Gene Transfer To Skeletal Muscle Research Articles

Five Years of Successful Inducible Transgene Expression Following Locoregional Adeno-Associated Virus Delivery in Nonhuman Primates with No Detectable Immunity.

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adeno-associated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a large-animal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart.

Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

The absorption enhancer sodium deoxycholate promotes high gene transfer in skeletal muscles

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. In addition, muscle is an attractive target tissue because it is easily accessible. However, very few synthetic vectors proved capable of surpassing naked DNA mediated muscle gene transfer. In fact, only neutral copolymers, in particular poloxamers, demonstrated capacities to increase transgene expression in skeletal muscles. Here, we studied in vitro and in vivo behaviour of different bile salts. We report that sodium deoxycholate (DOC) and derivatives thereof increase after intramuscular injection by more than 100-fold the levels of the reporter gene luciferase compared to naked DNA. Using a LacZ expression cassette, we found that more than 20% of the muscle fibers expressed the reporter gene. Prolonged expression of a secreted reporter gene derived from a natural murine alkaline phosphatase enzyme could be documented. Altogether, our results demonstrate that bile salts belong to the most efficient chemicals identified so far for skeletal muscle gene transfer. Importantly, since these compounds are naturally found in the body, there is no risk of immune response against them and in addition several bile salts are already used in human medicine. Bile salt mediated muscle gene transfer may thus have broad applications in gene therapy.

Insulin Therapy Improves Adeno-Associated Virus Transduction of Liver and Skeletal Muscle in Mice and Cultured Cells.

Adeno-associated virus (AAV) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions, that is, α1-antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhance transduction of liver and skeletal muscle, using these vectors, is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested whether insulin therapy improves liver and skeletal muscle gene transfer. In vitro studies demonstrated that the temporary coadministration (2, 8, and 24 hr) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers, but not of lung cells. In addition, there was a dose response related to this improved transduction. Interestingly, when insulin was not coadministered with the virus but given 24 hr afterward, there was no increase in the transgene product. Insulin receptor gene (INSR) expression levels were increased 5- to 13-fold in cultured liver cells and differentiated myofibers when compared with lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice, using a subcutaneously implanted insulin pellet or a high-carbohydrate diet. Insulin treatment began just before intramuscular delivery of AAV1-CMV-schFIX or liver-directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle- and liver-directed gene transduction in mice as seen by a 3.0- to 4.5-fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates that AAV transduction of liver or skeletal muscle can be improved by insulin therapy.

AAV-8 is more efficient than AAV-9 in transducing neonatal dog heart.

Adeno-associated virus serotype-8 and 9 (AAV-8 and 9) are the leading candidate vectors to test bodywide neonatal muscle gene therapy in large mammals. We have previously shown that systemic injection of 2-2.5×10(14) viral genome (vg) particles/kg of AAV-9 resulted in widespread skeletal muscle gene transfer in newborn dogs. However, nominal transduction was observed in the heart. In contrast, robust expression was achieved in both skeletal muscle and heart in neonatal dogs with 7.14-9.06×10(14) vg particles/kg of AAV-8. To determine whether superior cardiac transduction of AAV-8 is because of the higher vector dose, we delivered 6.14×10(14) and 9.65×10(14) vg particles/kg of AAV-9 to newborn puppies via the jugular vein. Transduction was examined 2.5 months later. Consistent with our previous reports, we observed robust bodywide transduction in skeletal muscle. However, increased AAV dose only moderately improved heart transduction. It never reached the level achieved by AAV-8. Our results suggest that differential cardiac transduction by AAV-8 and AAV-9 is likely because of the intrinsic property of the viral capsid rather than the vector dose.

A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

CpG-depleted adeno-associated virus vectors evade immune detection

Due to their efficient transduction potential, adeno-associated virus (AAV) vectors are leading candidates for gene therapy in skeletal muscle diseases. However, immune responses toward the vector or transgene product have been observed in preclinical and clinical studies. TLR9 has been implicated in promoting AAV-directed immune responses, but vectors have not been developed to circumvent this barrier. To assess the requirement of TLR9 in promoting immunity toward AAV-associated antigens following skeletal muscle gene transfer in mice, we compared immunological responses in WT and Tlr9-deficient mice that received an AAV vector with an immunogenic capsid, AAVrh32.33. In Tlr9-deficient mice, IFN-γ T cell responses toward capsid and transgene antigen were suppressed, resulting in minimal cellular infiltrate and stable transgene expression in target muscles. These findings suggest that AAV-directed immune responses may be circumvented by depleting the ligand for TLR9 (CpG sequences) from the vector genome. Indeed, we found that CpG-depleted AAVrh32.33 vectors could establish persistent transgene expression, evade immunity, and minimize infiltration of effector cells. Thus, CpG-depleted AAV vectors could improve outcome of clinical trials of gene therapy for skeletal muscle disease.

Single tyrosine mutation in AAV8 and AAV9 capsids is insufficient to enhance gene delivery to skeletal muscle and heart.

Site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of adeno-associated viral (AAV) capsids have been reported as a simple method to greatly enhance gene transfer in vitro and in vivo. To determine whether the Y-to-F mutation could also enhance AAV8 and AAV9 gene transfer in skeletal muscle and heart to facilitate muscular dystrophy gene therapy, we investigated four capsid mutants of AAV8 (Y447F or Y733F) and AAV9 (Y446F or Y731F). The mutants and their wild-type control AAV8 and AAV9 capsids were used to package reporter genes (luciferase or β-galactosidase) resulting in similar vector yields. To evaluate gene delivery efficiencies, especially in muscle and heart, the vectors were compared side by side in a series of experiments in vivo in two different strains of mice, the outbred ICR and the inbred C57BL/6. Because AAV8 and AAV9 are among the most effective in systemic gene delivery, we first examined the mutant and wild-type vectors in neonatal mice by intraperitoneal injection, or in adult mice by intravenous injection. To our surprise, no statistically significant differences in transgene expression were observed between the mutant and wild-type vectors, regardless of the reporter genes, vector doses, and the ages and strains of mice used. In addition, quantitative analyses of vector DNA copy number in various tissues from mice treated with mutant and wild-type vectors also showed similar results. Finally, direct intramuscular injection of the above-described vectors with the luciferase gene into the hind limb muscles revealed the same levels of gene expression between mutant and wild-type vectors. Our results thus demonstrate that a single mutation of Y447F or Y733F on capsids of AAV8, and of Y446F or Y731F on AAV9, is insufficient to enhance gene delivery to the skeletal muscle and heart.

Baculovirus‐mediated vascular endothelial growth factor‐DΔNΔC gene transfer induces angiogenesis in rabbit skeletal muscle

Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene.

A syringe‐focused ultrasound device for simultaneous injection of DNA and gene transfer

Although known for its success in ameliorating muscle disorders, ultrasound (US)-mediated gene transfer in skeletal muscle has recently shown potential for treating a wide range of therapeutic applications. The disadvantages of the technique, however, including its relatively low transfer efficiency and toxic effects resulting in tissue damage, have impeded clinical progress. We describe a novel syringe-focused US device of our design that combines simultaneous DNA injection and US irradiation to resolve these issues, achieving improved DNA uptake for high expression and lowering the incidence of tissue injury. The novel, syringe-focused US device was used to introduce purified plasmid DNA encoding human neprilysin (hNEP) into mouse hindlimb skeletal muscle concurrent with US protocol (1.7 MHz, 1 W/cm(2), 1-min exposure time). hNEP expression was measured in local skeletal muscle, blood serum and brain by immunoassay at different post-injection time points. The incidence of muscle fiber damage was subsequently assessed by Evans Blue dye (EBD) uptake using fluorescence microscopy. The syringe-US device at the first time enabled simultaneous combination of DNA transfer and US stimulation gives rise to two months of gene expression of the encoded polypeptide as it is measured, at the same time as withstanding minor tissue damage. In addition, DNA injection without US treatment failed to induce hNEP expression. This new technique provides a simple, safe and efficient nonviral gene delivery approach in skeletal muscle and shows promising applications for gene therapy of human disease, such as Alzheimer's disease.

Caveolin 3 Is Associated with the Calcium Release Complex and Is Modified via in Vivo Triadin Modification

The triadin isoforms Trisk 95 and Trisk 51 are both components of the skeletal muscle calcium release complex. To investigate the specific role of Trisk 95 and Trisk 51 isoforms in muscle physiology, we overexpressed Trisk 95 or Trisk 51 using adenovirus-mediated gene transfer in skeletal muscle of newborn mice. Overexpression of either Trisk 95 or Trisk 51 alters the muscle fiber morphology, while leaving unchanged the expression of the ryanodine receptor, the dihydropyridine receptor, and calsequestrin. We also observe an aberrant expression of caveolin 3 in both Trisk 95- and Trisk 51-overexpressing skeletal muscles. Using a biochemical approach, we demonstrate that caveolin 3 is associated with the calcium release complex in skeletal muscle. Taking advantage of muscle and non-muscle cell culture models and triadin null mouse skeletal muscle, we further dissect the molecular organization of the caveolin 3-containing calcium release complex. Our data demonstrate that the association of caveolin 3 with the calcium release complex occurs via a direct interaction with the transmembrane domain of the ryanodine receptor. Taken together, these data suggest that caveolin 3-containing membrane domains and the calcium release complex are functionally linked and that Trisk 95 and Trisk 51 are instrumental to the regulation of this interaction, the integrity of which may be crucial for muscle physiology.

Lack of Immunotoxicity After Regional Intravenous (RI) Delivery of rAAV to Nonhuman Primate Skeletal Muscle

In the absence of an immune response from the host, intramuscular (IM) injection of recombinant adeno-associated virus (rAAV) results in the permanent expression of the transgene from mouse to primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among these factors, the route of delivery is important, although poorly addressed in large animal models. Here, we compare the IM and the drug-free regional intravenous (RI) deliveries of rAAV in nonhuman primate (NHP) skeletal muscle monitoring the host immune response toward the transgene. We show that IM is consistently associated with immunotoxicity and the destruction of the genetically modified myofibers, whereas RI allows the stable expression of the transgene. This has important implications for the design of clinical trials for gene transfer in skeletal muscle.

Efficient gene transfer in skeletal muscle with AAV-derived bicistronic vector using the FGF-1 IRES

IRESs (internal ribosome entry sites) are RNA elements behaving as translational enhancers in conditions of global translation blockade. IRESs are also useful in biotechnological applications as they allow expression of several genes from a single mRNA. Up to now, most IRES-containing vectors use the IRES from encephalomyocarditis virus (EMCV), highly active in transiently transfected cells but long and not flexible in its positioning relative to the gene of interest. In contrast, several IRESs identified in cellular mRNAs are short and flexible and may therefore be advantageous in gene transfer vectors such as those derived from the adeno-associated virus (AAV), where the size of the transgene expression cassette is limited. Here, we have tested bicistronic AAV-derived vectors expressing two luciferase genes separated by the EMCV- or fibroblast growth factor 1 (FGF-1) IRES. We demonstrate that the AAV vector with the FGF-1 IRES, when administrated into the mouse muscle, leads to efficient expression of both transgenes with a stable stoechiometry, for at least 120 days. Interestingly, the bicistronic mRNA containing the FGF-1 IRES leads to transgene expression 10 times superior to that observed with EMCV, in vivo. AAV vectors featuring the FGF-1 IRES may thus be advantageous for gene therapy approaches in skeletal muscle involving coexpression of genes of interest.

Long-term Skeletal Muscle Protection After Gene Transfer in a Mouse Model of LGMD-2D

Limb girdle muscular dystrophy (LGMD) describes a group of inherited diseases resulting from mutations in genes encoding proteins involved in maintaining skeletal muscle membrane stability. LGMD type-2D is caused by mutations in alpha-sarcoglycan (sgca). Here we describe muscle-specific gene delivery of the human sgca gene into dystrophic muscle using an adeno-associated virus 1 (AAV1) capsid and creatine kinase promoter. Delivery of this construct to adult sgca(-/-) mice resulted in localization of the sarcoglycan complex to the sarcolemma and a reduction in muscle fiber damage. Sgca expression prevented disease progression as observed in vivo by T(2)-weighted magnetic resonance imaging (MRI) and confirmed in vitro by decreased Evan's blue dye accumulation. The ability of recombinant AAV-mediated gene delivery to restore normal muscle mechanical properties in sgca(-/-) mice was verified by in vitro force mechanics on isolated extensor digitorum longus (EDL) muscles, with a decrease in passive resistance to stretch as compared with untreated controls. In summary, AAV/AAV-sgca gene transfer provides long-term muscle protection from LGMD and can be non-invasively evaluated using magnetic resonance imaging.

Amphiphilic Poly[(propylene glycol)‐block‐(2‐methyl‐2‐oxazoline)] Copolymers for Gene Transfer in Skeletal Muscle

Amphiphilic triblock copolymers such as poly(ethylene glycol-b-propylene glycol-b-ethylene glycol) PE6400 (PEG(13)-PPG(30)-PEG(13)) have been recently shown to promote gene transfer in muscle. Herein we investigated the effect of a chemical change of the PEG moiety on the transfection activity of these compounds. We synthesized new amphiphilic copolymers in which the PEG end blocks are replaced by more hydrophilic poly(2-methyl-2-oxazoline) (PMeOxz) chains of various lengths. The resulting triblock PMeOxz-PPG-PMeOxz compounds were characterized by NMR, SEC, TGA, and DSC techniques and assayed for in vivo muscle gene transfer. The results confirm both the block structure and the monomer unit composition (DP(PG)/DP(MeOxz)) of the new PPG(34)-PMeOxz(41) and PPG(34)-PMeOxz(21) triblock copolymers. Furthermore, in vivo experiments show that these copolymers are able to significantly increase DNA transfection efficiency, despite the fact that their chemical nature and hydrophilic character are different from the poloxamers. Overall, these results show that the capacity to enhance DNA transfection in skeletal muscle is not restricted to PEG-PPG-PEG arrangements.

Skeletal muscle gene transfer and regulated expression of IGF‐I increases function of the failing heart

Current methods of cardiac gene transfer include intramuscular injection (im) into heart muscle or coronary delivery. When applied to rodents with heart failure, these surgical approaches are associated with high mortality, an impediment that would also complicate clinical studies. To circumvent this, we selected a transgene, Insulin-like Growth Factor-I (IGF-I), which may have favorable effects on heart function when secreted from a remote site. This study examines the feasibility of skeletal muscle injection of adeno-associated virus-5 encoding IGF-I under tet regulation (AAV5.IGFI-tet). Left coronary ligation was performed in rats and MI confirmed 1wk later by echocardiography. Rats with MI then received 2×1012 vp of AAV5.IGFI-tet in the anterior tibialis (im). Four wks later they were randomly assigned to receive doxycycline in the drinking water (IGF-On; n=5) or not (IGF-Off; n=3). Ten wks post MI anesthetized rats underwent a final echo and terminal study in which a 1.8F pressure-volume transducer was placed in the left ventricle (LV). IGF-On rats showed an increased in LV fractional area change (p=0.04), ejection fraction (p=0.04), and velocity of circumferential fiber shortening (p=0.04), and had smaller LV end-diastolic (p=0.002) and end-systolic (p=0.003) dimensions. LV function was improved with dobutamine infusion (Table; IGF-On, n=4; IGF-Off, n=3). Serum IGF-I was increased 5 wk after activation (IGF-On: 218±7 ng/ml; IGF-Off: 158±24 ng/ml; p=0.046). These data indicate that intramuscular injection of AAV5.IGFI-tet increases function of the failing heart.

974. Constitutive Expression of a Mutated Form of CTLA4Ig after Gene Transfer Is Well Tolerated and Prevents Immune Responses Against an Immunogenic Transgene in Nonhuman Primates

rAAV-mediated gene transfer in skeletal muscle of large animal models can sustain long-term expression of transgene, but when applied to genetic diseases, the newly expressed therapeutic protein often induces a deleterious immune response. We investigated whether such immune response could be prevented by treatment with a mutant form of CTLA4Ig with higher affinity for B7 molecules (LEA29Y), known to induce potent immunosuppression and currently used, in its recombinant form (Belatacept®), in phase II/ III clinical development.

Viral serotype and the transgene sequence influence overlapping adeno‐associated viral (AAV) vector‐mediated gene transfer in skeletal muscle

The overlapping approach was developed recently to expand the adeno-associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co-infected cells after homologous recombination. Despite the promising proof-of-principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate-limiting factors including AAV serotype and the transgene sequence. To study serotype effect, we delivered AAV-2, -5 and -6 overlapping vectors (5 x 10(8) vg particles of the upstream and the downstream vectors, respectively) and 5 x 10(8) vg particles of the intact gene vector to the tibialis anterior muscles of 7-week-old C57Bl/6 mice, respectively. To determine the effect of transgene sequence, we compared LacZ and alkaline phosphatase (AP) overlapping vectors. Transduction efficiency was quantified 6 weeks later by scoring the percentage of transgene-positive myofibers. AAV-2 overlapping vectors barely resulted in detectable transduction. Transduction efficiency was significantly improved in AAV-5 and AAV-6. The highest level was achieved in AAV-6 that reached 42% and 96% of that of the intact gene vector for the LacZ gene and the AP gene, respectively. Surprisingly, AAV-6 overlapping vector resulted in higher transduction than did AAV-2 and AAV-5 intact gene vectors. Our findings suggest that AAV serotype and the transgene sequence play critical roles in the overlapping approach. AAV-6 holds great promise for overlapping vector-mediated muscle gene therapy.

935. Magnetofection and Ultrasound To Increase “Naked” DNA Delivery to the Myocardium

We, and other have previously shown that using optimised formulations adenovirus (Ad) is several logs more efficient in myocardial gene transfer than “naked” plasmid DNA (pDNA). Here, we evaluated “magnetofection”, the use of magnetic force acting on vectors complexed to magnetic particles and ultrasound to increase non-viral gene transfer to the murine myocardium. C57BL6/J mice were intubated and a thoracotomy performed to expose the heart. The ability of the magnetic force to pass through tissue was tested ex vivo on a dissected lamb's heart. Iron filings sprinkled on the endocardial site moved and clustered together when the magnet was placed on the epicardial side of the left myocardial wall (n=3). A variety of magnetic particles have been described, here we used TransMAGPEI (Chemicell, Germany), a polyethyleneimine (PEI)–coated iron oxide particle. For the myocardial injection the total volume could not exceed 10 μl. The magnetic particle:DNA complexes were carefully optimised by altering the TransMAGPEI to pDNA w/w and v/v ratios to avoid precipitation. The complexes used for in vivo injections were made in the following way: 0.8 μl TransMAGPEI (60 μg/μl) was diluted with 41.7 μl water and mixed with 5 μl pDNA (5 μg/μl) carrying a luciferase reporter gene (pLux). 2.5 μl 0.9% NaCl (18%) was added after 15 minutes. 10 μl of this formulation containing 5 μg pLux were injected into the left ventricular wall. The hearts were exposed to the magnet (AlNiCo Rod magnet plus NeoDelta magnet, IBS) for 5 min straight after injection and for a further 10 min after re-sutering of the chest. The left ventricular wall was harvested 48 hrs after gene transfer and lux expression was measured. Although magnet exposed hearts expressed more lux than the non-magnet exposed heart, this difference was not significant (magnet: 268±124 RLU/mg protein, no magnet: 94.0±41.8 RLU/mg protein, n=8-10/group). It has previously been shown that ultrasound (US) enhances non-viral gene transfer in skeletal muscle probably through acoustic cavitation of the cell membrane. Here, US was applied for 5 minutes before and after myocardial injection of 10 μg pLux in 10 μl total volume containg 1:1 v/v Opitson microbubbles. For this purpose the chest cavity was filled with gel (Henleys Medical, Welwyn Garden City, UK) and a 0.5 cm2 probe massaged over the heart area. The Medi-Link system machine (Electro-Medical Supplies Ltd, Wantage, UK) was set to deliver 1MHz, 2 W/cm2 at 100% duty cycle continous wave. 48 hrs after gene transfer the left vetricular wall was harvested and lux expression measured. In a parallel experiment we demonstrated that ultrasound imaging (14 MHz) using a gel filled chest cavity is feasible and therefore concluded that ultrasound waves were reaching the heart. However, the US conditions described above did not increase lux expression in the mouse heart (US: 71.8±38.4 RLU/mg protein, no US:138±69.2 RLU/mg protein, n=10/group). In conclusion, although physical energy may be useful in improving non-viral gene transfer to the myocardium further optimisation of these techniques will be required.

Intramuscular SP1017‐formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues

The systemic administration of a therapeutic protein is a common approach for the treatment of multiple disorders. Intramuscular (i.m.) injection of plasmids, followed by electroporation, has been shown to facilitate naked DNA gene transfer in skeletal muscle allowing proteins to be produced and secreted at therapeutically relevant levels. Plasmid DNA, unformulated or formulated with the non-ionic carrier SP1017, was injected at the rat tibialis anterior muscle followed by the application of electric pulses. Follow-up of protein expression was measured. In our study we report that the non-ionic carrier SP1017 significantly increases transgene expression in rat muscle after the i.m. injection of a formulated-pCMVbeta plasmid followed by electroporation. Such increased expression was observed after delivering square-wave unipolar electric pulses at two different field strengths: low (110 V/cm) and high (175 V/cm). Moreover, elevated secreted placental alkaline phosphatase (SEAP) plasma levels were achieved with low-voltage (110 V/cm) electroporation. Our results also show that human hepatocyte growth factor (hHGF) can be produced from rat muscle and delivered to blood circulation at a biologically active level after a single i.m. injection of an SP1017-formulated plasmid (pCMV/hHGF) followed by electroporation. Tissue distribution studies mostly identified hHGF in the liver, but it was also found in the kidneys and lungs suggesting that here too the HGF could be of therapeutic benefit. Our results indicate that SP1017 enhances the expression of electrotransferred genes. Such a delivery approach could prove an efficient method for the systemic production of therapeutic proteins.