Muscle Transfection Research Articles

Induction of Bystander and Abscopal Effects after Electroporation-Based Treatments.

Simple SummaryThe delivery of electric field to tumor cells or nodules induces cell electroporation that allows intracellular delivery of cytotoxic agents and eventually inhibits tumor growth. In this study, we showed that intracellular delivery of calcium ions and anticancer drug bleomycin not only kills the cells but also has a negative bystander effect on indirectly treated cells. We also showed that, when directly applied to one tumor, these treatments can inhibit the growth of a second, non-electroporated tumor.Electroporation-based antitumor therapies, including bleomycin electrotransfer, calcium electroporation, and irreversible electroporation, are very effective on directly treated tumors, but have no or low effect on distal nodules. In this study, we aimed to investigate the abscopal effect following calcium electroporation and bleomycin electrotransfer and to find out the effect of the increase of IL-2 serum concentration by muscle transfection. The bystander effect was analyzed in in vitro studies on 4T1tumor cells, while abscopal effect was investigated in an in vivo setting using Balb/c mice bearing 4T1 tumors. ELISA was used to monitor IL-2 serum concentration. We showed that, similarly to cell treatment with bleomycin electrotransfer, the bystander effect occurs also following calcium electroporation and that these effects can be combined. Combination of these treatments also resulted in the enhancement of the abscopal effect in vivo. Since these treatments resulted in an increase of IL-2 serum concentration only in mice bearing one but not two tumors, we increased IL-2 serum concentration by muscle transfection. Although this did not enhance the abscopal effect of combined tumor treatment using calcium electroporation and bleomycin electrotransfer, boosting of IL-2 serum concentration had a significant inhibitory effect on directly treated tumors.

Temperature-Sensitive Amphiphilic Non-Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection.

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.

Integration of needle-free jet injection with advanced electroporation delivery enhances the magnitude, kinetics, and persistence of engineered DNA vaccine induced immune responses

The combination of optimized DNA constructs, improved formulations and advanced in vivo electroporation (EP) has been shown to generate potent and efficacious immune responses in the clinic. Needle-free jet injection has also been reported to improve DNA vaccine delivery over standard needle and syringe in clinical trials. Here we investigated the impact of combined jet injection and EP (Jet-EP) delivery on muscle transfection efficiency and DNA vaccine immunogenicity in rabbits and nonhuman primates (NHPs) compared to jet injection alone. Our results show that the addition of EP significantly enhanced in vivo DNA transfection efficiency of rabbit muscle over jet injection alone. Jet-EP delivery augmented the rate and magnitude of DNA vaccine induced humoral and cellular responses over jet injection alone in both rabbits and NHPs. Jet-EP delivery also resulted in higher proportions of polyfunctional antigen specific T cells producing IFNγ, IL-2, and/or TNFα. Elevated antibody levels were sustained nine months post immunization in NHPs immunized with a DNA vaccine using Jet-EP delivery, far outperforming jet delivery alone. Our results provide proof-of-concept that addition of advanced EP to needle-free jet injection delivery improves in vivo DNA transfection efficiency, increasing the magnitude, rate and duration of cellular and humoral immune responses to DNA vaccines. This combination likely has significant advantages in important vaccine and immunotherapy settings.

Data on macrophage mediated muscle transfection upon delivery of naked plasmid DNA with block copolymers.

The data contains 14 figures supporting the research article “Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers” [1]. The data explains the surgical procedure and histological characterization of Murine Hind Limb Ischemia. The data also shows the kinetics of luciferase gene expression, spread of GFP expression through muscle and the colocalization of GFP with cellular markers in ischemic muscles injected with pDNA alone or pDNA/Pluronic. Finally the data shows the effect of Pluronic Block Copolymer to enhance total gene expression (cmv-promoter driven luciferase gene) in coculture of DNA transfected MØs with muscle cells.

Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers

Intramuscular administration of plasmid DNA (pDNA) with non-ionic Pluronic block copolymers increases gene expression in injected muscles and lymphoid organs. We studied the role of immune cells in muscle transfection upon inflammation. Local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and expressed protein levels in ischemic muscles injected with pDNA/Pluronic. The systemic inflammation (MHLIM or peritonitis) also increased expression of pDNA/Pluronic in the muscles. When pDNA/Pluronic was injected in ischemic muscles the reporter gene, Green Fluorescent Protein (GFP) co-localized with desmin+ muscle fibers and CD11b+ macrophages (MØs), suggesting transfection of MØs along with the muscle cells. P85 enhanced (∼4 orders) transfection of MØs with pDNA in vitro. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells in MHLIM. Using a co-culture of myotubes (MTs) and transfected MØs expressing a reporter gene under constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) promoter we demonstrated that P85 enhances horizontal gene transfer from MØ to MTs. Therefore, MØs can play an important role in muscle transfection with pDNA/Pluronic during inflammation, with both inflammation and Pluronic contributing to the increased gene expression. pDNA/Pluronic has potential for therapeutic gene delivery in muscle pathologies that involve inflammation.

583. Nonionic Block Copolymers Guide Macrophages To Transfect Skeletal Muscle Cells Upon Direct i. m. Injections

Purpose: Skeletal muscle offers a clinically feasible site for i. m. delivery of genes for therapeutic proteins expression and prophylactic DNA vaccine purposes. Though much research has been focused on development of cationic lipids and polymers for encapsulating plasmid DNA (pDNA) for in vitro transfections, direct i. m. injection of pDNA still results in higher muscle transfection levels. In comparison to existing technologies such as viral vectors there still is a need to further improve muscle transfection levels with pDNA injections. Non-ionic block copolymers, Pluronic P85 and SP1017 (mixture of L61 and F127), when mixed with pDNA, enhance skeletal muscle transfection in euthymic (BALB/c and C57BL/6) mouse and not athymic (BALB/c nu/nu) mouse. Therefore, we studied role of immune cells in muscle transfection.Methods and Results: pDNA alone or an admix of pDNA+Pluronic were injected into tibialis anterior (TA) muscle in presence or absence of local inflammation to demonstrate the effect of immune cells in muscle transfection. Local-inflammation drastically enhanced DNA, RNA and gene expression levels when pDNA was co-delivered with P85 or SP1017 and not pDNA alone. Pluronics enhanced in vitro gene expression in MPs to upto 4 orders in magnitude and not muscle cells. Moreover, adoptive transfer of ex vivo transfected MPs with GFP DNA to ischemic mice resulted in GFP expression in muscle fibers along with MPs. Therefore, in vitro muscle inflammation model was developed by co-culturing DNA transfected macrophages (MPs) with myotubes (MTs) to see the effect of P85 on gene transfer to MTs. Treatment of co-culture (pDNA transfected MPs with MTs) with P85 resulted in increase in total and muscle specific gene expression ca. 30 and 120 times respectively, confirming pDNA transfer from MPs to MTs. Moreover, in vivo depletion of MPs resulted in significant decrease in gene expression confirming the role of MPs in Pluronic based muscle transfection. Finally, pre-injection with P85 on day 1.5 and not day 5 at subsequent sites of DNA injections, recruited MPs to the site of injections and further enhanced the gene expression levels of muscles injected with pDNA+P85 and not pDNA alone. View Large Image | Download PowerPoint SlideConclusions: Pluronic block copolymers utilize immune cells, specifically MPs, to deliver genes to otherwise very hard to transfect skeletal muscle fibers and hence constitute a novel class of biopharmaceuticals or a platform technology for in vivo gene delivery.

Imaging cytokine targeting to the tumor/bone microenvironment in vivo.

A major hurdle of systemic cytokine therapies is achieving therapeutic levels within the affected tissue, especially in the case of bone metastatic cancer. For instance, bone is the most common site for castrate-resistant prostate cancer metastasis, and the majority of patients with advanced disease will develop painful bone metastases and fractures, with a reduced quality of life and poor prognosis. Bone metastatic tumors are very difficult to treat since there may be multiple metastatic sites, and these may not be accessible to surgical or radiation interventions. Cytokine immunotherapies hold great promise since these are secreted molecules that can reach and treat distant bone metastatic tumors. Importantly, there remains a lack of therapeutics that can simultaneously and effectively treat the prostate tumor while restoring affected bone tissue. Our lab has developed several cytokine therapeutics specifically for treating the tumor/bone microenvironment. In order to deliver cytokines efficiently to tumor/bone metastases, we have devised a strategy based on C-terminal modification of secreted molecules for enabling their targeting and accumulation at metastatic sites. However, there are no current systems for measuring the delivery, binding, and accumulation of cytokines at the complex tumor/bone microenvironment. The present study thus examined the potential development of a reporter molecule or “cytokine model,” using a secreted luciferase (Gaussia Luc or GLuc), which we hypothesized would mimic therapeutic cytokine secretion, targeting, and accumulation in tumors. We used the gene delivery system called “sonodelivery” to deliver plasmid DNA (pDNA) to skeletal muscle of male mice bearing intratibial prostate tumors. Sonodelivery utilizes a novel oligolysine polymer (Parelkar et al., 2014) complexed to pDNA to create a nanoplex that is delivered along with microbubbles and sonicated to achieve ultrasound-enhanced muscle transfection (Fig. 1A) (Zolochevska et al., 2011, 2013). FIG. 1. Gaussia luciferase modified at the C-terminus with tumor or bone targeting peptides localize to intratibial prostate tumors. (A) Sonodelivery schematic and muscle secretion of tumor-targeted Gluc cytokine model. (B) TC2R-Luc-marked prostate tumor cells ... Using a tumor/bone metastasis model (Zolochevska et al., 2013), we labeled TRAMPC2Ras (TC2R) prostate tumor cells with firefly luciferase (FLuc) to show localization of tumor cells with luciferin substrate (Fig. 1B). Next, we sonodelivered a plasmid expressing GLuc to the hind thigh muscle. Seven days following sonodelivery, we detected accumulation of peptide-targeted but not wild-type GLuc at the tumor/bone metastatic sites when using coelenterazine substrate (Fig. 1C). The peptide-targeted Gluc were modified at the C-terminus by addition of peptide sequences that could specifically bind receptors upregulated in tumor (or remodeling bone) but not in other tissues. The peptides we have chosen bind IL6-Rα [pepTu; LSLITRL (Su et al., 2005)] or BMPR1b [pepB; AISMLYLDENEKVVL (Lin et al., 2005)] and thus are relevant for targeting therapeutic cytokines for tumor/bone retention. We used computational design to identify an optimal linker, 1x(Gly4Ser), between GLuc and the peptide to enhance the likelihood of native peptide folding, and showed improved Gluc binding up to 100-fold in tumor cells. These peptide-targeted GLuc retained functional activity and were able to localize and accumulate Gluc at intratibial tumors following sonodelivery at levels up to 123-fold higher than the control (Fig. 1D and E). Therefore, we present here the first data, to our knowledge, of utilizing GLuc as a cytokine model to assess targeting of C-terminal peptides to the prostate tumor/bone microenvironment in vivo.

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer

BackgroundWhen activated, NF-κB can promote the nuclear import and transcription of DNA possessing NF-κB consensus sequences. Here, we investigated whether NF-κB is involved in the plasmid electrotransfer process. MethodsMouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-κB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBα were studied as a marker of NF-κB activation. ResultsIntra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-κB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IκBα was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IκBα and thus of free NF-κB in the absence of any stimulation. General significancepDNA electrotransfer can increase transgene expression independently of NF-κB.The insertion of NF-κB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-κB in muscle might be sufficient to enhance the activity of pDNA bearing NF-κB consensus sequences.

Markedly enhanced skeletal muscle transfection achieved by the ultrasound-targeted delivery of non-viral gene nanocarriers with microbubbles

Our goal was to enhance ultrasound (US)-targeted skeletal muscle transfection through the use of poly(ethyleneglycol) (PEG)/polyethylenimine (PEI) nanocomplex gene carriers and adjustments to US and microbubble (MB) parameters. C57BL/6 mice received an intravenous infusion of MBs and either “naked” luciferase plasmid or luciferase plasmid condensed in PEG/PEI nanocomplexes. Pulsed ultrasound (1MHz; 0.6MPa or 0.8MPa) was applied to the right hindlimb for 12min. Luciferase activity in both hindlimbs was assessed at 3, 5, 7, and 10days post-treatment by bioluminescent imaging. When targeted to hindlimb using unsorted MBs and 0.6MPa US, 7days after treatment, we observed a >60-fold increase in luciferase activity in PEG/PEI nanocomplex‐treated muscles over muscles treated with “naked” plasmid DNA. Luciferase activity was consistently greater after treatment with PEG/PEI nanocomplexes at 0.6MPa as compared to 0.8MPa. The combination of small diameter MBs and 0.6MPa US also resulted in significantly greater gene expression when compared to concentration matched intramuscular injections, a control condition in which considerably more PEG/PEI nanocomplexes were present in tissue. This result suggests that, in addition to facilitating PEG/PEI nanocomplex delivery from the bloodstream to tissue, US enhances transfection via one or more secondary mechanisms, including increased cellular uptake and/or trafficking to the nucleus of PEG/PEI nanocomplexes. We conclude that PEG/PEI nanocomplexes may be used to markedly enhance the amplitude of US-MB-targeted skeletal muscle transfection and that activating “small” MBs with a moderate level (0.6MPa) of acoustic pressure can further enhance these effects.

A polymerase chain reaction-based methodology to detect gene doping

The non-therapeutic use of genes to enhance athletic performance (gene doping) is a novel threat to the world of sports. Skeletal muscle is a prime target of gene therapy and we asked whether we can develop a test system to produce and detect gene doping. Towards this end, we introduced a plasmid (pCMV-FAK, 3.8 kb, 50 μg) for constitutive expression of the chicken homologue for the regulator of muscle growth, focal adhesion kinase (FAK), via gene electro transfer in the anti-gravitational muscle, m. soleus, or gastrocnemius medialis of rats. Activation of hypertrophy signalling was monitored by assessing the ribosomal kinase p70S6K and muscle fibre cross section. Detectability of the introduced plasmid was monitored with polymerase chain reaction in deoxyribonucleic acids (DNA) from transfected muscle and serum. Muscle transfection with pCMV-FAK elevated FAK expression 7- and 73-fold, respectively, and increased mean cross section by 52 and 16% in targeted muscle fibres of soleus and gastrocnemius muscle 7 days after gene electro transfer. Concomitantly p70S6K content was increased in transfected soleus muscle (+110%). Detection of the exogenous plasmid sequence was possible in DNA and cDNA of muscle until 7 days after transfection, but not in serum except close to the site of plasmid deposition, 1 h after injection and surgery. The findings suggest that the reliable detection of gene doping in the immoral athlete is not possible unless a change in the current practice of tissue sampling is applied involving the collection of muscle biopsy close to the site of gene injection.

The Reverse Block Copolymer Pluronic 25R2 Promotes DNA Transfection of Skeletal Muscle

Muscle is an important and attractive target for gene therapy. Recent findings have shown that neutral amphiphilic triblock copolymers with a PEO-PPO-PEO arrangement significantly increase muscle transfection as compared to naked DNA. We were interested in evaluating whether reverse Pluronics (PPO-PEO-PPO) also possess transfection properties. Therefore, we measured the in vitro and in vivo transfection activity of 25R2 and 25R4, two copolymers that differ by their hydrophilic/hydrophobic balance. The results show that 25R2 significantly increases the transfection level in muscle compared to naked DNA. Taken together, this work demonstrates that the reverse Pluronic 25R2 possesses interesting properties for in vivo transfection.

Cardiospecific expression of AAV vectors

Adenoviral vectors have been set for cardiomyocyte gene therapy with considerable transfection rates. However, due to adenoviral immunogenicity, adenoviral transfected cells are frequently subject to specific cellular immune reactions leading to loss of cells and transgene expression. Non‐pathogenic adeno associated vectors lacking immunogenicity have been utilized alternately. Tropism of the most common AAV serotype 2, favoring liver of muscle transfection, has been altered by use of serotypes 1, 6 and 9.We used a model of chronic (4 weeks) regional myocardial transfection of the pig to investigate transfection efficacy of AAV vectors, which were applied to the target region by retroinfusion. Cardiac restriction was ensured by utilization of an MLC‐2v promoter‐CMV‐enhancer construct, fused to a luciferase reporter gene, the activity of which was analyzed.Compared to the wildtype (WT) AAV 2 vector, which barely exceeded background luciferase activity, a vector lacking the heparin binding site of the envelope (AAV2 ΔHep) improved expression only slightly. Substantial increase to 40 000 RLU/mg tissue) was achieved by the addition of VEGF‐A, which enhances vascular permeability. A further 15‐fold increase of luciferase activity was achieved by applying an equal amount of AAV6 virus particles to a pig heart, with a further 5–10 fold increase achieved by utilization of AAV serotype 9.Functionally relevant gene transfer was ensured by applying AAV9 hVEGF/PDGF (0.2/0.4 × 1013 virus particles, respectively). 4 weeks after retroinfusion into pig hearts suffering from chronic total LAD‐occlusion (induced by a reduction stent), we found a significant increase of blood flow and regional myocardial function.We conclude that AAV9 is a more efficient AAV vector when compared to WT‐ or ΔHep‐AAV2 or AAV6. Functionally relevant gene expression, as performed by VEGF‐A/PDGF transfection, opens the door for utilization of this vector system for xenotransplant organ priming.

Muscle transfection and permeabilization induced by electrotransfer or pluronic® L64: Paired study by optical imaging and MRI

Background Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein. Methods Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed. Results Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7 days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection. General significance It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.

Thrombospondin-2 therapy ameliorates experimental glomerulonephritis via inhibition of cell proliferation, inflammation, and TGF-β activation

We recently identified thrombospondin-2 (TSP-2) as an endogenous regulator of matrix remodelling and inflammation in experimental kidney disease by studying TSP-2-deficient mice. In this study, we asked whether systemic TSP-2 overexpression via thigh muscle transfection is able to ameliorate the time course of the anti-Thy1 glomerulonephritis model. After induction of anti-Thy1 nephritis, rats were transfected either with an overexpression plasmid for TSP-2 or lacZ as a control. Biopsies, urine, and blood samples were taken on days 1, 3, and 6 after disease induction. Muscular overexpression of TSP-2 reduced glomerular transforming growth factor (TGF)-beta activation and glomerular extracellular matrix formation as determined by collagen IV and fibronectin. In addition, activation of mesangial cells to the myofibroblast-like phenotype was also significantly decreased in TSP-2-overexpressing animals. TSP-2 overexpression inhibited both glomerular endothelial and mesangial cell proliferation, resulting in a reduced glomerular cell number and glomerular tuft area. The inflammatory response, as monitored by T cells and antigen-presenting cells, was reduced significantly by TSP-2 overexpression, but influx of macrophages was unchanged. These data demonstrate TSP-2 as a potential therapeutic agent to inhibit the glomerular proliferative and inflammatory response as well as TGF-beta activation and extracellular matrix accumulation in experimental mesangial proliferative glomerulonephritis.

770. Pharmacogenetics of Adenoviruses

Pharmacogenetics is the study of genotypic and phenotypic variation between individuals and how it affects drug response. Recombinant viral vectors and naked DNA are employed to deliver therapeutic transgenes in the liver, muscle, tumours and haematopoietic stem cells. However, to rationalise this delivery approach, the factors of variation between patients need to be identified. To study transgene expression variation, following muscle or liver transfection, we transfected various mouse and rat inbred strains with the luciferase transgene under CMV promotor encoded in a recombinant adenovirus serotype 5. It is considered that the differences observed between inbred strains reflect the differences expected between individual patients. We showed that, after liver or muscle transfection, the level of transgene expression depends on the transcription capacity of the animal strain. Indeed, the transcription efficiency varies up to 2 logs between one mouse or rat strains to the other. In order to translate this finding in human we transfected various cell lines of one breast cancer type, namely the ductal adenocarcinoma of the breast. It is believed that the difference observed between these cell lines of similar histological origin reflects the difference that one might encounter between cancer patients suffering from ductal breast cancer. Similarly, to the situation in liver and muscle, the variation of transgene expression was due to a polymorphism of the transcription activity in the cell lines. Further, this polymorphism is not restricted to Ad5 transfection, as we observed it also, in vitro and in vivo, with Ad35 and plasmid vectors. We also studied the influence of the promoter on this variation of transgene expression. The luciferase gene under MPL or modified CMV promoters also showed a polymorphism of expression. Consequently, a polymorphism of transgene expression might be expected in patient population after transfection with Ad5 Ad35, plasmid coding for transgenes under CMV or MLP promoter.

Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles

The aim of this study was to assess the relative efficacy and mechanism of gene transfection by ultrasound (US) destruction of plasmid-bearing microbubbles. Luciferase reporter plasmid was charge-coupled to cationic lipid microbubbles. Rat hindlimb skeletal muscle was exposed to intermittent high-power US during dose-adjusted intra-arterial (IA) or IV administration of plasmid-bearing microbubbles via the carotid artery or jugular vein, respectively. At 4 days, luciferase activity in US-exposed skeletal muscle was 200-fold greater with IA than with IV administration of plasmid-bearing microbubbles, and was similar to transfection achieved by IM injection of plasmid (positive control). No transfection occurred with US and IA injection of plasmid alone. Intravital microscopy of the cremaster muscle in mice following administration of microbubbles and US exposure demonstrated perivascular deposition of fluorescent plasmid, the extent of which was twofold greater for IA compared to IV injection. Electron microscopy demonstrated a greater extent of myocellular microporations in US-exposed muscle after IA injection of microbubbles. We conclude that muscle transfection by US destruction of plasmid-bearing cationic microbubbles is amplified by IA, rather than IV, injection of microbubbles due to greater extravascular deposition of plasmid and to greater extent of myocellular microporation. (E-mail: jlindner@virginia.edu)

Recipient intramuscular administration of naked plasmid TGF-β1 attenuates lung graft reperfusion injury

Background Gene therapy may be an effective strategy for modulating lung graft ischemia–reperfusion injury. We investigated whether recipient intramuscular (IM) naked plasmid gene transfer of transforming growth factor β1–active (TGF-β1–active) ameliorates lung graft ischemia–reperfusion injury. Methods Preliminary studies in F344 rats demonstrated that gastrocnemius muscle transfection of TGF-β1–active produced muscle and plasma protein expression at 24 and 48 hours after transfection. Recipients ( n = 8) received IM injection of naked plasmid-encoding chloramphenicol acetyl transferase (CAT), TGF-β1–latent or TGF-β1–active, respectively, at 24 or at 48 hours before left lung transplantation. We did not treat the control group before transplantation (18-hour cold ischemia). Donor lungs were flushed with low-potassium dextran–1% glucose and stored for 18 hours at 4°C. All groups were killed at 24 hours after transplantation. Immediately before killing the animals, we clamped the contralateral right hilum and assessed graft function. We measured wet-to-dry ratio (W/D), myeloperoxidase, pro-inflammatory cytokines (interleukin 1 [IL-1], tumor necrosis factor α [TNF-α], interferon-γ [INF-γ], and IL-2) and performed immunohistochemistry. Results Arterial oxygenation was greatest in the recipient group transfected with TGF-β1–active at 24 hours before transplantation compared with CAT, TGF-β1–latent, and 18-hour cold ischemia groups ( p < 0.01). The W/D ratio and myeloperoxidase decreased in both 24- and 48-hour groups, with TGF-β1–active compared with CAT, and 18-hour cold ischemia groups (W/D, p < 0.02 and p < 0.004, respectively; myeloperoxidase, p < 0.05 and p < 0.01, respectively). All pro-inflammatory cytokines decreased in the 24-hour TGF-β1–active group compared with CAT, TGF-β1–latent, 18-hour and 1-hour cold ischemia, and non-treated lung groups (IL-1β, p < 0.03; TNF-α, p < 0.02; IFN-γ, p < 0.001; IL-2, p < 0.0001). In 24- and 48-hour groups with TGF-β1–active, immunohistochemistry showed marked staining of Type I and Type II alveolar cells and of macrophages from the apical to the caudal sections of the lung grafts. Conclusions Recipient IM administration of naked plasmid encoding TGF-β1–active before transplantation ameliorates lung isograft reperfusion injury after prolonged ischemia.

Ultrasound Increases Plasmid-Mediated Gene Transfer to Dystrophic Muscles without Collateral Damage

Studies have shown that ultrasound, used either alone or in combination with microbubble contrast agents, can increase cell membrane permeability to plasmid DNA. Because ultrasound is a non-painful and well-established tool in clinical medicine, its potential to enhance DNA uptake into the muscles of patients with muscular dystrophy is conceptually attractive. Therefore, we evaluated the ability of ultrasound pulses (1 MHz; 1.5 W/cm2) to increase exogenous (LacZ) gene expression in normal wild-type and dystrophic Dmd(mdx/mdx) mice after plasmid DNA injection into muscle. We also ascertained whether co-injection of lipid-encapsulated perfluoropropane microbubbles (Definity) or pretreatment with hyaluronidase could further increase the level of gene transfer to ultrasound-treated muscles. The use of ultrasound did not increase transfection efficiency in normal mice. In contrast, dystrophic mice demonstrated an increase in the number of transfected fibers (threefold) as well as the amount of LacZ protein (22-fold) after ultrasound exposure, provided that Definity was also co-injected with the DNA. Pretreatment of muscles with hyaluronidase before ultrasound exposure was not effective in augmenting the level of gene transfer. Under the optimal conditions for dystrophic muscle transfection (ultrasound + Definity), there was no associated increase in muscle damage. Hence ultrasound may provide a safe and effective method for enhancing gene transfer to dystrophic muscles, thereby increasing the prospects for therapeutic application of naked DNA in muscular dystrophy patients.

Interleukin-10 expression after intramuscular DNA electrotransfer: kinetic studies

Transfected muscle can be used as a secreting tissue for therapeutic proteins. Skeletal muscle transfection is increased by suitable electric pulse application (electrotransfer). We and others had shown that electrotransfer of interleukin-10 encoding plasmid is an effective strategy in animal models of chronic diseases such as myocarditis, atherosclerosis, or rheumatoid arthritis. In the present work, we have studied murine interleukin-10 production and secretion after i.m. electrotransfer. In immunocompetent mice, serum and muscle mIL-10 levels were enhanced by electrotransfer. Serum mIL-10 concentration reached rapidly a peak level 2 days after electrotransfer. It then decreased to background at day 14. Muscle mIL-10 mRNA and protein remained more stable, being detectable up to 84 days after electrotransfer. A boost reinjection led to similar high level of circulating mIL-10. The fast decrease of serum mIL-10 was not observed in SCID mice.

The activity of exogenous genetic constructs introduced into cells by the technique of ballistic transfection in mouse ontogenesis

We used the method of particle bombardment (ballistic transfection) to introduce β-galactosidase and human dystrophin genes into mouse embryos and skeletal muscles of adult mice. We examined the mechanisms of DNA transfer into skeletal muscle cells, the biological processes accompanying and following this transfer, the susceptibility of various types of muscle cells to transfection, and the duration of expression of and conditions affecting the introduced genes. We have also developed an effective, convenient, and practical methods of skeletal muscle transfection.