumvent these problems (which ultimately result in low efficacy of gene transfer in vivo), improve vector production, reduce cytotoxicity and hence increase the overall efficiency of stable transgene expression, a number of hybrid viral vectors have been developed. These include gene delivery systems based on adenovirus/retrovirus (32–36). In the present study, we have used a hybrid adeno-retroviral vector to combine the high efficiency of infection of adenovirus with the capacity of the retrovirus to integrate its genome into transduced cells (37). Production of functional retroviral vector using this hybrid system is a two-step process; target cells are infected with adenoviruses expressing retrovirus structural genes and provirus sequences. Infected cells release functional retroviral vector, which then transduces neighbouring cells, resulting in stable integration of the therapeutic gene. Using adenovirus templates to produce retroviral vector in this manner offers an opportunity to produce retroviral vector in situ from autologous cells, reducing complement-mediated lysis, and increasing the efficiency of retroviral vector transduction at the target site. Vectors constructed using this approach have already been used in two rodent models of cancer, and were shown to transduce rapidly dividing cancer cells efficiently (32,35). One aspect of DMD pathology makes it an ideal target for gene therapy by in situ delivery of retroviral vector. Muscle fibres not expressing dystrophin degenerate and are subsequently replaced by proliferating myoblast stem cells during regeneration. If existing muscle fibres were allowed to act as a platform for retroviral production, myoblasts that are proliferating during the course of muscle regeneration could be transduced by newly produced retroviral vector in the surrounding milieu.
We have previously shown that both proliferating myoblasts and post-mitotic differentiated myotubes produce retroviral vector efficiently from hybrid adeno-retrovirus vector templates in vitro (38). As an extension to this study, we have examined whether in situ retroviral production from myofibres is feasible for the treatment of DMD. We show that myofibres from the tibialis anterior (TA) muscle of the mdx mouse act as efficient retroviral producer cells. In culture, colonies of retrovirally transduced myotubes appeared only from muscles injected with all the components required to produce retroviral vector. Retrovirally mediated expression of reporter construct increased the overall efficiency of muscle cell transduction compared with infection with adenoviral vector alone. Moreover, we have used a 3.7 kb dystrophin micro-gene to show that the hybrid adeno-retroviral vector approach is a feasible therapeutic strategy for the treatment of DMD. We show that expression of this construct restores expression of the dystrophin-associated glycoprotein complex and reduces muscle degeneration assayed by the degree of centro-nucleation. We also detected reverse-transcribed micro-dystrophin-expressing proviral sequences in genomic DNA isolated from transduced muscle, thus indicating that integration had occurred. Finally, we demonstrate that by using the hybrid system to express the micro-dystrophin gene stably in neonatal mdx muscle, we can restore expression of dystrophin in nearly 100% of muscle fibres by adulthood.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Construction of hybrid vectors expressing LacZ and 3.7 kb micro-dystrophin
We used the AdEasy system to construct hybrid adenoviruses expressing LacZ and micro-dystrophin, to reduce the time required to construct the hybrid vectors, given that the total size of each insert exceeded 7.5 kb. The retrovirus element of pLXIN (Clontech) was subcloned into pShuttle (Q-Biogene) to create pSh–LXIN. LacZ and micro-dystrophin genes were then cloned into this hybrid plasmid to create pSh–LZIN and pSh–LµDyIN, respectively. These plasmids were co-transformed with pAdEasy-1 (Q-Biogene) into Escherichia coli BJ5183, and analysis of several resulting colonies revealed that successful recombination had occurred to create pAdLZIN and pAdLµDyIN (data not shown). These new adeno-plasmids were linearized with PacI and transfected into 911 cells to generate AdLZIN and AdLµDyIN vectors, respectively (Fig. 1A).
|
AdLZIN and AdLµDyIN generate retroviral vector efficiently, in vitro
Before proceeding with in vivo experiments, we first analysed the ability of C2C12 myoblasts to produce functional retrovirus expressing LacZ and micro-dystrophin. C2C12 cells were seeded at a density of 105 cells per well of a six-well plate. The following day, myoblasts were infected with 500 p.f.u./cell AdLZIN or AdLµDyIN in the presence or absence of 500 p.f.u./cell AdGagPol and 500 p.f.u./cell Ad10A1. Forty-eight hours post adenoviral infection, medium was harvested, and cells infected with AdLZIN were stained with X-gal in order to assess the degree of infection (Fig. 1B, left panel). Cell lysates were also prepared from myoblasts infected with AdLµDyIN in order to detect expression of micro-dystrophin (Fig. 1C, lanes 1 and 2). The retrovirus-containing medium was then added, at a dilution factor of 1 : 10, to NIH3T3 cells, which were subsequently grown for several passages in the presence of 0.5 mg/ml G418. After 3 weeks in culture, transduced NIH 3T3 cells were stained with X-gal (Fig. 1B, right panel), or cell lysates were prepared and analysed by western blot (Fig. 1C, lane 3). Expression of LacZ and micro-dystrophin was only detected in 3T3 cells originally treated with medium harvested from C2C12 cells infected in the presence of AdGagPol and Ad10A1. NIH3T3 cells transduced with supernatant isolated from C2C12 myoblasts initially infected with AdLZIN or AdLµDyIN alone did not survive beyond the second passage (data not shown). We also determined the efficiency of retroviral vector production in vitro by counting the number of G418-resistant colonies of NIH 3T3 cells that formed after 2 weeks in selection. The LZIN retroviral vector was produced to titres approximating 104 c.f.u./ml and LµDyIN to titres in the region of 103 c.f.u./ml (data not shown). We therefore concluded that newly constructed AdLZIN and AdLµDyIN were able to act as templates for the production of retroviral vectors expressing LacZ and micro-dystrophin, respectively.
Efficient retroviral vector production in the tibialis anterior muscle of mdx mice
The data above and our previous studies have shown that cultures of myoblasts and differentiated myotubes are capable of producing retroviral vector to titres ranging between 103 and 105 c.f.u./ml in vitro (38). We next analysed if myofibres could produce retroviral vector in vivo, using a hybrid vector expressing ß-galactosidase (AdLZIN). TA muscles from 5-week-old mdx mice were injected with 109 viral particles of AdLZIN, in the presence or absence of 109 viral particles of AdGagPol and Ad10A1. After 1 week, the animals were sacrificed and the TA muscle was removed and used to establish a primary culture of myoblasts. After several days in culture, the myoblasts fused to form myotubes, which were subsequently stained with X-gal to detect ß-galactosidase activity. On average, 50 colonies of blue myotubes were detected in cultures isolated from TA muscle injected with all three viruses, whereas an average of one colony was detected in cultures isolated from TA muscle injected with AdLZIN alone (Fig. 2A–C). This significant increase (P<0.000004, Student's t-test, n=4) in the number of colonies detected in the presence of AdGagPol and Ad10A1 was mirrored by a further significant (9.2-fold) increase in the overall number of ß-galactosidase positive cells observed in the culture dish (P<0.0005, Student's t-test, n=4), compared with myotubes isolated from muscle injected with AdLZIN alone (Fig. 2C).
|
Retroviral vector production results in an increase in transduced muscle fibres
Before examining the effect of retroviral vector production on the efficiency of LacZ reporter gene expression in vivo, we determined the optimal dose of adenoviral vector to administer to each TA muscle without generating a severe inflammatory response. We injected 108, 109 and 1010 viral particles of AdLZIN into each TA muscle and counted the number of ß-galactosidase-positive fibres after 1 and 4 weeks. We found that injection of 109 particles gave the optimal expression of LacZ with minimal toxicity (data not shown). Initially, we compared the efficiency of LacZ expression in the TA of mice injected with AdLZIN only or with AdLZIN and AdGagPol, and found no difference in the number of fibres containing ß-galactosidase after one week (Table 1; P>0.54, student's t-test, n=5). Therefore, for all subsequent experiments, we used AdLZIN and AdGagPol as a negative control in order to equalize the overall viral load administered to each muscle. TA muscles from 4- to 5-week old mdx mice were then injected with 109 viral particles of AdLZIN and AdGagPol, or with 109 viral particles of AdLZIN, AdGagPol and Ad10A1. After 1 week in the absence of 10A1 envelope, an average of 39 muscle fibres were found to be positive for ß-galactosidase activity. This compared with an average of 85 positive muscle fibres in the presence of 10A1 envelope protein (Table 1; Fig. 3A and B). Interestingly, one animal injected with all three adenoviral vectors expressed ß-galactosidase in as many as 519 and 538 fibres per TA muscle, thus indicating efficient formation of several retroviral vector production foci in this animal (Fig. 3B, arrows). After 4 weeks of retroviral vector production, an average of 4 positive fibres were detected in the absence of 10A1 env expression, compared with a slight increase to 15 positive fibres in its presence (Table 1, Fig. 3C and D). This reduction in the number of LacZ-positive fibres in both the absence (P<0.02, Student's t-test, n=10) and presence (P<0.05, Student's t-test, n=12) of 10A1 env over 4 weeks was found to be significant, and was most probably due to an immune response against the transgene and/or adenoviral vector sequences. Therefore, in order to examine the effect of retroviral vector production over time in the absence of an immune response, we repeated the above experiment in mdx/nude mice.
|
|
Production of retroviral vector from muscle fibres in mdx mice had resulted in a 2-fold increase in ß-galactosidase-positive myofibres, but this increase was not shown to be statistically significant using Student's t-test. Therefore, to control for the large variation of ß-galactosidase-expressing fibres observed in individual animals, the left TA of each mdx/nude mouse was injected with AdLZIN and AdGagPol, whereas the right TA was injected with AdLZIN, AdGagPol and Ad10A1. Treating animals in this way allowed a direct comparison of any increase in positive fibres per TA of individual mice. After expression for 1 week, an average of 64 muscle fibres stained positive for ß-galactosidase activity in the absence of 10A1 env, compared with 66 fibres in its presence (Table 1, Fig. 3E and F). Therefore, at this time, there was no difference in expression of ß-galactosidase, indicating that insufficient regeneration of muscle fibres had occurred to allow retroviral transduction. By 4 weeks post injection, the average number of positive fibres in the absence of 10A1 env protein had remained at 61, whereas an average of 106 positive fibres were detected in its presence (Table 1, Fig. 3G and H). Because of the high variation between animals, the 4-week groups were not significantly different when examined with Student's t-test. However, by directly comparing the difference in the number of LacZ-expressing fibres between the TA muscles of individual mice, we found a statistically significant increase in ß-galactosidase-positive myofibres under conditions allowing for retroviral vector production (P=0.04, Mann–Whitney rank-sum test, n=12). The non-parametric test was used because the data were not normally distributed. Moreover, we found that, on average, TA muscles injected with AdLZIN, AdGagPol and Ad10A1 expressed LacZ in 4.6-fold more fibres than contralateral control TA muscle injected with AdLZIN and AdGagPol only. In 4 of 12 mice studied at the 4-week stage, the increase ranged from 1.3- to 1.9-fold, whereas in 5 of 12 mice examined, the observed increase ranged from 3.0- to 24.1-fold. From these data, we concluded that the TA muscle of mdx mice acts as an efficient platform for the production of retroviral vector. Furthermore, locally produced retroviral vector is able to complete a productive life cycle by infecting surrounding dividing myoblasts, resulting in more efficient transduction of muscle fibres.
We next examined whether the hybrid adeno-retroviral vector system could be applied to introduce a therapeutic gene stably into regenerating muscle.
AdLµDyIN expresses a functional dystrophin construct with therapeutic potential
In the present study, we have used first-generation (E1/E3-deleted) adenoviral vectors as templates for retroviral vector production. We were therefore limited by the size of exogenous DNA that could be inserted into the hybrid vector. In an attempt to correct the dystrophic phenotype observed in mdx mice, we chose to express a truncated 3.7 kb micro-dystrophin construct (Fig. 1A) (5). To establish that expression of this micro-dystrophin construct from a retroviral LTR promoter was sufficient to correct the mdx phenotype, we first injected the TA muscles of mdx mice with AdLµDyIN vector alone. Approximately 6x109 viral particles of AdLµDyIN vector were injected into the TA of 1-week-old mdx mice, and expression was allowed to occur for 4 weeks before animals were sacrificed. TA muscle isolated from treated animals was sectioned and stained with 6C5 antibody (a monoclonal antibody against the C terminus of full-length dystrophin). Some 69%±6% (SEM, n=6) of muscle fibres expressed the micro-dystrophin construct at the sarcolemmal membrane (Fig. 4A and D). The encircled area of the representative TA muscle presented in Figure 4A illustrates a large section of TA muscle fibres that stained negative for micro-dystrophin expression in animals injected with AdLµDyIN only. We then stained muscle sections with antibodies against ß-dystroglycan (Fig. 4I), 
-sarcoglycan (Fig. 4J) and ß-sarcoglycan (Fig. 4K). As has been reported previously, expression of this micro-dystrophin construct restored the correct localization of each of these members of the DAG complex (6). In order to examine the functional significance of expression of the micro-dystrophin construct and correct localization of the DAG complex at the sarcolemma, we next examined the degree of muscle degeneration in treated animals by measuring the extent of central nucleation (an indicator of regenerating muscle tissue). Compared with uninfected controls, the extent of central nucleation was reduced from 68%±6% to 34%±4% (Fig. 5), thus indicating that expression of the micro-dystrophin construct had significantly retarded the degeneration of muscle observed in mdx mice (P<0.0002, Student's t-test, n=6). Having established that expression of the 150 kDa micro-dystrophin gene partially corrected the mdx phenotype, we next examined if inclusion of AdGagPol and Ad10A1 would result in stable integration of the micro-dystrophin construct. Injection with 2x109 viral particles of all three adenoviral constructs significantly increased the level of micro-dystrophin expression to 92%±6% of muscle fibres after 4 weeks (P<0.04, Student's t-test, n=4, Fig. 4B), restoring correct localization of the DAG complex (data not shown). Note that almost all of the muscle fibres in the representative TA muscle section shown in Figure 4B stained positively for micro-dystrophin expression. After 12 weeks under these conditions, micro-dystrophin was still present in all the fibres of the TA muscle at high levels (Fig. 4H), whereas the amount of micro-dystrophin expression in animals injected with AdLµDyIN alone was substantially reduced (Fig. 4E).
|
|
Detection of integrated proviral DNA in muscle injected with adeno-retroviral hybrid vectors
In order to assess if the micro-dystrophin-expressing provirus had integrated into muscle DNA, we developed a novel PCR-based strategy to detect reverse-transcribed proviral elements isolated from injected muscles (Fig. 6A). Initially, a 670 bp segment of proviral template, between the left retroviral LTR and packaging signal, was amplified using forward primer P1 and reverse primer P2 (Fig. 6A). Detection of elevated amplicon in TAs injected with AdLµDyIN, AdGagPol and Ad10A1 over the negative control infected with AdLµDyIN alone indicated an increase in the number of templates, suggestive of efficient retroviral vector transduction (Fig. 6C; compare lanes 1 and 2). In order to confirm this supposition, we conducted a normalized, nested PCR using primer P3, which specifically annealed to the U3 region of the 3' long terminal repeat (LTR), and reverse primer P2. Hence, a 500 bp amplicon should only arise subsequent to the recombination event that occurs between the 5' and 3' retroviral LTRs during reverse transcription and integration. Low levels of amplicon were detected after 4 weeks in TA muscle treated with AdLµDyIN, AdGagPol and Ad10A1, but not in muscles from the negative control muscle treated with AdLµDyIN alone (Fig. 6B). By 3 months, increased levels of reverse-transcribed template were detected (Fig. 6B); indeed, we were able to detect integrated template at this stage by direct analysis of genomic DNA from treated tissues, without the need for nested PCR (data not shown). This indicates that the presence of reverse-transcribed integrated proviral DNA in the genome of muscle fibres producing retroviral vector may accumulate over time. Finally, in order to assess the potential spread of hybrid vector sequences, genomic DNA from liver, lung, kidney, ovaries and testes isolated from eight mice injected with AdLµDyIN, AdGagPol and Ad10A1 was analysed by PCR using primers P1 and P2. Under the conditions used to detect vector DNA in muscle (Fig. 6C, lanes 1 and 2), we were unable to detect vector sequences in the other tissues analysed (Fig. 6C, upper panel), although we were able to amplify a fragment of GAPDH DNA as a positive control (Fig. 6C, lower panel).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We have previously demonstrated that muscle satellite cells in vivo can be efficiently transduced by retroviral vector mediating long-term transgene expression (22,23). Moreover, retrovirally transduced satellite cells were shown to be mitotically active, indicating that these cells persist as progenitor stem cells. In this previous study, retroviral vector was delivered by implantation of retroviral vector producer cells – a procedure that led to a severe inflammatory response and the formation of palpable tumours (22). Unfortunately, intramuscular injection of neat retroviral vector results in inefficient transduction of muscle fibres, due most likely to a combination of low viral titres administered, low proportion of dividing myoblast stem cells and the genetic background of the treated mice (21). The recent development of hybrid adeno-retroviral vectors therefore provided the opportunity to exploit the high efficiency of retrovirally mediated muscle cell transduction observed subsequent to administration of retroviral vector producer cells, circumventing the associated problems and enabling retroviral vector production to occur in situ. Indeed, adenoviral vectors have been previously applied to enhance retrovirally mediated transduction of muscle. An adenoviral vector was used to deliver the retroviral vector receptor to myoblasts in order to increase the efficiency of retroviral infection (39). Similarly, adenovirus-mediated expression of the SV40 large T antigen was shown to promote myoblast proliferation and hence retroviral transduction (40). However, we are the first to employ the chimeric adeno-retroviral expression system in muscle to demonstrate the suitability of muscle fibres as in situ factories for retroviral vector production. Previously, we reported that postmitotic, differentiated myotubes in vitro were more efficient at generating retroviral vector than immature dividing myoblasts (38). Consequently, as mature myofibres are postmitotic, infection with adenoviral vectors expressing all the elements required for the production of a retroviral vector should result in the formation of a stable retroviral producing cell in vivo. Indeed, by introducing myoblasts from muscles injected with AdLZIN, AdGagPol and Ad10A1 into culture, we observed the formation of retrovirally transduced colonies of myofibres (Fig. 2). We proceeded to demonstrate that secondary retroviral transduction of dividing myoblasts within the infected TA muscle enhanced the efficiency of reporter gene expression 4.6-fold, following primary adenoviral infection (Fig. 3G and H). In these immune-compromised mdx/nude mice, the average number of muscle fibres expressing ß-galactosidase in the presence of AdLZIN, AdGagPol and Ad10A1 after 1 month was quite low (Table 1), representing some 8–9% of the total number of myofibres present in a TA muscle. However, we purposely chose to administer a low dose of AdLZIN in order to detect any change in transduction conferred by the addition of AdGagPol and Ad10A1. Nevertheless, it should be noted that some TAs contained as many as 30–33% ß-galactosidase-expressing fibres, thus indicating the formation of several successful retroviral vector-producing foci in muscles from these animals. Moreover, by examining the effects of retroviral production on the muscles from individual mice, we were able to control for the variation of transduction efficiency. There was a severe drop in transduced fibres after four weeks in muscles from immunocompetent mdx mice, in both the presence and absence of the 10A1 env gene (Table 1; Fig. 3C and D). This is most likely a consequence of an immune response against fibres infected with the first-generation adenoviral vectors employed in this study (16–18). This may be avoidable by utilizing second-generation, lacking the E2b region, or third-generation gutted adenoviral vectors instead, since immune responses to these vectors are significantly reduced when compared with first-generation E1/E3-deleted adenoviral vectors (41–44).
Our previous work had indicated that retroviral vectors had the potential to deliver truncated dystrophin constructs effectively to the muscle of mdx mice (20,23). We therefore proceeded to examine whether the hybrid adeno-retroviral vector system could be used as a feasible therapeutic strategy to treat DMD, by designing a chimeric vector that expressed a truncated 3.7 kb micro-dystrophin construct. In order to increase the efficiency of muscle fibre transduction, we chose to inject neonatal mice (<1 week), thereby maximizing transduction of muscle stem cells and minimizing the immune response against vector expressing transgene. We also administered a slightly higher dose of micro-dystrophin-expressing hybrid vector. As a result of these measures, the efficiency of micro-dystrophin expression was higher when compared with the LacZ data, and we were able to observe a significant increase in the number of micro-dystrophin-positive myofibres upon induction of retroviral production (Fig. 4A and B). We demonstrated that stable expression of this micro-dystrophin construct in mdx mice partially corrected the dystrophic phenotype, restoring sarcolemmal expression of the dystroglycan-associated complex in muscle fibres (Fig. 4). Moreover, the observed high-level expression of micro-dystrophin slowed muscle degeneration, as evidenced by a small, yet significant, reduction of centrally nucleated muscle fibres (Fig. 5). During the course of this study, Xiao and colleagues (7) rationally designed a series of 4.2 kb micro-dystrophin constructs that effectively prevented the accumulation of centrally nucleated myofibres that normally occurs as mdx mice age. Moreover, long-term muscle-specific expression of these constructs from AAV vectors protected against muscle damage induced by short-term vigorous exercise. The more efficient protection of muscle degeneration conferred by the Xiao constructs is likely to be a function of the five rod domains and two hinge regions incorporated in their design (7). The 3.7 kb construct employed in the present study contains only one rod domain and a single hinge region, thus explaining its reduced protective capacity. However, the hybrid adeno-retroviral system is not limited by the use of micro-dystrophin constructs for the treatment of DMD. Employment of second-generation adenoviral vectors (42–44) would enable the construction of a hybrid template capable of producing retroviral vectors expressing the therapeutically valid 6.3 kb Becker mini-dystrophin gene, thereby eliminating the need for partially functional micro-dystrophin constructs, whose application in the treatment of DMD is expected to be limited.
We designed a novel PCR-based system to detect reverse-transcribed proviral DNA in genomic DNA isolated from infected muscle, thus indicating that integration of the retroviral vector genome had occurred (Fig. 6A). Although PCR has been used before to detect retroviral sequences in genomic DNA from muscle (22), we had to design a new strategy to differentiate between unintegrated template sequences (from the adenoviral element) and integrated proviral elements. Using this technique, we detected reverse-transcribed sequences in genomic DNA isolated from TA muscle after 4 weeks, and subsequently observed an increase in the level of this template over 12 weeks (Fig. 6B). Although this method of detection can only be indicative of retroviral genomic integration, it also confirms the presence of retroviral vector templates within transduced myofibres. Furthermore, to demonstrate the safety of the hybrid adeno-retroviral vector approach, we analysed different tissues for distribution of vector sequences. As we were unable to detect any spread throughout the liver, lung, kidney, testes and ovaries, it is likely that this hybrid vector system will prove a safe and effective method for muscle-based gene transfer.
In summary, we have demonstrated that muscle constitutes an effective platform for the production of retroviral vector. Our results shown in Figure 2 suggest that each TA muscle contains a number of muscle progenitor stem cells transduced with retroviral vector, generating a sizeable pool of mature muscle fibres stably expressing transgene. This is evidenced by the 4.6-fold increase in transgene expression observed in TA muscles producing retroviral vector, compared with negative controls. We have also successfully applied this system to partially correct the dystrophic phenotype associated with the mdx model. We achieved efficient expression of a replacement dystrophin construct in all of the muscle fibres, a proportion of which contain integrated, reverse-transcribed retroviral vector sequences. Neonatal muscle proved a more efficient platform for retroviral production compared with juvenile tissue. This may be due to a less well-developed extracellular matrix allowing for increased adenoviral vector infection, the presence of a higher proportion of muscle stem cells, and the absence of immunity allowing tolerance to vector and transgene sequences to develop. Given that no one vector has proved an ideal means of introducing therapeutic transgene into disease tissue, the further development of hybrid vectors like the one employed in this study is critical for the successful progression of gene therapy.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Generation of adeno-retroviral constructs
Replication-defective hybrid adeno-retroviral vectors expressing ß-galactosidase (LacZ) and the 3.7 kb micro-dystrophin gene (µDy) were constructed by homologous recombination in E. coli BJ5813 (45). The LXIN retroviral genome was first excised from pLXIN (Clontech, Palo Alto, CA) with Bst1107I and Sac II, then treated with the large (Klenow) fragment of DNA polymerase I to generate blunt ends. The LXIN fragment was subsequently ligated into BglII-KpnI-digested pShuttle plasmid (Q-Biogene, Carlsbad, CA), also treated with Klenow. For the construction of a hybrid adeno-retroviral shuttle plasmid expressing ß-galactosidase (pSh–LZIN), the LacZ gene was removed from pCMVß by digestion with NotI, followed by Klenow-fragment treatment and ligation into HpaI-digested pSh–LXIN. A hybrid adeno-retroviral shuttle plasmid expressing 3.7 kb micro-dystrophin (pSh–LµDyIN) was constructed by removing the micro-dystrophin gene from p
DysM3 (6) by digestion with EcoRI, and ligation into Eco RI-digested pSh–LXIN. The plasmid pAdEasy-1 (Q-Biogene), containing the entire Ad5 genome, minus deletions in the E1 and E3 regions, was co-transformed together with either pSh–LZIN or pSh–LµDyIN into E. coli BJ5813, and the resultant colonies were analysed for recombinant adenoviral vector plasmid. Recombinant plasmids from positive clones were linearized with PacI and transfected into 911 cells (46). The resultant viral vectors (AdLZIN and AdLµDyIN) were plaque-purified and grown to a high-titre stock (1x1012 viral particles/ml, as determined by measuring optical density at 260 nm) (47). Adenoviruses expressing retroviral structural genes on separate viruses, AdGagPol and Ad10A1 (amphotrophic envelope protein), driven from CMV and EF1- promoters, respectively, have been described previously (36).
Cell culture and adenoviral infection
C2C12 cells are derived from a subclone of a mouse myoblast cell line and are used as a general model of muscle cell differentiation (48). C2C12 myoblasts were cultured in DMEM supplemented with Glutamine (Sigma, Poole, UK), anti-mycotic/antibiotic mix (Sigma) and 10% fetal calf serum (FCS; Sigma). For adenovirus infection, C2C12 cells were seeded at 1x105 cells/well in six-well plates. The next day, C2C12 cells were infected with hybrid adeno-retroviruses at a multiplicity of infection (MOI) of 500 p.f.u./cell for 3 h in 0.7 ml infection buffer (PBS, 70 mM CaCl2, 50 mM MgCl2). Infected cells were washed three times with PBS to remove residual adenovirus and incubated in 2 ml DMEM containing 10% FCS. Forty-eight hours later, supernatants were collected, filtered through a 0.45 µm filter, and titred for the presence of retroviral vector.
Determination of in vitro retroviral vector production
For detection of retroviral vector production, NIH 3T3 cells were seeded at 5x104 cells/well in six-well plates 1 day prior to retroviral transduction. Medium harvested from adenovirus-infected myoblasts was supplemented with polybrene (10 µg/ml), diluted 10-fold, and added to NIH 3T3 cells for 24 h. Transduced NIH 3T3 cells were then split into new six-well plates at a ratio of 1 : 20 and incubated in the presence of 0.5 mg/ml G418 (Invitrogen, Paisley, UK). Individual colonies of G418-resistant NIH 3T3 cells were picked using a cloning ring and cultured in the presence of G418 for 3 weeks. The resultant stable cell lines were fixed in 0.5% glutaraldehyde and stained with 1 mg/ml X-gal in 50 mM Tris–HCl pH 7.5, 25 mM K3Fe(CN)6, 25 mM K4Fe(CN)6, 15 mM NaCl and 1 mM MgCl2 to detect expression of ß-galactosidase, or cell lysates were prepared for western blotting to detect expression of micro-dystrophin.
Western blotting
Lysates from C2C12 cells were obtained in lysis buffer (50 mM Tris–HCl, pH 7.4, 250 mM sucrose, 14 mM ß-mercaptoethanol, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1 mM sodium orthovanadate, 0.2 mM PMSF, 10 µg/ml Leupeptin and 1% Triton). Proteins (20 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose Hybond membrane (Amersham Bioscience, Little Chalfont, UK). Protein transfer was confirmed by Ponceau-S stain, and blots were probed with 6C5 (a monoclonal antibody recognising an epitope in the C terminus of dystrophin; Novocastra Laboratories, Newcastle-upon-Tyne, UK). Anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) linked to peroxidase was used for ECL immunodetection (Amersham).
Primary culture of mdx muscles
Primary cultures of mdx mouse muscle were prepared using a modification of a method described previously (49). Briefly, TA muscles from injected mice were dissociated with 1 mg/ml collagenase and 1 mg/ml bovine serum albumin (Sigma-Aldrich, Poole, UK) in phosphate-buffered saline for four periods of 15 min. After mechanical disruption, mononuclear cells in suspension were passed through a nylon cell sieve with a pore size of 40 µm (BD, Oxford, UK), prior to centrifugation at 1000 g for 10 min. Cells were plated out at low density into six-well plates (Nunc, Roskilde, Denmark) that had been precoated with the growth matrix ECM gel (Sigma-Aldrich). Cultures were allowed to grow in DMEM containing 15% FCS for 5–7 days, before the medium was changed to one containing 1% FCS to induce terminal differentiation. Myotubes were fixed in 0.5% glutaraldehyde and incubated in LacZ staining solution (1 mg/ml X-gal in 50 mM Tris–HCl pH 7.5, 25 mM K3Fe(CN)6, 25 mM K4Fe(CN)6, 15 mM NaCl and 1 mM MgCl2) for 2 h to detect expression of ß-galactosidase. Colonies of retrovirally-transduced myotubes were identified as five or more myotubes expressing ß-galactosidase in direct proximity to each other.
Immuno- and enzyme-histochemistry of adenovirus-injected muscles
Approximately 25 µl of adenoviral vector solution (1x109 viral particles per TA) was directly injected using a 27-gauge needle into the TA muscle of anaesthetized mdx mice at 4–6 weeks of age. Neonatal mdx or mdx/nude mice were anaesthetized and injected with 5 µl of adenoviral vector solution containing an equivalent titre of virus. One, four or twelve weeks after injection, the muscle was isolated, coated with OCT compound and frozen in liquid-nitrogen-cooled isopentane. Cross-sections (10 µm for immunohistochemistry and 15 µm for enzyme histochemistry) from injected and non-injected mdx muscles were air-dried and stored at -80°C prior to use. To detect ß-galactosidase activity, muscle sections were fixed in a 0.5% solution of glutaraldehyde and stained overnight at 37°C in LacZ-staining solution. To assess the efficiency of transduction, the number of myofibres positive for ß-galactosidase activity in each TA muscle was counted. Detection of DAG complex components in mice injected with micro-dystrophin-expressing vector was achieved using monoclonal antibodies obtained from Novocastra Laboratories against micro-dystrophin (6C5), ß-dystroglycan (NCLBDG), -sarcoglycan (NCLASARC) and ß-sarcoglycan (NCLBSARC), with a Mouse On Mouse antibody kit (Vector Laboratories, Burlingame, CA, USA). To determine the efficiency of micro-dystrophin expression, images from whole muscle sections probed with 6C5 antibody were captured with a Color Coolview digital camera (Photonic Sciences, Staffordshire, UK) and quantification of the number of dystrophin-positive muscle fibres was achieved using the image analysis software Sigma Scan Pro5.
PCR-based detection of integrated provirus
Genomic DNA was prepared from tissue by grinding in liquid nitrogen followed by an overnight incubation at 55°C in 1 ml lysis buffer (10 mM Tris–HCl pH 8.2, 0.4 M NaCl, 2 mM EDTA, 0.7% SDS and 0.33 mg/ml proteinase K). DNA was extracted from lysate by extracting twice with phenol/chloroform/isoamyl alcohol and four times with chloroform/isoamyl alcohol prior to precipitation in an equal volume of propan-2-ol. Purified DNA was washed twice in 70% ethanol and stored at -80°C prior to use. Proviral vector template was initially amplified from 200 ng genomic DNA by PCR using forward primer 1 (5'-GCTAGCTTAAGTAACGCC-3'; Invitrogen, Paisley, UK) and reverse primer 2 (5'-ACATAGACACTAGACAATCGG-3'; Invitrogen) for 40 cycles annealing at 61°C. Integrated proviral template was subsequently detected by nested PCR (40 cycles) using a forward primer that specifically annealed at 54°C to the retroviral 3'-LTR (5'-GATGGAACAGCTGAATATGGG-3'; Invitrogen). The primers and conditions for GAPDH PCR have been described previously (50).
|
ACKNOWLEDGEMENTS |
|---|
This work was supported by the Muscular Dystrophy Campaign (UK), the Association Française contre les Myopathies (France) and the Muscular Dystrophy Association (USA).
|
FOOTNOTES |
|---|
* To whom correspondence should be addressed: Tel: +44 1784443545; Fax: +44 1784434326; Email: g.dickson{at}rhul.ac.uk
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Emery, A.E. (1993) Duchenne muscular dystrophy – Meryon's disease. Neuromuscul. Disord., 3, 263–266.[Medline]
2 Brenmen, J.E., Chao, D.S., Xia, H., Aldape, K. and Bredt, D.S. (1995) Nitrogen-oxide synthase complexed with dystrophin and absent from skeletal-muscle sarcolemma in Duchenne muscular-dystrophy. Cell, 82, 743–752.[Web of Science][Medline]
3
Grady, R.M., Grange, R.W., Lau, K.S., Maimone, M.M., Michol, M.C., Stull, J.T. and Sanes, J.R. (1999) Role for -dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies. Nat. Cell Biol., 1, 215–220.[Web of Science][Medline]
4 Bredt D.S. Knocking signalling out of the dystrophin complex. Nat. Cell Biol. 1999; 1, E89–E91.[Web of Science][Medline]
5 England, S.B., Nicholson, L.V.B., Johnson, M.A., Forrest, S.M., Love, D.R., Zubrzyckagaarn, E.E., Bulman, D.E., Harris, J.B. and Davies, K.E. (1990) Very mild muscular-dystrophy associated with the deletion of 46% of dystrophin. Nature, 343, 180–182.[Medline]
6 Yuasa, K., Miyagoe, Y., Yamamoto, K., Nabeshima, Y., Dickson, G. and Takeda, S. (1998) Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs. FEBS Lett., 425, 329–336.[Web of Science][Medline]
7
Wang, B., Li, J. and Xiao, X. (2000) Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc. Natl Acad. Sci. USA, 97, 13714–13719.
8
Quantin, B., Perricaudet, L.D., Tajbakhsh, S. and Mandel, J.L. (1992) Adenovirus as an expression vector in muscle cells in vivo. Proc. Natl Acad. Sci. USA, 89, 2581–2584.
9 Ascadi, G., Jani, A., Massie, B., Simoneau, M., Holland, P., Blaschuk, K. and Karpati, G. (1994) Cultured human myoblasts and myotubes show markedly different transducibility by replication-defective adenovirus recombinants. Gene Ther., 3, 579–584.
10 Alameddine, H.S., Quantin, B., Cartaud, A., Dehaupas, M., Mandel, J.L. and Fardeau, M. (1994) Expression of a recombinant dystrophin in mdx mice using adenovirus vector. Neuromusc. Disord., 4, 193–203.[Web of Science][Medline]
11 Ascadi, G., Lochmuller, H., Jani, A., Huard, J., Massie, B., Prescott, S., Simoneau, M., Petrof, B.J. and Karpati, G. (1996) Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer. Hum. Gene Ther., 7, 129–140.[Web of Science][Medline]
12 Clemens, P.R., Kochanek, S., Sunada, Y., Chan, S., Chen, H.H., Campbell, K.P. and Caskey, C.T. (1996) in vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes. Gene Ther., 3, 965–972.[Web of Science][Medline]
13 Petrof, B.J., Lochmuller, H., Massie, B., Yang, L., Macmillan, C., Zhao, J.E. Nalbantoglu, J. and Karpati, G. (1996) Impairment of force generation after adenovirus-mediated gene transfer to muscle is alleviated by adenoviral gene inactivation and host CD8+ T cell deficiency. Hum. Gene Ther., 7, 1813–1826.[Web of Science][Medline]
14 Floyd, S.S., Clemens, P.R., Ontell, M.R., Kochanek, S., Day, C.S., Yang, J., Haushka, S.D., Balkir, R., Morgan, J., Moreland, M.S. et al. (1998) Ex vivo gene transfer using adenovirus-mediated full-length dystrophin delivery to dystrophic muscles. Gene Ther., 5, 19–30.[Web of Science][Medline]
15 Van Deutekom, J.C.T., Cao, B., Pruchnic, R., Wickham, T.J., Kovesdi, I. and Huard, J. (1999) Extended tropism of an adenoviral vector does not circumvent the maturation-dependent transducibility of mouse skeletal muscle. J. Gene Med., 1, 393–399.[Web of Science][Medline]
16 Lochmuller, H., Petrof, B.J., Pari, G., Larochelle, N., Dodelet, V., Wang, Q., Allen, C., Prescott, S., Massie, B., Nalbabtoglu, J. et al. (1996) Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice. Gene Ther., 3, 706–716.[Web of Science][Medline]
17
Kafri, T., Morgan, D., Krahl, T., Sarvetnick, N., Sherman, L. and Verma, I. (1998) Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc. Natl Acad. Sci. USA, 95, 11377–11382.
18
Jooss, K., Yang, Y.P., Fisher, K.J. and Wilson, J.M. (1998) Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J. Virol., 72, 926–933.
19 Vincent, N., Ragot, T., Gilgenkrantz, H., Couton, D., Chafey, P., Gregoire, A., Briand, P., Kaplan, J.C., Kahn, A. and Perricaudet, M. (1993) Long-term correction of mouse dystrophic degeneration by adenovirus-mediated transfer of a minidystrophin gene. Nat. Genet., 5, 130–134.[Web of Science][Medline]
20
Dunckley, M.G., Wells, D.J., Walsh, F.S. and Dickson, G. (1993) Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo. Hum. Mol. Genet., 2, 717–723.
21 Fassati, A., Wells, D.J., Walsh, F. and Dickson, G. (1995) Efficiency of in vivo gene transfer using murine retroviral vectors is strain-dependent in mice. Hum. Gene Ther., 6, 1177–1183.[Web of Science][Medline]
22 Fassati, A., Wells, D.J., Walsh, F. and Dickson, G. (1996) Transplantation of retroviral producer cells for in vivo gene transfer into mouse skeletal muscle. Hum Gene Ther., 7, 595–602.[Web of Science][Medline]
23 Fassati, A., Wells, D.J., Serpente, P.A.S., Walsh, F.S., Brown, S.C., Strong, P.N. and Dickson, G. (1997) Genetic correction of dystrophin deficiency and skeletal muscle remodelling in adult mdx mouse via transplantation of retroviral producer cells. J. Clin. Invest., 100, 620–628.[Web of Science][Medline]
24 Hartigan-O'Connor, D. and Chamberlain, J.S. (2000) Developments in gene therapy for muscular dystrophy. Microsc. Res. Tech. 48, 223–238.[Web of Science][Medline]
25 Welsh, R.M., Cooper, N.R., Jenson, F.C. and Oldstone, M.B.A. (1975) Human serum lyses RNA tumor viruses. Nature, 257, 612–614.[Medline]
26 Cosset, F.L., Takeuchi, Y., Battini, J.L., Weiss, R.A. and Collins, M.K.L. (1995) High-titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol., 69, 7430–7436.[Abstract]
27
Ory, D.S., Neugeboren, B.A. and Mulligan, R.C. (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl Acad. Sci. USA, 93, 11400–11406.
28 Aoki, M., Morishita, R., Higaki, J., Moriguchi, A., Hayashi, S., Matsushita, H., Kida, I., Tornita, N., Sawa, Y., Kaneda, Y. et al. (1997) Survival of grafts of genetically modified cardiac myocytes transfected with FITC-labeled oligodeoxynucleotides and the ß-galactosidase gene in the non-infarcted area, but not the myocardial infracted area. Gene Ther., 4, 120–127.[Web of Science][Medline]
29 Kerr, W.G. (1998) Genetic modification of the hematolymphoid compartment for therapeutic purposes. Hematol. Oncol. Clin. North Am., 12, 503–519.[Web of Science][Medline]
30 Overturf, K., Al-Dhalimy, M., Manning, K., Ou, C.N., Finegold, M. and Grompe, M. (1998) Ex vivo hepatic gene therapy of a mouse model of hereditary tyrosinemia type I. Hum. Gene Ther., 9, 295–304.[Web of Science][Medline]
31
Culver, K.W., Ram, Z., Walbridge, S., Ishii, H., Oldfield, E.H. and Blease, R.M. (1992) In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science, 256, 1550–1552.
32 Feng, M., Jackson, W.H., Goldman, C.K., Rancourt, C., Wang, M., Dusing, S.K., Siegal, G. and Curiel, D.T. (1997) Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector. Nat. Biotechnol., 15, 866–870.[Web of Science][Medline]
33 Lin, X. (1998) Construction of new retroviral producer cells from adenoviral and retroviral vectors. Gene Ther., 5, 1251–1258.[Web of Science][Medline]
34 Ramsey, W.J., Caplen, N.J., Li, Q., Higginbotham, J.N., Shah, M. and Blaese, R.M. (1998) Adenovirus vectors as transcomplementing templates for the production of replication defective retroviral vectors. Biochem. Biophys. Res. Commun., 246, 912–919.[Web of Science][Medline]
35 Caplen, N.J., Higginbotham, J.N., Scheel, J.R., Vahanian, N., Yoshida, Y., Hamada, H., Blaese, R.M. and Ramsey, W.J. (1999) Adeno-retroviral chimeric viruses as in vivo transducing agents. Gene Ther., 6, 454–459.[Web of Science][Medline]
36 Duisit, G., Salvetti, A., Moullier, P. and Cosset, F.L. (1999) Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines. Hum. Gene Ther., 10, 189–200.[Web of Science][Medline]
37 Reynolds, P.N., Feng, M. and Curiel, D.T. (1999) Chimeric viral vectors – the best of both worlds? Mol. Med. Today, 5, 25–31.[Web of Science][Medline]
38 Roberts, M.L., Athanasopoulos, T., Pohlschmidt, M., Duisit, G., Cosset, F.L. and Dickson, G. (2001) Post-mitotic, differentiated myotubes efficiently produce retroviral vector from adeno-retrovirus templates. Gene Ther., 8, 1580–1586.[Web of Science][Medline]
39 Fujii, I., Suzuki, S., Igarashi, T., Matsukura, M., Miike, T., and Shimada, T. (2000) Targeted and stable gene delivery into muscle cells by a two-step transfer system. Biochem. Biophys. Res. Commun., 275, 931–935.[Web of Science][Medline]
40 Ito, M. and Kedes, L. (1997) Two-step delivery of retroviruses to postmitotic, terminally differentiated cells. Hum. Gene Ther., 8, 57–63.[Web of Science][Medline]
41 Haecker, S.E., Stedman, H.H., Balice-Gordon, R.J., Smith, D.B., Greelish, J.P., Mitchell, M.A., Wells, A., Sweeney, H.L. and Wilson, J.M. (1996) In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes. Hum. Gene Ther., 7, 1907–1914.[Web of Science][Medline]
42 Amalfitano, A., Hauser, M.A., Hu, H.M., Serra, D., Begy, C.R., and Chamberlain, J.S. (1998) Production and characterization of improved adenoviral vectors with E1, E2b and E3 genes deleted. J. Virol., 75, 4212–4223.
43 Hodges, B.L., Serra, D., Hu, H.M., Begy, C.A., Chaberlain, J.S., and Amalfitano, A. (2000) Multiply deleted [E1, polymerase-, and pTP-] adenovirus vector persists despite deletion of the preterminal protein. J. Gene Med., 2, 250–259.[Web of Science][Medline]
44
Hodges, B.L., Evans, H.K., Everett, R.S., Ding, E.V., Serra, D. and Amalfitano, A. (2001) Adenovirus vectors with the 100 K gene deleted and their potential for multiple gene therapy applications. J. Virol., 75, 5913–5920.
45
He, T.C., Zhou, S.B., da Costa, L.T., Yu, J., Kinzler, K.W. and Vogelstein, B. (1998) A simplified version for generating recombinant adenovirus. Proc. Natl Acad. Sci. USA., 95, 2509–2514.
46 Fallaux, F.J., Kranenburg, O., Cramer, S.J., Houweling, A., Van Ormondt, H., Hoeben, R.C., and van Der Eb, A.J. (1996) Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum. Gene Ther., 7, 215–222.[Web of Science][Medline]
47 Graham, F.L. and Prevec, L. (1995) Methods for construction of adenovirus vectors. Mol. Biotechnol., 3, 207–220.[Web of Science][Medline]
48 Yaffe, D. and Saxel, O. (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature, 270, 725–727.[Medline]
49
Brown, S.C., Fassati, A., Popplewell, L., Page, A.M., Henry, M.D., Campbell, K.P. and Dickson, G. (1999) Dystrophic phenotype induced in vitro by antibody blockade of muscle -dystroglycan–laminin interaction. J. Cell Sci., 112, 209–216.[Abstract]
50 De Geest, B., Van Linthout, S. and Collen, D. (2001) Sustained expression of human apo A-I following adenoviral gene transfer in mice. Gene Ther., 8, 121–127.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. S. Chamberlain Gene therapy of muscular dystrophy Hum. Mol. Genet., October 1, 2002; 11(20): 2355 - 2362. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||