Sustained muscle expression of dystrophin from a high-capacity adenoviral vector with systemic gene transfer of T cell costimulatory blockade.

Adenoviral vector (Ad)-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD). However, cellular and humoral immune responses induced by vector gene transfer limit the application of this approach. Blockade of the costimulatory interaction between na?ve T cells and antigen-presenting cells has proven to be a successful means to diminish immunity induced by gene transfer. In this study we explore the potential of supplementing dystrophin gene delivery to dystrophin-deficient Dmd mouse skeletal muscle with systemic gene delivery of CTLA4Ig and CD40Ig molecules to effect costimulatory blockade. We found that systemic administration of a high-capacity Ad (HC-Ad) vector carrying murine CTLA4Ig (AdmCTLA4Ig) either alone or codelivered with an HC-Ad vector carrying murine CD40Ig (AdmCD40Ig) provided sustained expression of recombinant full-length murine dystrophin from an HC-Ad vector carrying the dystrophin cDNA (AdmDys). The level of AdmDys vector genomes remained stable in animals cotreated with systemic delivery of vectors carrying molecules to block costimulation. In addition, muscle CD4(+) and CD8(+) T cell infiltrates and Th1 cytokine production by splenocytes were reduced. The production of neutralizing antibody against Ad vector was significantly inhibited in mice receiving systemic codelivery of both AdmCTLA4Ig and AdmCD40Ig, but not in the mice treated with AdmCTLA4Ig alone. The results suggested that coblockade of both CD28/B7 and CD40L/CD40 costimulatory pathways is required for effective inhibition of the Ad vector-induced humoral immune response in Dmd mice, whereas blockade of CD28/B7 alone by murine CTLA4Ig would be sufficient for prolonged dystrophin expression in treated muscle.     

Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells

In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude mice revealed no toxicity in liver, lung, kidney, or spleen. This study shows that infection with the second-generation adenovirus, ARR2PB.Bax.GFP, results in highly specific cytotoxicity in LNCaP cells, and that consequent overexpression of Bax in prostate carcinoma, even in the context of high levels of Bcl-2 protein, resulted in apoptosis. These results suggest that a second-generation adenovirus-mediated, prostate-specific Bax gene therapy is a promising approach for the treatment of prostate cancer.     

Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector

Dystrophin gene transfer using helper-dependent adenoviral vectors (HDAd) deleted of all viral genes is a promising option to treat muscles in Duchenne muscular dystrophy (DMD). Previously, we reported high-level dystrophin expression and functional correction of dystrophin-deficient (mdx) mouse muscle 60 days after gene transfer with an HDAd encoding two full-length murine dystrophin cDNAs (referred to as HDCBDysM). In the present study, we tested the long-term efficacy of HDCBDysM by examining muscle contractility parameters and the stability of dystrophin expression 1 year after injection into neonatal mdx muscles. At this point, HDCBDysM-treated muscles averaged 52% dystrophin-positive fibers. Treated muscles also displayed significantly greater isometric force production as well as greater resistance to the force deficits and damage caused by eccentric contractions. The level of protection against eccentric contraction-induced force deficits correlated with the percentage of dystrophin-positive fibers. Furthermore, HDCBDysM treatment restored the dystrophin-glycoprotein complex (DGC) to the sarcolemma and improved other aspects of mdx muscle histopathology examined (central nucleation, muscle hypertrophy, and mononuclear [phagocytic] cell infiltration). These improvements occurred despite the induction of a humoral response against murine dystrophin. Our results indicate that major therapeutic benefits of HDCBDysM are maintained for a long period of the animals' lifespan and suggest that HDCBDys holds promise for treating DMD by gene therapy.     

CTLA4Ig adenoviral gene transfer induces long-term islet rat allograft survival,without tolerance,after systemic but not local intragraft expression

Genetic engineering using recombinant adenoviruses offers an opportunity to modify islet grafts in order to prevent allograft rejection. We have used an adenovirus coding for CTLA4Ig to compare its efficacy in preventing islet rejection depending on local or systemic production after gene transfer either into the islets or intramuscularly, respectively. Islet allograft survival was also evaluated using recombinant CTLA4Ig administered intraperitoneally or incubated ex vivo with islets prior to transplantation. Transduction of islets with 10(3) or 10(4) plaque-forming units (pfu) per islets of AdCTLA4Ig prolonged islet survival (mean +/- standard deviation [SD] days = 19.5 +/- 5.8 and 19.5 +/- 5.6, respectively, vs. 10.6 +/- 2.4 in control islets, p < 0.001), with low levels of circulating CTLA4Ig. In contrast, long-term survival (>60 days) was obtained after intramuscular injection of AdCTLA4Ig that resulted in sustained high levels of circulating CTLA4Ig. Islets incubated in vitro with CTLA4Ig did not show prolonged survival (10.3 +/- 2.5 days). Graft rejection was delayed after one injection of CTLA4Ig (23 +/- 7.6 days, p < 0.003 vs. control). Recipients of long-term surviving grafts after intramuscular AdCTLA4Ig gene transfer were not tolerant because second islet grafts of donor origin were rejected. These recipients also had a strong inhibition of humoral responses against nominal antigens, whereas animals receiving transduced islets showed normal responses. These data demonstrate that local production of CTLA4Ig after gene transfer was as efficient as a single injection of CTLA4Ig in preventing graft rejection. Furthermore, intramuscular gene transfer of CTLA4Ig was the most efficient way to induce long-term islet graft survival but no donor-specific tolerance was induced.     

A non-immunogenic adenoviral vector,coexpressing CTLA4Ig and bilirubin-uridine-diphosphoglucuronateglucuronosyltransferase permits long-term,repeatable transgene expression in the Gunn rat model of Crigler-Najjar syndrome

Host immune responses limit the duration of expression of transgenes introduced by recombinant adenoviruses, preclude gene transfer upon vector readministration and cause liver injury. CTLA4Ig inhibits immune response by blocking the co-stimulatory interaction between CD28 on T cells and B7 on antigen-presenting cells. We have constructed a recombinant adenovirus, Ad-hUGT1A1-CTLA4Ig that coexpresses human bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (hUGT1A1) and soluble murine CTLA4Ig, both driven by CMV immediate-early promoters. After intravenous injection of this vector (6 x 10(11) p.f.u.) into UGT1A1-deficient jaundiced Gunn rats, serum CTLA4Ig levels peaked at 1.8-2.0 mg/ml on day 7 and declined thereafter to 0.2 mg/ml by day 180. Serum bilirubin declined from mean preinjection levels of 8.0 mg/dl to 0.48-0.6 mg/dl in 3 days, remained normal for 28 weeks, and then gradually increased to 8 mg/dl by day 350. A second injection of Ad-hUGT1A1-CTLA4Ig normalized serum bilirubin. In two rats in this group that were followed longer, serum bilirubin increased to 3.1 and 3.5 mg/dl in 40 weeks, but was normalized again after a third injection. The antibody and cytotoxic lymphocyte (CTL) responses were negligible, and liver biopsy showed no inflammatory cell infiltration. Rats receiving a tertiary challenge with Ad-LacZ (expressing E. coli beta-galactosidase) (5 x 10(11) p.f.u.), 2 months after the second dose of Ad-hUGT1A1-CTLA4Ig, showed beta-galactosidase expression in over 80% of hepatocytes. In contrast, after Ad-hUGT1A1 (which expresses UGT1A1 alone) injection, serum bilirubin remained normal for only 4 weeks, and returned to preinjection levels by day 120. Bilirubin levels did not decline upon reinjection, and beta-galactosidase was not expressed after Ad-LacZ. High levels of adenovirus-specific antibodies and CTL, and hepatic inflammation were found. This is the first demonstration that coexpression of CTLA4Ig permits prolonged expression and repeatable gene transfer by an adenoviral vector.     

Comparison of high-capacity and first-generation adenoviral vector gene delivery to murine muscle in utero

In utero gene delivery could offer the advantage of treatment at an early stage for genetic disorders such as Duchenne muscular dystrophy (DMD) in which the inevitable process of muscle degeneration is already initiated at birth. Furthermore, treatment of fetal muscle with adenoviral (Ad) vectors is attractive because of a high density of Ad receptors, easy vector accessibility due to immaturity of the basal lamina and the possibility of treating stem cells. Previously, we demonstrated the efficient transduction of fetal muscle by high-capacity Ad (HC-Ad) vectors. In this study, we compared HC-Ad and first-generation Ad (FG-Ad) vectors for longevity of lacZ transgene expression, toxicity and induction of immunity after direct vector-mediated in utero gene delivery to fetal C57BL/6 mice muscle 16 days after conception (E-16). The total amount of beta-galactosidase (betagal) expressed from the HC-Ad vector remained stable for the 5 months of the study, although the concentration of betagal decreased due to muscle growth. Higher survival rates that reflect lower levels of toxicity were observed in those mice transduced with an HC-Ad vector as compared to an FG-Ad vector. The toxicity induced by FG-Ad vector gene delivery was dependent on mouse strain and vector dose. Animals treated with either HC-Ad and FG-Ad vectors developed non-neutralizing antibodies against Ad capsid and antibodies against betagal, but these antibodies did not cause loss of vector genomes from transduced muscle. In a mouse model of DMD, dystrophin gene transfer to muscle in utero using an HC-Ad vector restored the dystrophin-associated glycoproteins. Our results demonstrate that long-term transgene expression can be achieved by HC-Ad vector-mediated gene delivery to fetal muscle, although strategies of vector integration may need to be considered to accommodate muscle growth.     

Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse

Here, we report the isolation of a human multipotent adipose-derived stem (hMADS) cell population from adipose tissue of young donors. hMADS cells display normal karyotype; have active telomerase; proliferate >200 population doublings; and differentiate into adipocytes, osteoblasts, and myoblasts. Flow cytometry analysis indicates that hMADS cells are CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1(-), CD34-, CD15-, CD117-, Flk-1(-), gly-A(-), CD133-, HLA-DR(-), and HLA-I(low). Transplantation of hMADS cells into the mdx mouse, an animal model of Duchenne muscular dystrophy, results in substantial expression of human dystrophin in the injected tibialis anterior and the adjacent gastrocnemius muscle. Long-term engraftment of hMADS cells takes place in nonimmunocompromised animals. Based on the small amounts of an easily available tissue source, their strong capacity for expansion ex vivo, their multipotent differentiation, and their immune-privileged behavior, our results suggest that hMADS cells will be an important tool for muscle cell-mediated therapy.     

Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres.

Patterns of dystrophin and beta-galactosidase expression were examined in mdx mice after i.m. injections of synthetic microspheres (MF-2) loaded with full-length (pHSADy) or mini-dystrophin gene (pSG5dys) cDNA plasmid constructs or with LacZ marker gene (pCMV-LacZ). A single injection of 25 microg pHSADy into quadriceps femoris muscle resulted in 6.8% of dystrophin positive myofibers (DPM) in a given muscle; 8.4% of DPM in glutaeus muscle and 4.3% of DPM in quadriceps femoris muscle of contralateral limb on day 21 after exposure compared with only 0.6% DPM in intact (non-injected) mdx mice. A high proportion of DPM (17.6% and 10.8%, respectively) was registered in both injected and contralateral muscles after mini- gene cDNA administration. MF-2/dystrophin cDNA particles were detected by FISH analysis in about 60-70% of myofiber nuclei in muscles of injected and contralateral limbs 7 days after application. The presence of human dystrophin cDNA and its products in all skeletal muscles and in different internal organs was proven by PCR and RT-PCR analysis. Patches of beta-galactosidase expression were abundant in injected muscle, and frequent in the contralateral and other skeletal muscles as well as in diaphragm, heart and lungs. High levels of dystrophin cDNA expression, and an efficient distant transfection effect with preferential intranuclei inclusion of MF-2 vehicle, are very encouraging for the development of a new constructive strategy in gene therapy trials of DMD.     

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.     

Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.     

Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response.     

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.     

Early manifestation of alteration in cardiac function in dystrophin deficient mdx mouse using 3D CMR tagging

Background

Duchenne muscular dystrophy (DMD) is caused by the absence of the cytoskeletal protein, dystrophin. In DMD patients, dilated cardiomyopathy leading to heart failure may occur during adolescence. However, early cardiac dysfunction is frequently undetected due to physical inactivity and generalized debilitation. The objective of this study is to determine the time course of cardiac functional alterations in mdx mouse, a mouse model of DMD, by evaluating regional ventricular function with CMR tagging.

Methods

In vivo myocardial function was evaluated by 3D CMR tagging in mdx mice at early (2 months), middle (7 months) and late (10 months) stages of disease development. Global cardiac function, regional myocardial wall strains, and ventricular torsion were quantified. Myocardial lesions were assessed with Masson''s trichrome staining.

Results

Global contractile indexes were similar between mdx and C57BL/6 mice in each age group. Histology analysis showed that young mdx mice were free of myocardial lesions. Interstitial fibrosis was present in 7 month mdx mice, with further development into patches or transmural lesions at 10 months of age. As a result, 10 month mdx mice showed significantly reduced regional strain and torsion. However, young mdx mice showed an unexpected increase in regional strain and torsion, while 7 month mdx mice displayed similar regional ventricular function as the controls.

Conclusion

Despite normal global ventricular function, CMR tagging detected a biphasic change in myocardial wall strain and torsion, with an initial increase at young age followed by progressive decrease at older ages. These results suggest that CMR tagging can provide more sensitive measures of functional alterations than global functional indexes in dystrophin-related cardiomyopathies.     

Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion

Allograft rejection is a T cell-dependent process. Productive T cell activation by antigen requires antigen engagement of the T cell receptor as well as costimulatory signals delivered through other T cell surface molecules such as CD28. Engagement of CD28 by its natural ligand B7 can be blocked using a soluble recombinant fusion protein, CTLA4Ig. Administration of CTLA4Ig blocks antigen-specific immune responses in vitro and in vivo, and we have shown that treatment of rats with a 7-d course of CTLA4Ig at the time of transplantation leads to prolonged survival of cardiac allografts (median 30 d), although most grafts are eventually rejected. Here, we have explored additional strategies employing CTLA4Ig in order to achieve long-term allograft survival. Our data indicate that donor-specific transfusion (DST) plus CTLA4Ig can provide effective antigen-specific immunosuppression. When DST is administered at the time of transplantation followed by a single dose of CTLA4Ig 2 d later, all animals had long-term graft survival (> 60 d). These animals had delayed responses to donor-type skin transplants, compared with normal rejection responses to third-party skin transplants. Furthermore, donor-matched second cardiac allografts were well tolerated with minimal histologic evidence of rejection. These data indicate that peritransplant use of DST followed by subsequent treatment with CTLA4Ig can induce prolonged, often indefinite, cardiac allograft acceptance. These results may be clinically applicable for cadaveric organ and tissue transplantation in humans.     

Quantification of high-capacity helper-dependent adenoviral vector genomes in vitro and in vivo, using quantitative TaqMan real-time polymerase chain reaction

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host's immune system. Another advantage is their large cloning capacity (up to approximately 35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials.     

Transduction of myogenic cells by retargeted dual high-capacity hybrid viral vectors: robust dystrophin synthesis in duchenne muscular dystrophy muscle cells.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), making it amenable to gene- or cell-based therapies. Another possible treatment entails the combination of both principles by transplantation of autologous myogenic cells after their genetic complementation. This approach requires efficient and stable transduction of these cells with recombinant DMD. Recently, we generated a dual high-capacity (hc) adenovirus (Ad)-adeno-associated virus (AAV) hybrid vector (HV) that can deliver two full-length dystrophin-encoding modules into target cells. We showed that HV transduction of human cells containing AAV Rep proteins leads to the insertion of foreign DNA into the AAVS1 locus. Here, we improved HV entry into muscle cells from DMD patients. After having verified that these cells barely express the coxsackie B virus and Ad receptor (CAR), which constitutes the attachment molecule for Ad serotype 5 (Ad5) fibers, we equipped dual hcAd/AAV HV particles with Ad serotype 50 fiber domains to achieve CAR-independent uptake. These retargeted vectors complemented much more efficiently the genetic defect of dystrophin-defective myoblasts and myotubes than their isogenic counterparts with conventional Ad5 fibers. Importantly, the accumulation of beta-dystroglycan along the membranes of vector-treated DMD myotubes indicated proper assembly of dystrophin-associated glycoprotein complexes.     

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.     

Helper-dependent adenoviral vector achieves prolonged, stable expression of interleukin-10 in rabbit carotid arteries but does not limit early atherogenesis

Vascular gene therapy could potentially complement or replace current therapies for human atherosclerosis, while avoiding their side effects. However, development of vascular gene therapy is limited by lack of a useful vector. Helper-dependent adenovirus (HDAd) shows promise to overcome this barrier because, unlike first-generation adenovirus, HDAd achieves durable transgene expression in the artery wall with minimal inflammation. To begin to test whether HDAd, delivered to the artery wall, can limit atherosclerosis we constructed HDAd that expresses rabbit interleukin (IL)-10, a potent atheroprotective cytokine, and tested its activity in a rabbit model of early carotid atherogenesis. HDAd expressed immunoreactive, active IL-10 in vitro. In contrast to other HDAd-expressed transgenes, IL-10 expression from HDAd increased significantly between 3 days and 2 weeks after infusion and remained stable for at least 8 weeks. Rising, persistent IL-10 expression was associated with relative persistence of HDAdIL-10 genomes 4 weeks after infusion, compared with HDAdNull genomes. Surprisingly, IL-10 expression had no significant effects on atherosclerotic lesion size, macrophage content, or expression of either adhesion molecules or atherogenic cytokines. These results might be due to inadequate protein expression in vivo or lack of suitability of this rabbit model to reveal IL-10 therapeutic effects. IL-10 remains a promising agent for vascular gene therapy and HDAd remains a promising vector; however, proof of efficacy of HDAdIL-10 is elusive. Future preclinical studies will be aimed at increasing IL-10 expression levels and improving the sensitivity of this animal model to detect atheroprotective effects.