Intracerebroventricular delivery of glucocerebrosidase reduces substrates and increases lifespan in a mouse model of neuronopathic Gaucher disease.

Gaucher disease is caused by a deficit in the enzyme glucocerebrosidase. As a consequence, degradation of the glycolipids glucosylceramide (GluCer) and glucosylsphingosine (GluSph) is impaired, and their subsequent buildup can lead to significant pathology and early death. Type 1 Gaucher patients can be treated successfully with intravenous replacement enzyme, but this enzyme does not reach the CNS and thus does not ameliorate the neurological involvement in types 2 and 3 Gaucher disease. As one potential approach to treating these latter patients, we have evaluated intracerebroventricular (ICV) administration of recombinant human glucocerebrosidase (rhGC) in a mouse model of neuronopathic Gaucher disease. ICV administration resulted in enzyme distribution throughout the brain and alleviated neuropathology in multiple brain regions of this mouse model. Treatment also resulted in dose-dependent decreases in GluCer and GluSph and significantly extended survival. To evaluate the potential of continuous enzyme delivery, a group of animals was treated ICV with an adeno-associated viral vector encoding hGC and resulted in a further extension of survival. These data suggest that ICV administration of rhGC may represent a potential therapeutic approach for type 2/3 Gaucher patients. Preclinical evaluation in larger animals will be needed to ascertain the translatability of this approach to the clinic.

Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity.

Recombinant serotype 5 adenovirus (Ad5) vectors lacking E1 expression induce robust immune responses against encoded transgenes in pre-clinical models, but have muted responses in human trials because of widespread pre-existing anti-adenovirus immunity. Attempts to circumvent Ad5-specific immunity by using alternative serotypes or modifying capsid components have not yielded profound clinical improvement. To address this issue, we explored a novel alternative strategy, specifically reducing the expression of structural Ad5 genes by creating E1 and E2b deleted recombinant Ad5 vectors. Our data show that [E1-, E2b-]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1-] vectors. These pre-clinical studies with E1 and E2b-deleted recombinant Ad5 vectors suggest that anti-Ad immunity will no longer be a limiting factor, and that clinical trials to evaluate their performance are warranted.

Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors.

The systemic delivery of [E1(-)] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1(-)]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1(-)]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1(-), E2b(-)]Ad vector. In this study, we confirmed that [E1(-), E2b(-)]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1(-), E2b(-)]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.

Liver toxicities typically induced by first-generation adenoviral vectors can be reduced by use of E1, E2b-deleted adenoviral vectors.

Adenoviral vectors from which the E1 region has been deleted ([E1(-)] Ad) are known to induce strong immune responses after systemic delivery. In this study we have evaluated liver toxicities in mice after intravenous injection with high doses of [E1(-)] or modified [E1(-), E2b(-)] Ad vectors (both expressing the bacterial beta-galactosidase [lacZ] marker gene) in C57BL/6, BALB/c, and SCID mice. Our data demonstrate a marked reduction in maximal liver toxicities and pathologies (typically noted at 21 days postinjection) with the use of the [E1(-), E2b(-)] modified vector in all strains of mice tested. Our data also demonstrated that despite the use of the [E1(-), E2b(-)] Ad vector, significant liver toxicities were still observed. To address this issue and the fact that the lacZ gene was perceived as a foreign antigen in the immune-competent C57BL/6 and BALB/c mice, we similarly injected mice tolerant of LacZ (lacZ-TG). In contrast to our studies in C57BL/6 and BALB/c mice, LacZ-TG mice exhibited virtually no evidence of hepatotoxicity after intravenous injection with the [E1(-), E2b(-)] vector, in contrast to use of the [E1(-)] Ad vector. Our results demonstrate that the [E1(-), E2b(-)] Ad vector class can reduce liver toxicities typically ascribed to Ad vector-mediated gene transfer after transfer of a highly immunogenic or foreign gene, whereas transfer of a transgene that is perceived as nonforeign by the host can be delivered with virtually no evidence of toxicity. On the basis of a careful review of the literature, these improvements in vector safety rival those noted with other, more significantly modified Ad vectors described to date.

A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function.

The loss of cochlear hair cells, or the loss of their capacity to transduce acoustic signals, is believed to be the underlying mechanism in many forms of hearing loss. To develop viral vectors that allow for the introduction of genes directly into the cochleae of adult animals, replication-deficient (E1(-), E3(-)) and replication-defective (E1(-), E3(-), pol(-)) adenovirus vectors were used to transduce the bacterial beta-galactosidase gene into the hair cells of the guinea pig cochlea in vivo. Distortion product otoacoustic emissions, which monitor the functional status of outer hair cells, were measured throughout the viral infection periods to identify hair cell ototoxicity. The results demonstrated that the use of the (E1(-), E3(-)) adenovirus vectors containing CMV-driven LacZ, compromised cochlear function when gradually introduced into scala tympani via an osmotic pump. However, when (E1(-), E3(-), pol(-)) adenoviral vectors containing CMV-driven LacZ were used to transduce cochlear hair cells, there was no loss of cochlear function over the frequency regions tested, and beta-galactosidase (beta-gal) was detected in over 80% of all hair cells. Development of a viral vector that infects cochlear hair cells without virus-induced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for otoprotective strategies to prevent hair cell losses to treat progressive hearing disorders. Moreover, in vivo (E1(-), E3(-), pol(-)) adenovirus mediated gene-transfer techniques applied to adult guinea pig cochleae may be useful in testing several hypotheses concerning what roles specific genes play in normal cochlear function.

Long-term efficacy after [E1-, polymerase-] adenovirus-mediated transfer of human acid-alpha-glucosidase gene into glycogen storage disease type II knockout mice.

Glycogen storage disease type II (GSD-II) is a lethal, autosomal recessive metabolic myopathy caused by a lack of acid-alpha-glucosidase (GAA) activity in the cardiac and skeletal muscles. Absence of adequate intralysosomal GAA activity results in massive amounts of glycogen accumulation in multiple muscle groups, resulting in morbidity and mortality secondary to respiratory embarrassment and/or cardiomyopathy. In a mouse model of GSD-II, we demonstrate that infection of the murine liver with a modified adenovirus (Ad) vector encoding human GAA (hGAA) resulted in long-term persistence of the vector in liver tissues for at least 6 months. Despite both a rapid shutdown of hGAA mRNA expression from the vector, as well as the elicitation of anti-hGAA antibody responses (hGAA is a foreign antigen in this model), the hGAA secreted by the liver was taken up by all muscle groups analyzed and, remarkably, persisted in them for at least 6 months. The persistence of the protein also correlated with long-term correction of pathologic intramuscular glycogen accumulations in all muscle groups tested, but most notably the cardiac tissues, which demonstrated a significantly decreased glycogen content for at least 190 days after a single vector injection. The results suggest that gene therapy strategies may have the potential to significantly improve the clinical course for GSD-II patients.

Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications.

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K-]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1-,100K-]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K-]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K-]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1-,100K-]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K-]Ad vectors suggest that they may have utility in a variety of gene therapy applications.

Mdx mice inducibly expressing dystrophin provide insights into the potential of gene therapy for duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by the lack of expression of the dystrophin protein in muscle tissues. We genetically engineered a mouse model (mdx) of DMD that allowed for the high level and inducible transcription of a dystrophin mini-gene. This was achieved via the tetracycline-responsive transactivator (tTA) system. Multiple analyses confirmed that dystrophin expression in the mice was: (i) tTA dependent; (ii) correctly localized to the sarcolemmal membranes; (iii) capable of preventing the onset of dystrophy; and (iv) effectively blocked by the oral administration of tetracyclines. The model allowed us to somatically extinguish or induce dystrophin gene transcription. Somatic induction of dystrophin transcription prevented the onset of muscular dystrophy in some muscle groups. The levels of phenotypic rescue were influenced, however, by the age of the animals at the time of dystrophin induction. We also found that despite somatic termination of dystrophin gene transcription, the dystrophin protein was found to be associated with the sarcolemmal membrane for at least 26 weeks. Persistent detection of dystrophin was also accompanied by a prolonged protection of the muscle cells from the onset of dystrophy. The findings demonstrated that somatic transfer of the dystrophin gene not only may allow for the prevention of muscular dystrophy in multiple muscle groups, but also may be accompanied by persistent efficacy, secondary to the long-term functional stability of the dystrophin protein in vivo. This model should be useful in future studies concerning the potential of genetic therapy for DMD, as well as other muscle disorders.

Multiply deleted [E1, polymerase-, and pTP-] adenovirus vector persists despite deletion of the preterminal protein.

BACKGROUND: The inherent limitations of [E1-]Ad vectors as gene therapy vehicles suggest that further modifications may improve their overall performance profiles. However, Ad vector modifications can have untoward effects on their basic biology, e.g., some helper-virus dependent Ad vectors have been found to be unstable without the presence of preterminal protein (pTP) activities. Despite this concern, we generated a new class of helper-virus independent Ad vector that was multiply deleted for the E1, polymerase, and pTP genes, and investigated the ramifications of these deletions upon several vector performance parameters. METHODS: The construction and propagation of an [E1-, polymerase-, pTP-]Ad vector was achieved with the use of trans-complementing cells co-expressing the Ad E1, polymerase and pTP genes. RESULTS: High titer production of the [E1-, polymerase-, pTP-]Ad vector was successfully accomplished via conventional Ad purification techniques. This unique class of Ad vector was capable of long-term gene transfer in vivo (despite lacking pTP functions) that was concomitant with a significantly decreased hepatic toxicity. CONCLUSIONS: Previous studies had suggested that Ad genome persistence in vivo may be dependent upon the presence of low level vector genome replication and/or pTP functions. Our results suggest that [E1-, polymerase-, pTP-]Ad vectors can overcome these barriers. The further benefits afforded by the use of this class of Ad vector (increased cloning capacity, high level growth, decreased propensity to generate replication competent Ad (RCA), decreased toxicity) suggests that they will be highly beneficial for use in several aspects of human gene therapy.

A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering.

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin.

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.

Altered expression of the alpha7beta1 integrin in human and murine muscular dystrophies.

The alpha7beta1 integrin is the primary laminin receptor on skeletal myoblasts and adult myofibers. It has distinct functions during muscle development and contributes to muscle structural integrity. We have studied this integrin in cases where expression of dystrophin or laminin are compromised. Immunofluorescence demonstrates an increase in alpha7beta1 in patients with Duchenne muscular dystrophy and in mdx mice that lack dystrophin. Analysis of RNA from mdx mice and from patients with Duchenne and Becker muscular dystrophies indicates that the increase in the alpha7beta1 integrin is regulated at the level of alpha7 gene transcription. In contrast, the levels of alpha7beta1 integrin are severely diminished in patients with laminin alpha2 chain congenital dystrophy muscular dystrophy and in dy/dy mice that also do not make the alpha2 laminin chain. Analysis of RNA from the hindlimbs of dy/dy mice demonstrated that in the absence of laminin alpha7 gene transcription is inhibited and limited to specific alternatively spliced isoforms. We suggest that the increased expression of alpha7beta1 integrin in the absence of dystrophin compensates for the reduced dystrophin-mediated linkage of fibers with the basal lamina and modulates the development of pathology associated with these diseases. The decrease in alpha7beta1 integrin and its transcripts in the absence of laminin likely contributes to the severe myopathy that results from laminin alpha2 chain deficiency and suggests that laminin-2 regulates expression of the alpha7 integrin gene. The role of the alpha7beta1 integrin in muscle integrity also suggests that compromised expression of this receptor may underlie as yet undefined myopathies.

Inhibitors of nitric oxide synthesis antagonize nitrous oxide antinociception in mice and rats.

Administration of the anesthetic gas N2O evoked an antinociceptive effect in two rodent antinociception paradigms. In the mouse abdominal constriction test, pretreatment with the nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine (L-NOARG) caused dose-related antagonism of the antinociceptive effect of N2O but not of either morphine or trans(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulfonate. This antinociceptive effect was also antagonized by systemic pretreatment with the NOS inhibitors L-NG-nitroarginine methyl ester (L-NAME) and L-NG-monomethylnitroarginine. The antagonism of N2O by L-NOARG and L-NAME was completely reversed by i.c.v. administration of L-arginine but not D-arginine. In the absence of NOS inhibition, N2O antinociception was potentiated by i.c.v. treatment with L-arginine but not D-arginine. The i.c.v. pretreatment with L-NAME also reduced N2O antinociception; this antagonism was also stereospecifically reversed by L-arginine. In the rat hot plate test, the antinociceptive response to 70% N2O was antagonized in dose-related manner by i.c.v. pretreatment with L-NOARG or L-NAME. N2O antinociception was restored by i.c.v. treatment with L-arginine but not D-arginine. However, neither L-arginine nor D-arginine alone affected N2O antinociception. These results implicate a key role for NO in the mediation of the antinociceptive effects of N2O in both mice and rats.

Antagonism of nitrous oxide antinociception in the rat hot plate test by site-specific mu and epsilon opioid receptor blockade.

The analgesic property of the anesthetic gas N2O has long been known and used to treat pain in clinical medicine and dentistry. The present study was conducted to identify by subtype and possible location the brain opioid receptors that mediate N2O antinociception in rats. A 5-min exposure to 70% N2O consistently evoked an antinociceptive effect in the hot plate test. This drug effect was partly antagonized in dose-related fashion by i.c.v. pretreatment with naltrexone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 and beta-endorphin, which block multiple, mu and epsilon opioid receptors, respectively. However, the N2O-evoked antinociception was unaffected by i.c.v. pretreatment with either the delta opioid antagonist naltrindole or the kappa opioid antagonist nor-binaltorphimine. When D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was administered intracerebrally directly into the periaqueductal gray, N2O antinociception was partly antagonized in a dose-dependent manner. The antinociceptive response to N2O was uninfluenced by beta-endorphin administered into the periaqueductal gray. The findings of these pharmacological antagonism studies are consistent with the hypothesis that exposure to N2O causes a neuronal release of beta-endorphin. These results indicate that supraspinal mu and epsilon opioid receptors mediate N2O antinociception in the rat hot plate paradigm and that one central site of such mu but not epsilon opioid receptors is the periaqueductal gray.

Benzodiazepine receptor-mediated behavioral effects of nitrous oxide in the rat social interaction test.

The present study was conducted to ascertain whether an anxiolytic effect of nitrous oxide was demonstrable in rats using the social interaction test and whether this drug effect might be mediated by benzodiazepine receptors. Compared to behavior of vehicle-pretreated, room air-exposed rats, rat pairs exposed to nitrous oxide showed a generally inverted U-shaped dose-response curve with the maximum increase in social interaction encounters occurring at 25% and significant increase in time of active social interaction at 15-35%; higher concentrations produced a sedative effect that reduced social interaction. Treatment with 5.0 mg/kg of the anxiolytic benzodiazepine chlordiazepoxide also increased social interaction. Pretreatment with 10 mg/kg of the benzodiazepine receptor blocker flumazenil, which alone had no effect, significantly antagonized the social interaction-increasing effects of both nitrous oxide and chlordiazepoxide. In summary, these findings suggest that nitrous oxide produces a flumazenil-sensitive effect comparable to that of chlordiazepoxide and implicate central benzodiazepine mechanisms in mediation of the anxiolytic effect of nitrous oxide.