Biomedical subjects

P R Clemens

Publications and source records attributed to P R Clemens.

At least 19 recordsLinked to original sources

PubMed2005

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.

Adenoviridae↗

PubMed2004

CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle.

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.

Abatacept↗

PubMed2003

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Adenoviridae↗

PubMed2001

Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection.

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca(2+)-dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophin-deficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wild-type cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.

Animals↗

PubMed2001

Clinical evaluator reliability for quantitative and manual muscle testing measures of strength in children.

Measurements of muscle strength in clinical trials of Duchenne muscular dystrophy have relied heavily on manual muscle testing (MMT). The high level of intra- and interrater variability of MMT compromises clinical study results. We compared the reliability of 12 clinical evaluators in performing MMT and quantitative muscle testing (QMT) on 12 children with muscular dystrophy. QMT was reliable, with an interclass correlation coefficient (ICC) of >0.9 for biceps and grip strength, and >0.8 for quadriceps strength. Training of both subjects and evaluators was easily accomplished. MMT was not as reliable, and required repeated training of evaluators to bring all groups to an ICC >0.75 for shoulder abduction, elbow and hip flexion, knee extension, and ankle dorsiflexion. We conclude that QMT shows greater reliability and is easier to implement than MMT. Consequently, QMT will be a superior measure of strength for use in pediatric, neuromuscular, multicenter clinical trials.

Child↗

PubMed2001

Local high-capacity adenovirus-mediated mCTLA4Ig and mCD40Ig expression prolongs recombinant gene expression in skeletal muscle.

Multiple forms of muscular dystrophy are due to the absence of cytoskeletal muscle proteins that normally protect the integrity of muscle cells. The lack of any adequate treatments for these devastating diseases propels research toward the development of strategies for gene delivery to skeletal muscle. High-capacity adenoviral vectors (HC-AdV) devoid of all viral coding sequences have been developed to avoid expression of viral proteins by the gene therapy vector. However, the capsid proteins that are an essential component of the input viral vector and any residual helper virus in the vector preparation could induce an immune response. Furthermore, the therapeutic protein provided by a gene transfer vector presents the potential to induce an immune response in a patient who does not express a normal cellular protein due to genetic mutation. Therefore, we hypothesize that some immune suppression will be required with therapeutic gene delivery designed for the treatment of patients with inherited muscle diseases. In this study, we constructed and rescued three HC-AdVs expressing murine CTLA4Ig, murine CD40Ig, or both. The backbone vector without a gene insert was rescued as a negative control vector. The production of relevant proteins from each vector was determined in vitro. In vivo function of each of the immunosuppressant vectors was assayed by co-injection with an enhanced green fluorescent protein (EGFP)-expressing first-generation adenoviral vector (AdEGFP) into the tibialis anterior muscle of C57BL/10 mice. Higher levels of muscle EGFP expression were observed in animals receiving an immunosuppressant vector. Furthermore, the production of total anti-AdV and anti-EGFP antibodies was reduced in mice treated with each of the three immunosuppressant vectors. A second intramuscular administration of AdEGFP alone 4 weeks after the initial co-injection was successful in all immunosuppressant vector-treated groups, but not in the negative control vector-treated group. All groups had a high antibody response to adenoviral proteins after the second injection of AdEGFP alone, indicating that the initial co-injection did not tolerize against vector capsid antigens.

Abatacept↗

PubMed2001

Transfer of full-length Dmd to the diaphragm muscle of Dmd(mdx/mdx) mice through systemic administration of plasmid DNA.

Mutations in the gene encoding dystrophin, a large cytoskeletal protein in muscle, lead to Duchenne muscular dystrophy (DMD). Affected individuals often die of respiratory failure resulting primarily from diaphragm muscle degeneration. Here we report a new procedure to transfer the full-length dystrophin cDNA into the diaphragm muscle of Dmd(mdx/mdx) mice, which carry a mutation in the dystrophin gene (Dmd). Significant gene transfer was found after intravenous injection of naked plasmid DNA followed by a brief (eight second) occlusion of blood flow at the vena cava. This is the first demonstration of gene transfer into the diaphragm muscle through systemic administration of naked plasmid DNA. The approach has potential application for treatment of DMD.

Animals↗

PubMed2001

Progress in gene therapy for Duchenne muscular dystrophy.

Gene transfer research for Duchenne muscular dystrophy (DMD) has brought the goal of successful treatment of this devastating, inherited disease closer to being a reality. Although gene therapeutic approaches for DMD patients are not yet in clinical use, recent advances using DMD animal models are encouraging. Progress in vector design, such as high-capacity adenoviral vectors, targeted adenoviral vectors, and heterodimerization of DNA delivered by adeno-associated virus (AAV) vectors have advanced the field considerably. The recent studies into the pharmacologic-induced read-through of stop codons, the increased study of utrophin and its upregulation, and the introduction of point mutation correction using chimeric oligonucleotides have expanded the field, providing new avenues of inquiry.

Anti-Bacterial Agents↗

PubMed1999

Polylysine modification of adenoviral fiber protein enhances muscle cell transduction.

Adenoviral vectors (ADVs) are used widely for gene delivery to different tissues including muscle. One particularly promising use for ADVs is in the transfer of the dystrophin gene to the muscle of patients with Duchenne muscular dystrophy (DMD). However, studies in different animal models of DMD suggest that ADVs inefficiently transduce mature skeletal muscle. In this article we test whether AdZ.F(pK7), a genetically modified ADV that expresses a polylysine moiety on the end of the fiber protein, could enhance transduction of muscle cells and circumvent the maturation-dependent loss of muscle infectivity by ADVs. The efficiency of transduction was tested at different levels of muscle maturation. In vitro, AdZ.F(pK7) showed a higher level of transduction at all stages of differentiation including myoblasts, myotubes, and single muscle fibers. In vivo, mature skeletal muscle was transduced fourfold better by AdZ.F(pK7) than by the unmodifled vector (AdZ.F). Together, these observations demonstrate improved ADV transduction of skeletal muscle by modifying ADV tropism, and provide a proof-of-principle that modification of ADVs to target muscle-specific molecules could result in tissue-specific transfer of skeletal muscle tissue as well.

Adenoviruses, Human↗

PubMed1999

DNA from both high-capacity and first-generation adenoviral vectors remains intact in skeletal muscle.

Previous studies of the use of adenoviral vectors in animal models of gene therapy have focused on the immune response against transduced cells as the major limiting factor to long-term transgene expression. In this study we eliminated the variable of immunity induced by expression of the transgene in order to investigate vector DNA stability of both first-generation and high-capacity adenoviral vectors after gene transfer to skeletal muscle. Transgene expression from a high-capacity adenoviral vector remained at a high level for at least 20 weeks and was accompanied by persistence of intact vector genomes. In contrast, transgene expression from a first-generation adenoviral vector markedly diminished by 6 weeks after gene transfer and was accompanied by mild and variable inflammatory cell infiltrates. Surprisingly, despite this loss of transgene expression, the first-generation adenoviral vector genomes persisted like the high-capacity adenoviral vector genomes. Therefore, in the absence of immunity to transgene proteins, loss of expression from the first-generation vector was due to inhibition of transgene expression rather than to the elimination of vector-containing cells. DNA stability and persistent expression of the high-capacity adenoviral vector supports the potential of this vector for clinical applications of muscle gene transfer.

Adenoviridae↗

PubMed1998

Ex vivo gene transfer using adenovirus-mediated full-length dystrophin delivery to dystrophic muscles.

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle disease characterized by a lack of dystrophin expression. Myoblast transplantation and gene therapy have the potential of restoring dystrophin, thus decreasing the muscle weakness associated with this disease. In this study we present data on the myoblast mediated ex vivo gene transfer of full-length dystrophin to mdx (dystrophin deficient) mouse muscle as a model for autologous myoblast transfer. Both isogenic primary mdx myoblasts and an immortalized mdx cell line were transduced with an adenoviral vector that has all viral coding sequences deleted and encodes beta-galactosidase and full-length dystrophin. Subsequently, these transduced myoblasts were injected into dystrophic mdx muscle, where the injected cells restored dystrophin, as well as dystrophin-associated proteins. A greater amount of dystrophin replacement occurred in mdx muscle following transplantation of mdx myoblasts isolated from a transgenic mouse overexpressing dystrophin suggesting that engineering autologous myoblasts to express high amounts of dystrophin might be beneficial. The ex vivo approach possesses attributes that make it useful for gene transfer to skeletal muscle including: (1) creating a reservoir of myoblasts capable of regenerating and restoring dystrophin to dystrophic muscle; and (2) achieving a higher level of gene transfer to dystrophic muscle compared with adenovirus-mediated direct gene delivery. However, as observed in direct gene transfer studies, the ex vivo approach also triggers a cellular immune response which limits the duration of trans-gene expression.

Adenoviridae↗

PubMed1997

Persistence in muscle of an adenoviral vector that lacks all viral genes.

Genetic correction of inherited muscle diseases, such as Duchenne muscular dystrophy, will require long term expression of the recombinant protein following gene transfer. We have shown previously that a new adenoviral vector that lacks all viral genes expressed both full-length dystrophin and beta-galactosidase in mdx (dystrophin-deficient) mouse muscle. We observed a significant histologic improvement of vector-transduced mdx muscle before the eventual loss of vector-encoded transgene expression. In this study, we investigated whether an immunological response against vector-encoded beta-galactosidase contributed to the loss of vector expression and affected vector persistence in muscle. Intramuscular vector injection in control normal mice resulted in an early and complete loss of beta-galactosidase expression accompanied by predominantly CD4+ and CD8+ lymphocytic infiltration and a significant loss of vector DNA. In contrast, intramuscular vector injection in lacZ transgenic mice resulted in persistent expression of beta-galactosidase for at least 84 days with no evidence of inflammation or significant loss of vector DNA. Our studies demonstrate that, in the absence of an immune response induced by beta-galactosidase expression, an adenoviral vector lacking all viral genes is stably maintained in muscle.

Adenoviridae↗

PubMed1997

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.

Adenoviridae↗

PubMed1996

A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.

Adenoviral vector-mediated gene transfer offers significant potential for gene therapy of many human diseases. However, progress has been slowed by several limitations. First, the insert capacity of currently available adenoviral vectors is limited to 8 kb of foreign DNA. Second, the expression of viral proteins in infected cells is believed to trigger a cellular immune response that results in inflammation and in only transient expression of the transferred gene. We report the development of a new adenoviral vector that has all viral coding sequences removed. Thus, large inserts are accommodated and expression of all viral proteins is eliminated. The first application of this vector system carries a dual expression cassette comprising 28.2 kb of nonviral DNA that includes the full-length murine dystrophin cDNA under control of a large muscle-specific promoter and a lacZ reporter construct. Using this vector, we demonstrate independent expression of both genes in primary mdx (dystrophin-deficient) muscle cells.

Adenoviridae↗

PubMed1996

HyperCKemic, proximal muscular dystrophies and the dystrophin membrane cytoskeleton, including dystrophinopathies, sarcoglycanopathies, and merosinopathies.

Of the various muscular dystrophies, the dystrophinopathies are the most common, accounting for the majority of male muscle disease patients and for about 10% of female patients referred for the evaluation of muscular dystrophy or persistent high serum creatine kinase values (hyperCKemia). The approach to diagnosis and family genetic counseling for the dystrophinopathies is now well established, and implementation of carrier detection and prenatal diagnosis has dramatically decreased the incidence of familial cases. With the decreasing observation of a positive family history in newly ascertained cases, molecular genetic and protein studies become imperative for accurate diagnosis. Genetic counseling in families of isolated cases can still be problematic. There is a wide range of opinions regarding the management of Duchenne muscular dystrophy, with surgical interventions (eg, tendon lengthenings and spinal fusion), steroid use, and extent of respiratory support actively debated. There has been progress in defining the underlying cause of disease for some patients of muscular dystrophy who have normal dystrophin findings. Nearly all patients with proximal, hyperCKemic muscular dystrophy who have normal dystrophin show no family history of the disorder, consistent with autosomal recessive disease. Approximately 5% of both boys and girls with childhood-onset dystrophy and normal dystrophin have been found to have mutations in one of the four sarcoglycan proteins identified to date. Also, approximately half of the patients with congenital muscular dystrophy show deficiency of a component of the muscular extracellular matrix. (merosin/laminin-alpha 2). In this review, we give a short primer on relevant muscle structure and function, followed by a series of case reports on patients referred for molecular testing that illustrate the diagnostic protocols, range of clinical presentations, and genetic counseling options in the work-up of proximal muscular dystrophy and hyperCKemia.

Child↗

PubMed1996

Cognitive dysfunction as the major presenting feature of Becker's muscular dystrophy.

We report four patients, currently aged 15, 17, 19, and 42 years, with X-linked dystrophinopathy who presented with mental retardation (IQ range, 60-68) and psychiatric disturbance in the absence of muscle weakness. All patients had elevated serum creatine kinase and dystrophic changes on muscle biopsy. There were alterations in the size and abundance of dystrophin on immunohistochemistry and immunoblotting in all cases, consistent with a molecular diagnosis of Becker's muscular dystrophy. Two patients had deletions of the dystrophin gene on DNA analysis. These findings suggest that Becker's muscular dystrophy may be associated with a predominantly neuropsychiatric presentation and that dystrophinopathy should be considered in the differential diagnosis of unexplained cognitive or psychiatric disturbance in males. Serum creatine kinase may provide an adequate screening test in this clinical situation.

Adolescent↗

PubMed1996

In vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes.

Duchenne muscular dystrophy (DMD) is an important target for gene transfer because of the disease's high frequency and devastating course. To date, adenoviral vector-mediated gene transfer for DMD has been unavailable because (1) adenoviral vectors were unable to accommodate the full-length dystrophin cDNA (14 kb); and (2) adenoviral vectors induced inflammatory reactions in the gene transfer recipient. We addressed both problems with a novel adenoviral vector that contains no viral genes and encodes 28.2 kb of foreign DNA including both the full-length dystrophin cDNA with the muscle creatine kinase promoter for transcriptional control and a lacZ marker gene. This report presents the in vivo expression of dystrophin and beta-galactosidase from this vector in skeletal muscle of the mdx mouse, a mutant mouse that lacks dystrophin. Somatic delivery of the vector by intramuscular injection in 6-day-old mice resulted in the expression of full-length, recombinant dystrophin at the muscle membrane. Dystrophin-associated proteins were restored in muscle fibers expressing recombinant dystrophin. Mdx muscle injected with our vector showed a decrease in the proportion of fibers with nuclei located centrally; centrally placed nuclei in muscle fibers are characteristic of cycles of degeneration and regeneration suffered by dystrophin-deficient muscle tissue. These results are strong evidence that adenoviral vector-mediated full-length dystrophin delivery provides substantial somatic function.

Adenoviridae↗

PubMed1995

Recombinant truncated dystrophin minigenes: construction, expression, and adenoviral delivery.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder for which there is currently no effective treatment. Although clinical application of adenoviral vector-mediated gene transfer has not been fully developed, it shows promise for the treatment of DMD. One significant problem posed by adenoviral vector-mediated gene transfer for DMD is that currently available adenoviral vectors cannot accommodate the entire 14-kb dystrophin cDNA. To address this problem, we selectively deleted regions of the murine dystrophin cDNA to produce truncated constructs. We created three constructs, each with an in-frame deletion of a segment (3.0, 4.4, and 5.7 kb) of the spectrin-like repeat region of dystrophin. As an additional modification, we removed the majority of the 3' untranslated region of the cDNA in expression vectors encoding some of these truncated constructs. Comparative quantitative expression studies after transfection into COS and C2C12 mouse muscle cells demonstrate variations in the level of expression with different deletions in the spectrin-like repeat region. Furthermore, deletion of the 3' untranslated region was tested for one recombinant construct and resulted in a reduction in the level of expression in both cell culture systems. Toward the ultimate goal of gene transfer therapy for DMD, we created an adenoviral vector from one of our truncated constructs. Using this vector, we demonstrated truncated dystrophin expression in vitro in primary mdx (dystrophin-deficient) muscle cells and in vivo in mdx mouse muscle. In vivo, recombinant dystrophin was properly localized to the muscle membrane.

Adenoviridae↗