Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells
Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cellsRajendra Kumar-Singh and Jeffrey S. Chamberlain*
Department of Human Genetics, University of Michigan Medical School, 1150 West Medical Center Drive, Med Sci II, Rm 3726, Ann Arbor, MI 48109-0618, USA
Received February 23, 1996;Revised and Accepted April 4, 1996
Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of ~8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (~14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a[beta]-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1+E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (>108/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.
Duchenne and Becker muscular dystrophy (DMD/ BMD) are X-linked recessive disorders characterized by progressive muscle weakness, cardiomyopathy and early death. Prevalence estimates for DMD are of the order of 1 in 3500 newborn males with one-third of these cases being sporadic. Patients typically begin using a wheelchair by the age of 8-11 years due to progressive degeneration of limb and axial musculature and usually die in their early twenties as a result of respiratory and/or cardiac failure (1 ).
DMD and the milder version of the disease, BMD, are due to mutations in the gene encoding dystrophin, a 427 kDa sarcolemma-associated protein that is also expressed as multiple isoforms in non-muscle tissues (2 ). The mdx mouse, an animal model for DMD, has a point mutation in exon 23 of dystrophin eliminating the 427 kDa muscle and brain isoforms (3 ,4 ). We have previously shown that expression of full-length dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity, suggesting that gene therapy may be an effective treatment for this debilitating disorder (5 ). We and others have also demonstrated that truncated dystrophin minigenes can almost completely prevent the appearance of dystrophic symptoms in mdx mice; however, this incomplete correction may not be optimal for clinical trials (6 ,7 ).
Methods to deliver dystrophin in an efficient manner to muscle tissues are currently being investigated in many laboratories. Human adenovirus (Ad) has many favorable characteristics that make it a promising vector for gene delivery to muscle. These characteristics include a high tropism for muscle tissue, no proven integration into the host genome and convenient mass production yielding high titer stocks (8 ,9 ). However, current generation Ad-based gene therapy vectors are of limited use due to their relatively small cloning capacity (~8.5 kb) and their induction of an inflammatory response that leads to transient gene expression in vivo (10 -12 ). This small cloning capacity restricts the inclusion of gene regulatory elements or large gene cassettes such as dystrophin. Although current versions of Ad vectors have greatly diminished replication capacities in vivo due to deletions of the viral genes E1a and E1b, low level expression of the remaining viral genes is believed to alert the host's immune system, triggering a cytotoxic T-cell-mediated response that attacks Ad-infected cells (13 ,14 ). Initial steps to address these problems have demonstrated that temperature-sensitive viruses partially defective in E2A (DNA binding protein) significantly prolong transgene expression by limiting viral replication and gene expression (14 ,15 ).
In this report we describe the construction of an encapsidated adenovirus minichromosome (EAM) consisting of an infectious encapsidated linear genome containing Ad origins of replication, packaging signal elements, a [beta]-galactosidase reporter gene cassette and a full-length (14 kb) dystrophin cDNA regulated by a muscle-specific enhancer/promoter. EAMs are generated by co-transfecting 293 cells with supercoiled plasmid DNA (pAd5[beta]dys) containing an embedded inverted origin of replication (and the remaining above elements) together with linear DNA from E1-deleted virions expressing human placental alkaline phosphatase (hpAP). All proteins necessary for the generation of EAMs are provided in trans from the hpAP virions and the two can be separated from each other on equilibrium CsCl gradients. Such EAMs may prove useful for gene transfer to a variety of cell types both in vitro and in vivo.
To establish a vector system capable of delivering full-length dystrophin cDNA clones, we combined the minimal region of Ad5 needed for replication and packaging with a conventional plasmid carrying both dystrophin and a [beta]-galactosidase reporter gene. The resultant vector, pAd5[beta]dys (Fig. 1 ) contains 2.1 kb of adenovirus DNA, together with a 14 kb murine dystrophin cDNA under the control of the mouse muscle creatine kinase enhancer/promoter, as well as a [beta]-galactosidase gene regulated by the human cytomegalovirus (CMV) enhancer/promoter (see Materials and Methods). We reasoned that such a plasmid should be packageable into an encapsidated minichromosome when grown in parallel with an E1-deleted virus due to the inclusion of both inverted terminal repeats (ITRs) and the major Ad packaging signals in the plasmid. The ITRs and packaging signals were derived from pFG140, a plasmid that generates E1-defective Ad particles upon transfection of human 293 cells (16 ). hpAP is an E1-deleted Ad5 containing the human placental alkaline phosphatase gene (17 ). This virus was chosen to provide the helper functions so that we would be able to monitor the titer of the helper virus throughout serial passages by quantitative alkaline phosphatase assays. Co-transfection of 293cells with supercoiled pAd5[beta]dys and linear hpAP DNA produced Ad5[beta]dys EAMs (the encapsidated version of pAd5[beta]dys) ~6-12 days post-transfection, as evidenced by the appearance of a cytopathic effect (CPE). Initially, the amount of Ad5[beta]dys EAMs produced was significantly lower than that of hpAP virions, so the viral suspension was used to re-infect fresh cultures, from which virus was isolated and used for serial infection of additional cultures (Fig. 2 ). The rate of increase in titer of Ad5[beta]dys EAMs between transfection and serial passage 6 is ~100 times greater than that for hpAP virions. This result indicates that Ad5[beta]dys has a replication advantage over the helper virus, probably due to the shorter genome length (a difference of ~8 kb) and hence an increased rate of packaging.
We have examined the possibility of separating Ad5[beta]dys EAMs from hpAP virions based on their buoyancy difference (due presumably to their different genome lengths) on CsCl gradients. Repeated fractionation of the viral lysate (see Materials and Methods) allows small differences in buoyancy to be resolved. Results of the physical separation between Ad5[beta]dys EAMs and hpAP virions are shown in Figure 4 . Purification of the band fractions containing predominantly Ad5[beta]dys EAMs from 20% of the lysate from passage 6 and analyses of contamination by hpAP virions in the various fractions by infection of 293cells is shown in Figure 5 . The maximum ratio of transducing Ad5[beta]dys EAMs to hpAP virions reproducibly achieved in this study was 24.8-a contamination with helper virus corresponding to ~4% of the final viral isolate.
To determine if the Ad5[beta]dys EAMs were able to express [beta]-galactosidase and dystrophin in muscle cells, we infected mouse mdx myogenic cultures with CsCl-purified EAMs. Western blot analysis of EAM-infected mdx myoblasts and myotubes (3 days post-fusion) indicated that EAMs were able to infect both of these cell types (Fig. 6 ). Expression of [beta]-galactosidase was detected in both the infected mdx myoblasts and myotubes, indicating that the CMV promoter was active at both these early stages of differentiation in muscle cells. However, only infected mdx myotubes produced protein detected by dys-2, an antibody recognizing the 17 C-terminal amino acids of dystrophin (Fig. 6 ). No dystrophin expression was detected in infected myoblasts by western analysis, indicating that the muscle creatine kinase promoter functions minimally, if at all, within the Ad5[beta]dys EAM prior to terminal differentiation of these cells. Dystrophin expression in mdx cells infected with EAMs was confirmed by immunofluorescence studies using N-terminal dystrophin antibodies. In agreement with the western analysis, dystrophin expression from the muscle creatine kinase (MCK) promoter was detected only in differentiated mdx myotubes infected by Ad5[beta]dys EAMs (Fig. 7 ).
Several genetic complementation strategies have been proposed as potential therapeutic treatments for DMD, including the transplantation of donor myoblasts (22 ), delivery of recombinant dystrophin expression cassettes by intramuscular injection of naked DNA (23 ) or using retroviral (24 ) and adenoviral vectors (25 ). Each strategy has met with severe limitations. Although 30-80% of muscle fibers of nude/mdx mice are dystrophin-positive following myoblast transplantation, few dystrophin-positive fibers have been obtained using these methods in human trials (22 ,26 -28 ). Intramuscular injection of plasmid DNA transduces only 1-3% of myofibers in the quadriceps of mdx mice, a scenario unlikely to be effective in clinical trials (23 ). Retroviral vectors only infect dividing cells, limiting their application to post-mitotic muscle fibers and neurons, the target tissues for DMD (29 ). Adenovirus-mediated transfer of a minidystrophin gene to newborn mdx quadriceps has produced a reduction of dystrophic degeneration (25 ). However, at high doses of virus administration to immunocompetent animals, an inflammatory response results from the low-level expression of viral proteins (14 ,15 ). Hence, considerable improvement in vector technology will be needed to overcome the limitations of cloning capacity and viral gene expression before clinical trials of gene therapy for DMD could be considered.
The approach described in this study makes use of the positive characteristics of adenovirus, i.e. a high infectivity rate and hence efficient transduction of target cells while eliminating the viral genome, expression of which contributes to the development of a strong host immune response. Our strategy depends on the observation that Ad DNA is packaged into virions in a polar, left-to-right fashion and is dependent on cis-acting packaging elements. The Ad5 packaging domain extends from nucleotide 194 to 358 and is composed of five distinct elements that are functionally redundant (30 ,31 ). Theoretically, any molecule containing the Ad5 origin of replication and packaging elements should replicate and be packaged into mature virions in the presence of non-defective helper virus. It has already been shown that Ad origins of replication are functional on autonomously replicating minichromosomes (32 ). Minichromosomes containing two cloned inverted Ad termini require non-defective Ad as a helper. DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear minichromosomes from circular molecules (16 ). Furthermore, it has been shown previously that viruses rendered defective by replacement of a small part of the genome with a reporter gene cassette can be rescued in a helper-dependent manner (33 ).
These data suggest that an artificial, infectious virus could be constructed containing no viral genes yet maintaining a high infection efficiency due to the viral capsid. To test this hypothesis, we have constructed encapsidated adenovirus minichromosomes comprised of a full-length dystrophin cDNA under the control of a 3.3 kb MCK enhancer/promoter, a bacterial [beta]-galactosidase ([beta]-gal) gene under control of a CMV enhancer/promoter and the Ad origin of replication together with cis-acting viral packaging signals. EAMs were propagated and amplified by serial passage in 293 cells with the use of a trans-acting E1-deleted helper virus. The obvious advantage of these vectors is that they should theoretically have a cloning capacity up to 35 kb and yet be unable to express any virally encoded genes.
The choice of hpAP as a helper virus has allowed monitoring of the relative populations of helper to EAMs at the various stages of serial propagation. We have shown that the smaller genome of the EAM (27.8 kb) has a packaging advantage over that of the helper virus (~36 kb) as evidenced by the net accumulation of EAMs at a rate higher than that of the helper virus after several rounds of replication. Separation of EAMs from helper virus was achieved by repeated centrifugation of viral lysates on a continuous CsCl gradient. Similar conditions have been used to achieve partial separation of viruses differing in size by only 200 bp (33 ). Our results indicate that EAMs can be propagated efficiently to high titers in the presence of hpAP as a helper and that EAMs can be separated from helper virus on CsCl gradients. We have shown that the lysate can by fractionated to yield an enriched fraction for EAMs, which before CsCl purification represented a minor amount of the total viral preparation.
Temporal specific expression of the recombinant genes has been examined by comparing expression of [beta]-galactosidase and dystrophin in mdx myogenic cultures at various stages of differentiation following infection by Ad5[beta]dys EAMs. The endogenous MCK promoter has been shown previously to be inactive in myoblasts, and is transcriptionally activated only upon terminal differentiation of myoblasts into myocytes and myotubes (34 ,35 ). Since the Ad5[beta]dys EAMs contain the E1 enhancer, it was of interest to determine whether the MCK promoter/enhancer would be active in myoblasts. However, we did not detect dystrophin in myoblast cultures following infection with Ad5[beta]dys. In contrast, dystrophin synthesis was readily observed following infection of differentiated mdx myofibers, using both indirect immunofluorescence and western analysis with dystrophin antibodies.
A similar strategy to deliver a full-length dystrophin cDNA has been reported recently by Kochanek and colleagues (36 ). However, in that study, the ITRs were ligated onto a linear plasmid immediately prior to co-transfection of 293 cells with helper viral DNA. Our approach incorporates the ITRs directly into the plasmid vector, simplifying preparation of EAMs by direct plasmid propagation.
Several key issues need to be addressed prior to the introduction of EAMs into mdx mice or other animal models of human disease. Firstly, contamination of EAMs with helper virions will be deleterious by contributing towards adenoviral gene expression. Secondly, the emergence of defective virions other than EAMs at later passages allows for the possibility of co-migration of EAMs and defectives in the CsCl gradient. Such defectives may contain parts of the Ad genome capable of expressing some or all of the adenoviral genes by trans complementation. During the course of this study, we have been able to obtain fractions with a maximum contamination by helper of 4%. Reduction in the size of pAd5[beta]dys by removal of the [beta]-galactosidase gene cassette should further increase the physical separation between the two types of virions and hence enhance the purity of Ad5[beta]dys EAMs. The vector prepared by Kochanek and colleagues incorporated a slightly larger amount of DNA than our vector, yet those authors were able to isolate viral fractions with reduced amounts of helper (36 ). The difference in helper contamination obtained in the two studies probably reflects differences in CsCl purification protocols, although it will be important to determine the effect of genome size on both vector stability and separation from helper virus. A systematic test of materials with potentially better resolving powers than CsCl at [rho] = 1.3, such as rubidium chloride and potassium bromide (37 ), may result in further enhancement of the purity of Ad5[beta]dys EAMs. Furthermore, although Ad5[beta]dys EAMs seem to have a packaging advantage over hpAP, titers of the Ad5[beta]dys EAM might be increased further by using several strategies including the use of helper viruses that approach the packaging limits of Ad5 (10 ), the use of viruses with mutations in E4 or E2 genes (38 -40 ), inclusion of non-lethal mutations in the packaging signals of hpAP virions (41 ) or a combination of these strategies. Strategies may also be developed where fractions enriched for Ad5[beta]dys EAMs are used for co-propagation of the two viruses, reducing the total multiplicity of infection and hence the probability of recombination events.
Purification of high titer EAM stocks will enable in vivo experiments to test the potential immunogenicity of these vectors. Previous studies documenting transient gene expression from adenoviral vectors suggest that a major limitation of these delivery systems originates from low-level viral gene expression resulting in an immune response against infected cells (10 -12 ). The deletion of almost the entire Ad genome from these viruses removes the possibility of Ad gene expression in vivo and could result in a greatly reduced or absent host immune response as compared with current generation Ad vectors. However, detailed experiments in animals will be needed to characterize fully the potential immunogenicity of these vectors.
In summary, we have shown that embedded inverted Ad origins of replication coupled to an encapsidation signal can convert circular DNA molecules to linear forms in the presence of helper virus and that these genomes can be encapsidated efficiently and propagated to high titers. Such viruses can be purified on a CsCl gradient and maintain their ability to transduce cells in vitro, and their increased cloning capacity allows the inclusion of large genes and tissue-specific gene regulatory elements. We have also shown that the dystrophin gene is expressed in cells transduced by such viruses and that the protein product is correctly localized to the cell membrane. Our method for preparing EAMs theoretically enables virtually any gene of interest to be inserted into an infectious minichromosome by conventional cloning in plasmid vectors, followed by co-transfection with helper viral DNA in 293 cells. This approach should prove useful for a variety of gene transfer studies in vitro. While considerable work remains to optimize the propagation and purification of Ad minichromosome vectors, the observation that vectors completely lacking viral genes can be used to transfer a full-length dystrophin cDNA into myogenic cells indicates the potential for this method to contribute to the development of effective gene therapy for DMD.
Cloning and in vitro mutagenesis were carried out according to standard techniques (42 ). A modified version of BluescriptII KS+ (Stratagene) was used as the backbone for construction of pAd5[beta]dys. The inverted fused Ad5 origin of replication and five encapsidation signals were excised as a PstI-XbaI fragment from pAd5ori, a plasmid containing the 6 kb HindIII fragment from pFG140 (16 ). This strategy also introduces a 290 bp fragment from Ad5 corresponding to map units 6.97-7.77 adjacent to the right inverted repeat (see Fig. 1 ). The Escherichia coli [beta]-galactosidase gene regulated by the human CMV immediate early promoter/enhancer expression cassette was derived as an EcoRI-HindIII fragment from pCMV[beta] (43 ). The murine dystrophin expression cassette was derived as a BssHII fragment from pCVAA, and contains a 3.3 kb MCK promoter/enhancer element (6 ).
Low passage 293 cells (Microbix Biosystems) were grown and passaged as suggested by the supplier. pAd5[beta]dys and hpAP DNA (5 and 0.5 [mu]g, respectively) were dissolved in 70 [mu]l of 20 mM HEPES buffer (pH 7.4) and incubated with 30 [mu]l of DOTAP (BMB) for 15 min at room temperature. This mixture was resuspended in 2 ml of DMEM supplemented with 2% fetal calf serum (FCS) and added dropwise to a 60 mm plate of 293 cells at 80% confluency. Four hours post-transfection the media was replaced by DMEM with 10% FCS. CPE was observed 6-12 days post-transfection.
Lysate from one 60 mm plate of transfected 293 cells was prepared by vigorously washing the cells from the plate and centrifuging at 1000 r.p.m. in a clinical centrifuge. Cells were resuspended in DMEM and 2% FCS, freeze-thawed in a dry ice-ethanol bath, cell debris removed by centrifugation, and ~75% of the crude lysate was used to infect 293 cells in DMEM supplemented with 2% FCS for 1 h and then supplemented with 10% FCS thereafter. Infection was allowed to proceed for 18-20 h before harvesting the virus. The total number of cells infected in each serial passage is indicated in Figure 2 .
Approximately 25% of the lysate prepared from various passages during serial infections was used to purify virions. Freeze-thawed lysate was centrifuged to remove the cell debris. The cleared lysate was extracted twice with 1,1,2 trichlorotrifluoroethane (Sigma) and applied to CsCl step and self-forming gradients. Purification of virus was initially achieved by passing it twice through CsCl step gradients with densities of [rho] = 1.45 and [rho] = 1.20 in a SW28 rotor. After isolation of the major band in the lower gradient, the virus was passed through a self-forming gradient (initial [rho] = 1.334) at 37 000 r.p.m. for 24 h followed by a relaxation of the gradient by reducing the speed to 10 000 r.p.m. for 10 h in a SW41 rotor at 12oC (44 ). The upper band from the gradient (composed mainly of Ad5[beta]dys virions) was isolated using an 18 gauge needle, reloaded on a fourth CsCl gradient ([rho] = 1.334) and purified at 37 000 r.p.m. for 24 h followed by 10 000 r.p.m. for 10 h at 12oC. The Ad5[beta]dys-containing CsCl band was removed in 100 [mu]l fractions from the top of the centrifugation tube and CsCl was removed by chromatography on Sephadex G-50. Aliquots from each fraction were used to infect 293 cells followed by [beta]-galactosidase and alkaline phosphatase assays to quantitate the level of contamination by hpAP virions in the final viral isolate.
For detection of alkaline phosphatase, infected 293 cells on Petri dishes were rinsed twice with phosphate-buffered saline (PBS) and fixed for 10 min in 0.5% glutaraldehyde in PBS. Cells were again rinsed twice with PBS for 10 min followed by inactivation of endogenous alkaline phosphatase activity at 65oC for 1 h in PBS prior to the addition of the chromogenic substrate BCIP (5-bromo-4-chloro-3-indolyl phosphate) at 0.15 mg/ml and nitro blue tetrazolium at 0.3 mg/ml. Cells were incubated at 37oC in darkness for 3-24 h. For [beta]-galactosidase assays, the cells were fixed and washed as above, then assayed as described previously (45 ).
MM14 and mdx myogenic cell lines were kindly provided by S. Hauschka (University of Washington) and were cultured as previously described (46 ). Myoblasts or differentiated myotubes (3 days post-switching) were infected at a multiplicity of infection of 2.2 Ad5[beta]dys EAMs per cell. Fractions containing minimal contamination with hpAP virions (3, 4 and 5 of passage 6) were used for western and immunofluorescence analysis. Infection was allowed to proceed for 3 days for both the myoblasts and myotubes before harvesting cells.
For protein extraction, muscle cells were briefly trypsinized, transferred to a microcentrifuge tube, centrifuged at 14 000 r.p.m. for 3 min at room temp and resuspended twice in PBS. After an additional centrifugation, the cell pellet was resuspended in 80 [mu]l of RIPA homogenate (42 ). The sample was sheared briefly using a 22 gauge needle to reduce viscosity, and total protein concentration assayed using the Coomassie Plus protein assay reagent (Pierce, Rockford, IL). Expression of full-length dystrophin or [beta]-galactosidase in infected mdx and MM14 myoblasts or myotubes was analyzed by electrophoresis of 40 [mu]g of total protein extract on a 6% SDS-PAGE gel (in 25 mM Tris, 192 mM glycine, 10 mM [beta]-mercaptoethanol, 0.1% SDS). After transferring to Gelman Biotrace NT membrane (in 25 mM Tris, 192 mM glycine, 10 mM [beta]-mercaptoethanol, 0.05% SDS, 20% methanol), the membrane was blocked with 5% non-fat milk and 1% goat-serum in Tris-buffered saline-Tween (TBS-T) for 12 h at 4oC. Immunostaining was done according to the protocol for the ECL western blotting detection reagents (Amersham Life Sciences, Buckingham, UK). The primary antibodies used were Dys-2 (Vector laboratories) and anti-[beta]-galactosidase (BMB, Indianapolis, IN) with a horseradish peroxidase-conjugated anti-mouse secondary antibody.
Approximately 1.5 * 106 MM14 or mdx myoblasts were plated on poly-L-lysine (Sigma) coated glass slides (7*3 cm) which previously had been etched with a 0.05% chromium potassium sulfate and 0.1% gelatin solution. For myotube analysis, the cultures were switched to differentiation media (47 ) 48 h after plating, immediately infected and then allowed to fuse for 3 days, whereas myoblasts were propagated continuously in proliferation media (47 ). Cells were washed three times with PBS at room temperature and fixed in 3.7% formaldehyde. For immunostaining, cells were incubated in 0.5% Triton X-100, blocked with 1% normal goat serum and incubated with an affinity-purified antibody against the N-terminus of murine dystrophin (K. Corrado and J.S.C., in preparation) for 2 h followed by extensive washing in PBS and 0.1% Tween-20 with gentle shaking. Cells were incubated with a 1:200 dilution of biotin-conjugated anti-rabbit antibody (Pierce) for 1 h and washed as above. Cells were incubated further with a 1:300 dilution of streptavidin-fluorescein isothiocyanate conjugate (Vectorlabs) for 1 h and washed as above, followed by extensive washing in PBS.
We thank M. A. Hauser and A. Amalfitano for helpful discussions, and S. D. Hauschka for providing the MM14 and mdx myogenic cell lines. This work was supported by grants from the Muscular Dystrophy Association (USA), Harmonize for Hope fund and the Ades family fund.
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