AdV-mediated Gene Transfer Research Articles

Current Targeting Strategies for Adenovirus Vectors in Cancer Gene Therapy

Adenovirus vectors (Adv) are the most frequently used vectors in gene therapy research, especially in cancer gene therapy. However, despite encouraging preclinical and early clinical results, the successful clinical utility of gene therapy has not yet been fully realized. Challenges to clinical trial success for targeted Adv include inefficient Adv-mediated gene transfer (because many tumor cells lack Adv receptors), poor transduction in tumor tissues after systemic administration, accumulation and undesirable transgene expression in the liver. This review summarizes current targeting strategies for Adv to overcome these obstacles. Strategies include transductional selectivity through genetic modification of viral coat proteins, transcriptional selectivity by means of tumor-specific promoters, and selective biodistribution from conjugation with targeting ligands or polymers such as polyethylene glycol (PEG). Furthermore, combining selective biodistribution and active targeting ligands such as proteins, antibodies and peptides is an intriguing and promising approach that will also be covered in this review. These studies have provided new insights into our understanding of the utility of Adv in cancer gene therapy.

Implantation of adult bone marrow-derived mesenchymal stem cells transfected with the neurotrophin-3 gene and pretreated with retinoic acid in completely transected spinal cord

Implantation of marrow-derived mesenchymal stem cells (MSCs) is the most promising therapeutic strategy for the treatment of spinal cord injury (SCI), especially because of their potential for clinical application, such as the avoidance of immunologic rejection, their strong secretory properties, and their plasticity for developing into neural cells. However, the recovery from SCI after MSC implantation is minimal due to their limited capacity for the reduction of cystic cavitation, for the axonal regeneration and their uncertain neural plasticity in the spinal cord. We previously pretreated MSCs with all -trans retinoic acid (RA) in vitro. Then we genetically modified them to overexpress neurotrophin-3 (NT-3) via a recombinant adenoviral vector (Adv). This combined treatment not only permitted more neuronal differentiation of MSCs, but stimulated more NT-3 secretion prior to grafting, according to our previous and present results. When these cells were implanted into the transected spinal cord of rats, the animals had some improvement (both functionally and structurally), including the recovery of hindlimb locomotor function, shown by the highest Basso, Beattie, and Bresnahan (BBB) scores, as well as dramatically reduced cavity volume, clear axonal regeneration and more neuronal survival. In contrast, simple MSC implantation is not a very effective therapy for spinal transection. However, the neuronal differentiation of MSCs after treatment with a combination of Adv-mediated NT-3 gene transfer and RA was only mildly improved in vivo.

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.

Modulation of coxsackie and adenovirus receptor expression for gene transfer to normal and dystrophic skeletal muscle

Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle. To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the target muscle, prior to administration of AdV, through the use of recombinant adeno-associated virus (AAV2) expressing CAR. Lastly, we assessed the effect of histone deacetylase inhibitors on CAR and AdV transduction efficiency in myoblasts and mdx muscle. Although somewhat higher rates of transduction can be achieved in adult mdx mice than in normal mice as a result of ongoing muscle regeneration in these animals, CAR expression in the mdx background (mdx/CAR transgenics) still markedly improved the susceptibility of mature muscle to AdV-mediated gene transfer of dystrophin. Prior administration of AAV2-CAR to normal muscle led to significantly increased transduction by subsequent injection of AdV. The histone deacetylase inhibitor valproate increased CAR transcript and protein levels in myoblasts and mdx muscle, and also increased AdV-mediated gene transfer. We have developed a method of increasing CAR levels in both normal and regenerating muscle.

913. Histone Deacetylase Inhibitors Improve Gene Transfer To Mature Skeletal Muscle

AdV-mediated gene transfer occurs inefficiently in adult skeletal muscle due to downregulation of the primary adenovirus receptor CAR during muscle maturation. We have shown previously that AdV-mediated gene transfer is enhanced in CAR transgenics, indicating that prior modulation of CAR expression results in very efficient AdV transducibility of mature skeletal muscle. We hypothesized that transient CAR expression could be achieved through activation of the transcription of the endogenous CAR gene using histone deacetylase (HDAC) inhibitors. HDACs are known to regulate gene expression via the modulation of chromatin structure by histone acetylation and deacetylation.

Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy

Adenovirus vector (AdV)-mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (alphavbeta3 and alphavbeta5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, alphavbeta3, and alphavbeta5 expression. A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

623. Receptor-Independent Cell Entry of Adenovirus-Polylactide Nanoparticle Composites Leads to an Increased Gene Expression in Endothelial and Smooth Muscle Cells

Background: Adenovirus is a promising gene vector for therapeutic applications. However its effective use is compromised, since AdV-mediated gene transfer is optimal in cell types expressing high amounts of Coxsackie-AdV receptor (CAR). Our goal was to investigate immobilization of AdV on biodegradable particles platform as a means to achieve improved gene transfer independent of the receptor-dependent pathway, and characterize the efficacy of this approach in vitro.

Efficient IL-18 and IL-18BPc adenoviral gene transfer to cultured murine submandibular gland epithelial cells and successful murine retrograde submandibular duct cannulation to modulate IL-18 function in the salivary gland of animal models of Sjogren's syndrome

We recently demonstrated that the proinflammatory and immuno-regulatory cytokine IL-18 is strongly upregulated in salivary glands (SG) of Sjogren's syndrome (SS) patients. IL-18 gain or loss of function experiments in animal models of human diseases have provided evidence of the pathogenic role of this cytokine in chronic inflammation. However, in SS the pathogenic relevance of IL-18 in the development of murine autoimmune sialoadenitis has not as yet been evaluated. Here we describe in vitro experiments in which murine SG epithelial cells were tested for transfection efficiency with IL-18 and IL-18BPcAdV gene transfer. In addition, in preparation for in vivo SG modulation of IL-18 function by AdV-mediated gene transfer, we optimised retrograde SG cannulation in mice.

The Short MCK1350 Promoter/Enhancer Allows for Sufficient Dystrophin Expression in Skeletal Muscles of mdx Mice

First-generation adenovirus vectors (AdV) have been used successfully to transfer a human dystrophin minigene to skeletal muscle of mdx mice. In most studies, strong viral promoters such as the cytomegalovirus promoter/enhancer (CMV) were used to drive dystrophin expression. More recently, a short version of the muscle creatine kinase promoter (MCK1350) has been shown to provide muscle-specific reporter gene expression after AdV-mediated gene delivery. Therefore, we generated a recombinant AdV where dystrophin expression is controlled by MCK1350 (AdVMCKdys). AdVMCKdys was injected by the intramuscular route into anterior tibialis muscle of mdx mice shortly after birth. Dystrophin expression was assessed at 20, 30, and 60 days after AdV-injection. At 20 days, muscles of AdVMCKdys-injected mdx mice showed a high number of dystrophin-positive fibers (mean: 365). At 60 days, the number of dystrophin-positive fibers was not only maintained, but increased significantly (mean: 600). In conclusion, MCK1350 allows for sustained dystrophin expression after AdV-mediated gene transfer to skeletal muscle of newborn mdx mice. In contrast to previous studies, where strong viral promoters were used, dystrophin expression driven by MCK1350 peaks at later time points. This may have implications for the future use of muscle-specific promoters for gene therapy of Duchenne muscular dystrophy.

Forced myofiber regeneration promotes dystrophin gene transfer and improved muscle function despite advanced disease in old dystrophic mice.

Duchenne muscular dystrophy (DMD) is caused by defects in the dystrophin gene. In young dystrophic mdx mice, immature regenerating myofibers represent the principal substrate for adenovirus vector (AdV)-mediated dystrophin gene transfer. However, in DMD patients immature regenerating myofibers are generally sparse. Such a situation also exists in old mdx mice, which may represent a more realistic model. Therefore, here we have used old mdx mice (of 14- to 17 months of age) to test the hypothesis that one-time administration of a myonecrotic agent can transiently re-establish a population of immature myofibers susceptible to AdV-mediated dystrophin gene transfer. This strategy led to upregulation of the coxsackie/adenovirus attachment receptor by means of induction of regenerating myofibers, significantly augmented AdV-mediated dystrophin gene expression, and enhanced force-generating capacity. In addition, it led to an increased resistance to contraction-induced injury compared with untreated controls. The latter protective effect was positively correlated with the number of dystrophin-expressing myofibers (r=0.83, P<0.05). Accordingly, the risk:benefit ratio associated with the sequential use of forced myofiber regeneration and AdV-mediated dystrophin gene transfer was favorable in old mdx mice despite advanced disease. These findings have implications for the potential applicability of AdV-mediated gene therapy to DMD and other muscle diseases in which immature regenerating myofibers are lacking.

Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle.

Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 +/- 121 in transgenic mice versus 8 +/- 4 in nontransgenic littermates) as well as the 25-fold increase in overall beta-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer-chicken beta-actin gene promoter), differential transducibility was still evident (893 +/- 149 versus 153 +/- 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 +/- 130 versus 14 +/- 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.

Combinational immunotherapy for established tumors with engineered tumor vaccines and adenovirus-mediated gene transfer

There are currently extensive studies relating to cancer vaccines using tumor cells engineered to express immunogenes and cancer gene therapy using adenovirus (AdV)-mediated gene transfer. In this study, a mouse tumor cell line, VKCK, was cotransfected with genes coding for tumor necrosis factor-alpha (TNF-alpha) and costimulatory B7-1 molecule to enhance immunogenicity. The transfectant cell line VKCK-TNF-alpha/B7-1 showed reduced tumorigenicity and tumor regression. Its inoculation further induced protective immunity; both CD4+ and CD8+ T cells were involved in the induction phase, whereas only CD8+ T cells mediated the effector phase. Susceptible mice bearing VKCK tumors developed a T helper type 2-dominant response, whereas resistant mice with VKCK-TNF-alpha/B7-1 tumor regression developed a T helper type 1-dominant response to VKCK, indicating that the tumor regression was related to a shift in the cytokine profile of the host from type 2 to type 1. Vaccination of VKCK-TNF-alpha/B7-1 cells inhibited tumor formation derived from a single dose of 3 x 10(6) VKCK cells and eradicated 3-day tumors but not 10-day tumors. AdV-mediated TNF-alpha gene transfer by intratumoral injection of AdV-TNF-alpha significantly inhibited tumor growth but failed to eradicate any well-established tumors. However, combinational immunotherapy with vaccination of VKCK-TNF-alpha/B7-1 cells and AdV-mediated TNF-alpha gene transfer not only significantly inhibited tumor growth but also eradicated 10-day VKCK tumors in three of eight mice. Therefore, the present study may be useful not only in understanding the mechanisms responsible for an efficient antitumoral immunity, but also in establishing a more effective immunotherapeutic approach for cancer patients.

Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells

Objective Based on their differentiation properties and facilely of ex vivo expansion, human bone marrow mesenchymal progenitor cells (MPC), are considered as attractive targets to deliver foreign genes to the bone marrow or other mesenchymal tissues. In this study we investigated the feasibility of transduce MPC with adenoviral vectors (Adv). Methods MPC were expanded ex vivo and transduced with replication-defective Adv-containing reporter genes (lacZ or GFP) under the control of CMV promoter. Transfection efficiency was assessed by microscopical scoring or by flow cytometry. Expression and involvement of Adv-attachment (CAR) and Adv-internalization (integrins αv) receptors were evaluated by flow cytometric studies. Results Transgene expression analysis showed that only 19% ± 3% of cells expressed the transgenes at high levels. MPC express the attachment and internalization receptors required for Adv infection. While integrins αv β3 and αv β5 are expressed by all MPC, CAR is solely expressed by a fraction of low size cells. Antibodies against CAR and αv β5, but not against αv β3, blocked Adv-mediated gene transfer into MPC, showing that CAR and αv β5 are required for infection. Because αv β5, as compared with CAR, is overexpressed in MPC, the results suggest that the efficiency of Adv-mediated gene transfer into MPC depends on the level of CAR expression. Conclusion These findings demonstrate that Adv may be useful to engineer a subpopulation of ex vivo expanded human mesenchymal progenitors, with a high level of transgene expression.

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route.

Duchenne muscular dystrophy (DMD) and other inherited myopathies lead to progressive destruction of most skeletal muscles in the body, including those responsible for maintaining respiration. DMD is a fatal disorder caused by defects in the dystrophin gene. Recombinant adenovirus vectors (AdV) are considered a promising means for therapeutic delivery of a functional dystrophin gene to DMD muscles. If AdV-mediated dystrophin gene replacement in DMD is to be successful, development of a systemic delivery method for targeting the large number of diseased muscles will be required. In this study we investigated two major factors preventing efficient AdV-mediated gene transfer to skeletal muscles of adult animals after intravascular AdV administration: (1) an inability of AdV particles to breach the endothelial barrier and enter into contact with myofibers, and (2) a relatively nonpermissive myofiber population for AdV infection due at least in part to insufficient levels of the coxsackie/adenovirus attachment receptor (CAR). On the basis of established principles governing the transendothelial flux of macromolecules, we further hypothesized that an alteration in Starling forces (increased hydrostatic and decreased osmotic pressures) within the intravascular compartment would facilitate AdV transendothelial flux via convective transport. In addition, experimental muscle regeneration was employed to increase the prevalence of immature myofibers in which CAR expression is upregulated. Here we report that by employing the above-described strategy, high-level heterologous reporter gene expression was achievable in hindlimb muscles of normal rats as well as dystrophic (mdx) mice (genetic homolog of DMD) after a single intraarterial injection of AdV. Microsphere studies confirmed enhanced transport into muscle of fluorescent tracer particles in the size range of AdV, and there was a high concordance between CAR upregulation and myofiber transduction after intraarterial AdV delivery. Furthermore, in mdx mice examined 10 days after intraarterial AdV delivery, the aforementioned procedures had no adverse effects on the force-generating capacity of targeted muscles. These findings have implications for eventual AdV-mediated gene therapy of generalized skeletal muscle diseases such as DMD using a systemic intraarterial delivery approach.

Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer.

Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of alphavbeta3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.

Combinatorial blockade of calcineurin and CD28 signaling facilitates primary and secondary therapeutic gene transfer by adenovirus vectors in dystrophic (mdx) mouse muscles.

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Adenovirus-mediated dystrophin minigene transfer improves muscle strength in adult dystrophic (MDX) mice.

Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx) are both due to absence of the subsarcolemmal protein dystrophin. Recombinant adenovirus vectors (AdV) are considered a promising means for delivering a functional dystrophin gene to muscle. However, the usefulness of AdV for this purpose is limited by vector toxicity as well as immune-mediated elimination of infected fibers. In addition, studies to date of AdV-mediated dystrophin gene transfer have either failed to examine effects on muscle strength or been performed in immunologically immature neonatal animals with little baseline abnormality of force-generating capacity. In the present study, AdV-mediated dystrophin gene transfer was performed in adult mdx mice with pre-existent dystrophic pathology and muscle weakness. The main findings are as follows: (1) acute myofiber toxicity and gene transfer efficiency are both AdV dose-dependent, such that the therapeutic margin of safety is fairly narrow; (2) immunosuppressive therapy (FK506) prevents immune-mediated elimination of dystrophin-positive fibers but not the dose-dependent toxic effects; (3) at the optimal vector dosage and with effective immunosuppression, AdV-mediated dystrophin minigene transfer is capable of alleviating the loss of force-generating capacity as well as histopathological evidence of disease progression normally seen in adult mdx muscles over a 2-month period. These findings have important implications for the eventual application of AdV-mediated dystrophin gene transfer in DMD patients.

Respiratory muscles as a target for adenovirus-mediated gene therapy.

The protein dystrophin is absent in the muscles of patients with Duchenne muscular dystrophy (DMD) as well as dystrophin-deficient mice with muscular dystrophy (mdx mice). The mdx mouse diaphragm closely resembles the human DMD phenotype and thus provides a useful model for studies of dystrophin gene replacement. Recombinant adenovirus vectors (AdVs) hold promise as a means for delivering a functional dystrophin gene to muscle. As an initial step toward this goal, we have determined the efficiency and functional consequences of AdV-mediated reporter gene transfer to the diaphragm in both normal and mdx adult mice. At 1 week after AdV administration, there was a high level of transgene expression in the diaphragm. One month later, however, elimination of transgene expression was observed along with a significant decrease in force production by both normal and mdx diaphragms. Immunosuppression with cyclosporine did not augment the level of transgene expression, but a beneficial effect on diaphragm force-generating capacity was observed in both groups of animals. In order to further elucidate the cellular mechanisms underlying these findings, the effects of AdV gene inactivation (by ultraviolet (UV) irradiation) and interference with host T-lymphocyte subsets were examined. Both UV-inactivation of AdV and CD8+ T-cell deficiency were found to significantly alleviate AdV-induced reductions in diaphragm force-generating capacity. Brief (2 day) administration of a neutralizing antibody against host CD4+ T-cells also produced a trend towards mitigation of AdV-induced contractile dysfunction. In addition, transgene expression one month after AdV delivery was significantly enhanced with inhibition of either CD4+ or CD8+ T-cell function. The data suggest two major sources of reduced force generation after recombinant adenovirus vector-mediated gene transfer to muscle: 1) a cytotoxic component associated with recombinant adenovirus vector transcriptional activity; and 2) an immune-based component of more delayed onset that is primarily dependent upon CD8+ T-cell activity. These results have important implications for the design of future generation vectors and the potential need for immunosuppressive therapy after recombinant adenovirus vector mediated dystrophin gene transfer to Duchenne muscular dystrophy patients.

Impairment of force generation after adenovirus-mediated gene transfer to muscle is alleviated by adenoviral gene inactivation and host CD8+ T cell deficiency.

Recombinant adenovirus vectors (AdV) hold promise as a means of delivering therapeutic genes to muscle in diseases such as Duchenne muscular dystrophy (DMD). However, we have previously shown that the use of AdV is hampered by the development of reduced force-generating capacity, which occurs within 1 week and is progressive up to at least 1 month after AdV delivery in immune-competent animals. Determinations of muscle force production provide a sensitive and clinically important measure of potential adverse effects of AdV-mediated gene transfer on muscle cell function. In the present study, we investigated the role of AdV-related gene expression and host T lymphocyte responses in the genesis of muscle dysfunction following AdV injection of muscle. We report that UV-irradiation of AdV particles, which reduced AdV transcriptional activity without impairing infectivity (as confirmed by in situ polymerase chain reaction), significantly reversed early (4 days post-injection) AdV-induced contractile impairment in immune-competent mice as well as in mice lacking effective CD8+ T cell activity. The superimposed additional reduction in force-generating capacity normally found between 4 and 30 days post-AdV delivery in immune-competent mice, along with the associated loss of transgene (beta-galactosidase) expression, was largely abrogated by the absence of an intact CD8+ T lymphocyte response. Furthermore, short-term administration of a neutralizing antibody against CD4+ T cells significantly prolonged transgene expression and showed a trend toward mitigation of AdV-induced reductions in force-generating capacity. Cellular infiltration and humoral immune responses against the vector and transgene product were also blunted to varying degrees in the setting of CD8+ or CD4+ T cell deficiency. We conclude that AdV-related gene expression has an early negative (probably toxic) effect on muscle cell function that is independent of CD8+ T cell-mediated immunity. In contrast, further progression of contractile impairment and the accompanying loss of transgene expression from AdV-injected muscle are largely dependent upon the activity of CD8+ T cells. These results have implications for the design of future generation vectors and the potential need for immunosuppressive therapy after AdV-mediated gene transfer to muscle.