Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase -- increased expression with reduced muscle damage

The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.     

Expression of Dog Microdystrophin in Mouse and Dog Muscles by Gene Therapy

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.     

Optimizing plasmid-based gene transfer for investigating skeletal muscle structure and function.

Intramuscular injection of naked plasmid DNA is a less cytotoxic alternative to viral vectors for delivering genetic material to skeletal muscle in vivo. However, the low efficiency of plasmid-based gene transfer limits its potential therapeutic efficacy and/or its use for many experimental applications. Current strategies to enhance transfection efficiency (i.e., electroporation) can cause significant muscle damage, confounding physiological assessments such as muscle contractility. Optimizing protocols to limit damage is critical for accurate physiological, biochemical, and molecular measurements. Following extensive testing, we developed an electroporation protocol that enhances transfection efficiency in skeletal muscles without causing muscle damage. Pretreating mouse tibialis anterior muscles with hyaluronidase and electroporation at 75 V/cm (using 50% vol/vol saline as a vehicle for plasmid DNA) resulted in 22 +/- 5% of the muscle fibers expressing a reporter gene. This protocol did not compromise contractile function of skeletal muscles assessed at both the intact (whole) muscle and the cellular (single fiber) level. Furthermore, ectopic expression of insulin-like growth factor I to levels that induced muscle fiber hypertrophy without causing tissue damage or compromising muscle function highlights the therapeutic potential of these methods for myopathies, muscle wasting disorders, and other pathophysiologic conditions.     

Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles.

Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.     

Hyaluronidase increases electrogene transfer efficiency in skeletal muscle

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.     

Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.     

Immune responses to dystropin: implications for gene therapy of Duchenne muscular dystrophy

Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse full-length or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be tolerant due to the presence of rare 'revertant' dystrophin-positive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of 'revertant' dystrophin-positive muscle fibres.     

In vivo liver electroporation: optimization and demonstration of therapeutic efficacy

Adverse effects (death and leukemogenesis) from viral vector-mediated gene therapy have renewed interest in plasmids as safer, more scalable, simple, and cost-effective vectors. Electroporation and hydrodynamic delivery are two techniques that improve the efficiency of plasmid-mediated gene transfer. The liver is a good tissue platform for targeted transfer of therapeutically relevant genes for correction of metabolic disorders, for example, hemophilia A. However, in vivo electroporation of liver has not yet been shown to achieve therapeutic efficacy of systemically active, secreted transgenic proteins. We have investigated the effect of field strength, pulse duration, pulse number, electrical waveforms, electrode contact area, plasmid administration routes, and injection technique on the efficiency of in vivo electrotransfer of naked plasmid to liver. Plasmid injection into a systemic vein was superior to intrahepatic injection. Unlike in vivo muscle electroporation, high-voltage pulses and microsecond pulses offered no advantage. Optimal electroporation conditions were 8-10 uni- or bipolar pulses of 20 msec, each at 250 V/cm. Using a nonhydrodynamic technique that greatly enhanced electrotransfer efficiency with minimal tissue injury, we demonstrate for the first time that liverdirected in vivo electroporation of factor VIII cDNA achieved significant phenotypic correction in hemophilic mice.     

Efficient whole-body transduction with trans-splicing adeno-associated viral vectors.

Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.     

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

One of the possible therapies for Duchenne muscular dystrophy (DMD) is the introduction of a functional copy of the dystrophin gene into the patient. For this approach to be effective, therapeutic levels and long-term expression of the protein need to be achieved. However, immune responses to the newly expressed dystrophin have been predicted, particularly in DMD patients who express no dystrophin or only very truncated versions. In a previous study, we demonstrated a strong humoral and cytotoxic immune response to human dystrophin in the mdx mouse. However, the mdx mouse was tolerant to murine dystrophin, possibly due to the endogenous expression of dystrophin in revertant fibres or the other nonmuscle dystrophin isoforms. In the present study, we delivered human and murine dystrophin plasmids by electrotransfer after hyaluronidase pretreatment to increase gene transfer efficiencies. Tolerance to murine dystrophin was still seen with this improved gene delivery. Tolerance to exogenous recombinant full-length human dystrophin was seen in mdx transgenic lines expressing internally deleted versions of human dystrophin. These results suggest that the presence of revertant fibres may prevent the development of serious immune responses in patients undergoing dystrophin gene therapy.     

Hyaluronidase and collagenase increase the transfection efficiency of gene electrotransfer in various murine tumors

One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy.     

AAV-mediated Overexpression of Human α7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.     

Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.     

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.     

Enhanced expression of recombinant dystrophin following intramuscular injection of Epstein-Barr virus (EBV)-based mini-chromosome vectors in mdx mice.

Gene transfer by direct intramuscular injection of naked plasmid DNA has been shown to be a safe, simple but relatively inefficient method for gene delivery in vivo. Eukaryotic plasmid expression vectors incorporating the Epstein-Barr virus (EBV) origin of replication (oriP) and EBNA1 gene have been shown to act as autonomous episomally replicating gene transfer vectors which additionally provide nuclear matrix retention functions. Prolonged expression of a LacZ reporter gene and recombinant human dystrophin was shown using EBV-based plasmid vectors transfected into C2C12 mouse myoblast and myotube cultures. Intramuscular injection of EBV-based dystrophin expression plasmids into nude/mdx mice resulted in significant enhancement in the number of muscle fibres expressing recombinant dystrophin compared with a conventional vector. This effect was observed for over 10 weeks after a single administration. These results indicate the potential advantage of EBV-based expression vectors for focal plasmid-mediated gene augmentation therapy in Duchenne muscular dystrophy (DMD) and a range of other gene therapeutic applications.     

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.     

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, beta-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product. We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. AAV vector administration, however, evoked substantial immune responses in CVBA3' muscle. Importantly, AAV vector using muscle-specific MCK promoter also elicited responses in mdx muscle, but at a considerably later period. These results suggested that neo-antigens introduced by AAV vectors could evoke immune reactions in mdx muscle, since increased permeability allowed a leakage of neo-antigens from the dystrophin-deficient sarcolemma of muscle fibers. However, resident antigen-presenting cells, such as myoblasts, myotubes and regenerating immature myofibers, might also play a role in the immune response.     

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.     

Human apolipoprotein E expression from mouse skeletal muscle by electrotransfer of nonviral DNA (plasmid) and pseudotyped recombinant adeno-associated virus (AAV2/7)

Plasma apolipoprotein E (apoE) has multiple atheroprotective actions. However, although liver-directed adenoviral gene transfer of apoE reverses hypercholesterolemia and inhibits atherogenesis in apoE-deficient (apoE(-/-)) mice, safety considerations have revived interest in nonviral DNA (plasmid) and nonpathogenic adeno-associated viral (AAV) vectors. Here, we assess the effectiveness of these two delivery vehicles by minimally invasive intramuscular injection. First, we constructed AAV2-based expression plasmids harboring human apoE3 cDNA, driven by two muscle-specific promoters (CK6 and C5-12) and one ubiquitous promoter (CAG); each efficiently expressed apoE3 in transfected cultured C2C12 mouse myoblasts, although muscle-specific promoters were active only in differentiated multinucleate myotubes. Second, a pilot study verified that electrotransfer of the CAG-driven plasmid (p.CAG.apoE3) into tibialis anterior muscles, pretreated with hyaluronidase, of apoE(-/-) mice significantly enhanced (p < 0.001) local intramuscular expression of apoE3. However, in a 7-day experiment, the CK6- and C5-12-driven plasmids produced less apoE3 in muscle than did p.CAG.apoE3 (0.61 +/- 0.38 and 0.45 +/- 0.38 vs. 13.38 +/- 7.46 microg of apoE3 per muscle, respectively), but plasma apoE3 levels were below our detection limit (<15 ng/ml) in all mice and did not reverse the hyperlipidemia. Finally, we showed that intramuscular injection of a cross-packaged AAV serotype 7 viral vector, expressing human apoE3 from the CAG promoter, resulted in increasing levels of apoE3 in plasma over 4 weeks, although the concentration reached (1.40 +/- 0.35 microg/ml) was just below the threshold level needed to reduce the hypercholesterolemia. We conclude that skeletal muscle can serve as an effective secretory platform to express the apoE3 transgene, but that improved gene transfer vectors are needed to achieve full therapeutic levels of plasma apoE3 protein.     

Detecting Degenerative Changes in Myotonic Murine Models of Duchenne Muscular Dystrophy Using High‐Frequency Ultrasound

Objective. Ultrasound imaging is an economical and noninvasive technique for studying musculoskeletal diseases such as Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy results from the loss of the cytoskeletal protein dystrophin. This in turn increases muscle susceptibility to injury, resulting in myofiber membrane leakage, inflammation, and degeneration. The purpose of this study was to detect dystrophic changes in muscle noninvasively. High‐frequency ultrasound (HFU; 40 MHz) was used to obtain a resolution of 80 μm, which is not achievable with lower‐frequency clinical scanners. Methods. Using HFU, we were able to visualize musculoskeletal abnormalities as hyperechoic lesions within the dystrophic muscle. To validate the imaging findings, fiducial markers were placed in close proximity to lesions under HFU guidance. The nature of the lesion was then investigated histologically. This was repeated in the lower limbs of 10 mdx (mutated dystrophin gene) mice, a transgenic murine model of DMD. Results. The abnormalities in the dystrophic muscle consisted of large influxes of leukocytic infiltrates, fibrotic scars, and calcified lesions. Conclusions. Although macrophages and fibrosis are commonly noted in DMD, to our knowledge, the presence of intramuscular calcific necrosis in dystrophic muscle has not been reported. This novel dystrophic feature of muscle degeneration may be useful in longitudinal studies of murine DMD and regenerative studies.