Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells.

CD40 ligation has been shown to promote antigen-presenting functions of dendritic cells, which express CD40 receptor. Here we reported significantly altered biodistribution and immune responses with the use of CD40-targeted adenovirus. Compared with unmodified adenovirus 5, the CD40-targeted adenovirus following intravenous administration (i.v.) resulted in increased transgene expressions in the lung and thymus, which normally do not take up significant amounts of adenovirus. Intradermal injection saw modified adenovirus being mainly processed in local draining lymph nodes and skin. Following intranasal administration (i.n.), neither unmodified nor targeted viruses were found to be in the liver or spleen, which predominantly took up the virus following i.v. administration. However, inadvertent infection of the brain was found with unmodified adenoviruses, with the second highest gene expression among 14 tissues examined. Importantly, such undesirable effects were largely ablated with the use of targeted vector. Moreover, the targeted adenovirus elicited more sustained antigen-specific cellular immune responses (up to 17-fold) at later time points (30 days post boosting), but also significantly hampered humoral responses irrespective of administration routes. Additional data suggest the skewed immune responses induced by the targeted adenoviruses were not due to the identity of the transgene but more likely a combination of overall transgene load and CD40 stimulation.

Synthesis and evaluation of novel dipeptide-bound 1,2,4-thiadiazoles as irreversible inhibitors of guinea pig liver transglutaminase.

Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 microM(-1) min(-1).

Intravitreal adenoviral gene transfer evokes an immune response in the retina that is directed against the heterologous lacZ transgene product but does not limit transgene expression.

Recombinant E1-deleted adenoviral vectors (DeltaE1-Ad) are promising tools for in vivo gene transfer into the mammalian CNS including the retina. However, the duration of transgene expression is limited, and this limitation has partly been attributed to an immune response directed against vector-derived proteins. Here, we employed immunocytochemistry to assess the immune response to intravitreously injected DeltaE1-Ad encoding the lacZ gene or various neurotrophins (NTs). beta-Galactosidase was expressed by retinal cells for up to 4 weeks. Following intravitreal inoculation of AdCMV-lacZ, microglial and T cells were detected with a panel of antibodies in the retinal cell layers after 2 days (D2). The inflammatory response reached a maximum between D7 and D14. In contrast, no immune response was seen following injection of Ad encoding NTs. Yet, like with Ad-CMV-lacZ, their expression was also limited to approximately 4 weeks. Thus, beta-galactosidase seems to trigger a host immune response following intravitreal adenoviral lacZ gene transfer, but immune responses are not the cause of limited NT transgene expression from the CMV promoter in the inner retina.

In vivo adenoviral transduction of the neonatal rat cochlea and middle ear.

Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a beta-galactosidase (beta-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of beta-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of beta-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of beta-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express beta-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.

Adenovirus-mediated expression of ciliary neurotrophic factor (CNTF) rescues axotomized rat retinal ganglion cells but does not support axonal regeneration in vivo.

Rat optic nerve (ON) transection leads to mainly apoptotic cell death of about 85% of the retinal ganglion cell (RGC) population within 14 days after lesion. In the present study, we tested the effect of adenovirally delivered CNTF (Ad-CNTF) on survival and regeneration of axotomized adult RGCs in vivo. Single intravitreal Ad-CNTF injection led to stable CNTF mRNA and protein expression for at least 18 days and significantly enhanced RGC survival by 155% when compared to control animals 14 days after ON transection. ON stump application of Ad-CNTF also resulted in an increased number of surviving RGCs. Ad-CNTF injection led to better preservation of intraretinal RGC axons but did not support regeneration of axotomized RGCs into a peripheral nerve graft. Thus, adenovirus-mediated neurotrophic factor supply is a suitable approach for reducing axotomy-induced RGC death in vivo and may constitute a relevant strategy for clinical treatment of traumatic brain injury.

Protection by synergistic effects of adenovirus-mediated X-chromosome-linked inhibitor of apoptosis and glial cell line-derived neurotrophic factor gene transfer in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical, and neuropathological changes reminiscent of those occurring in idiopathic Parkinson's disease (PD). Here we show that a peptide caspase inhibitor, N-benzyloxy-carbonyl-val-ala-asp-fluoromethyl ketone, or adenoviral gene transfer (AdV) of a protein caspase inhibitor, X-chromosome-linked inhibitor of apoptosis (XIAP), prevent cell death of dopaminergic substantia nigra pars compacta (SNpc) neurons induced by MPTP or its active metabolite 1-methyl-4-phenylpyridinium in vitro and in vivo. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites does not differ between AdV-XIAP- and control vector-treated mice, this protection is not associated with a preservation of nigrostriatal terminals. In contrast, the combination of adenoviral gene transfer of XIAP and of the glial cell line-derived neurotrophic factor to the striatum provides synergistic effects, rescuing dopaminergic SNpc neurons from cell death and maintaining their nigrostriatal terminals. These data suggest that a combination of a caspase inhibitor, which blocks death, and a neurotrophic factor, which promotes the specific function of the rescued neurons, may be a promising strategy for the treatment of PD.

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.

Extra neurofilament NF-L subunits rescue motor neuron disease caused by overexpression of the human NF-H gene in mice.

Previous studies demonstrated that transgenic mice overexpressing human neurofilament heavy (hNF-H) protein develop a progressive motor neuron disease characterized by the perikaryal accumulations of neurofilaments resembling those found in amyotrophic lateral sclerosis (ALS). To further investigate this neurofilament-induced pathology, we generated transgenic mice expressing, solely or concomitantly, the hNF-H and the human neurofilament light (hNF-L) proteins. We report here that the motor neuron disease caused by excess hNF-H proteins can be rescued by overexpression of hNF-L in a dosage-dependent fashion. In hNF-H transgenic mice, the additional hNF-L led to reduction of perikaryal swellings, relief of axonal transport defect and restoration of axonal radial growth. A gene delivery approach based on recombinant adenoviruses bearing the hNF-L gene also demonstrated the possibility to reduce perikaryal swellings after their formation in adult mice. The finding that extra NF-L can protect against NF-H-mediated pathogenesis is of potential importance for ALS, particularly for cases with NF-H abnormalities.

Excess target-derived brain-derived neurotrophic factor preserves the transient uncrossed retinal projection to the superior colliculus.

During early postnatal development, a widespread ipsilateral projection to the superior colliculus is secondarily restricted to a small topographically defined region by elimination of ipsilaterally projecting retinal ganglion cells. Brain-derived neurotrophic factor (BDNF) has been proposed as the target-derived neurotrophic factor for retinal ganglion cells in several studies. Here we investigated the long-term effects of excess BDNF in the retinal ganglion cell target on naturally occurring retinal ganglion cell (RGC) elimination and on the restriction of the ipsilateral projection. To this end, sustained overexpression of BDNF was achieved in the postnatal superior colliculus using an adenoviral vector. While the total number of retinal ganglion cells in the adenovirus-BDNF treated animals was unchanged, a much higher proportion of RGCs retained a projection to the ipsilateral superior colliculus. We conclude that an excess of target-derived BDNF does not reduce the net amount of naturally occurring cell death in the retino-collicular system, but prevents the negative selection of retinal ganglion cells making inappropriate topographic connections.

Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse.

Several mutations causing both photoreceptor degeneration and malfunction have been identified in humans and animals. Although intraocular injection of trophic factors has been shown to reduce photoreceptor death in a few conditions of rapid photoreceptor loss, it is unclear whether long-term beneficial changes in functional properties of affected photoreceptors can be obtained by treatment with these factors. The rds/rds mouse is a spontaneous mutant bearing a null mutation in the rds/peripherin gene, which is linked to many forms of dominant retinal degenerations in humans. Here, we report that intraocular adenovirus-mediated gene transfer of ciliary neurotrophic factor (CNTF) in this mutant reduces photoreceptor loss, causes a significant increase in the length of photoreceptor segments, and results in a redistribution and an increase in the retinal content of the photopigment rhodopsin. These effects are accompanied by a significant increase in the amplitude of the a- and b-waves of the scotopic electroretinogram. These results suggest that continuous administration of CNTF could potentially be useful for the treatment of some forms of retinal degeneration.

Short communication: protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo.

Following intraorbital transection of the optic nerve (ON) in rats, more than 80% of the retinal ganglion cell (RGC) population die by apoptosis within 14 days. Repeated intraocular injection of brain-derived neurotrophic factor (BDNF) has been efficient in enhancing RGC survival following ON axotomy. The present study was designed to define a potential survival-promoting effect of adenovirally administered BDNF on axotomized RGCs. A single injection of an adenoviral vector expressing the human BDNF gene from a CMV promoter/enhancer (Ad-BDNF) enhanced RGC survival 14 days after axotomy by 40.3%. Moreover, a combinatory treatment regimen consisting of intraocular Ad-BDNF administration and systemic application of the free radical scavenger, N-tert-butyl-(2-sulphophenyl)-nitrone (S-PBN), enhanced RGC survival by 63.0%. Our data demonstrate that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced RGC death. Further, as shown for S-PBN, therapeutic regimens that combine local virus-mediated gene delivery with systemic administration of protective compounds, may offer promising strategies for future treatment also in human neurodegenerative conditions.

Adenoviral gene transfer of ciliary neurotrophic factor and brain-derived neurotrophic factor leads to long-term survival of axotomized motor neurons.

The neurotrophic factors ciliary neurotrophic factor and brain-derived neurotrophic factor can prevent motor neuron cell death during development and after nerve lesion in neonatal rodents. However, local and systemic application of these factors to newborn rats with damaged motor nerves rescues motor neurons only transiently during the first two weeks after axotomy. In order to test the effect of continuous delivery of these factors, the effect of localized injection of CNTF- or BDNF-transducing recombinant adenoviruses into the lesioned nerves was investigated. Under such conditions, survival of axotomized motor neurons is maintained for at least 5 weeks. This way of delivery corresponds to the physiological situation in adult rodents, under which endogenous CNTF is present in the cytosol of Schwann cells and BDNF expression is upregulated after nerve lesion, making these factors available to the damaged motor neurons. Recent results show that overexpression of muscle-derived neurotrophin-3 prevents degeneration of axons and motor endplates, but has only little effect on the number of motor neuron cell bodies in a murine animal model of motor neuron disease. Therefore, techniques suitable for tonic exposure to both nerve- and muscle-derived neurotrophic factors may have implications for the design of future therapeutic strategies against human motor neuron disease.

Inflammatory damage following first-generation replication-defective adenovirus controlled by anti-LFA-1.

First-generation replication-defective adenoviruses have been reported to lead to transient reporter gene expression due to a specific immune reaction involving T and B lymphocytes. Some recent reports have also demonstrated the presence of a nonspecific inflammatory reaction involving macrophages and neutrophils after both intramuscular injections and viral vectors transduction. To further investigate this nonspecific inflammatory reaction, deltaE1/E3a adenoviruses were injected intramuscularly in immunocompetent mice. Some of these mice were treated with anti-LFA-1. The adenovirus-injected muscles showed abundant CD4+, CD8+, LFA-1+, and Mac-1+ cell infiltration 3 days after the deltaE1/E3a injection. The anti-LFA-1 monoclonal antibody was able to block the nonspecific inflammatory damage due mostly to neutrophils and macrophages. The anti-LFA-1 did not produce this effect by reducing the muscle infiltration by LFA-1+ cells. It may instead have blocked the direct interaction between LFA-1 and ICAM-1 thus preventing the damage produced by the respiratory burst of neutrophils. Blocking the resulting damage of this inflammatory reaction with anti-LFA-1 in animals also treated with FK506, a powerful immunosuppressant for gene therapy, largely increased the long-term transgene expression compared with mice only treated with FK506.

Adenovirus-mediated gene transfer of ciliary neurotrophic factor can prevent photoreceptor degeneration in the retinal degeneration (rd) mouse.

Mutations in the beta-subunit of the cGMP phosphodiesterase gene (betaPDE) can cause photoreceptor degeneration leading to blindness in humans, dogs, and mice. Since intravitreal administration of trophic factors has been shown to reduce photoreceptor death in the Royal College of Surgeons rat and the light overexposure models of photoreceptor degeneration, we have tested the possibility of rescuing photoreceptors in the rd mouse using ciliary neurotrophic factor (CNTF). Following intravitreal injection of an adenoviral vector encoding a ngf/cntf fusion gene into one eye of rd/rd mice, many strong CNTF-immunoreactive profiles are detected in various cell types of the injected eyes. Semiquantitative analysis of the corresponding retinae reveals that the photoreceptor-containing layer (outer nuclear layer, or ONL) retains significantly more rows of photoreceptor nuclei than that of eyes treated with a control (LacZ) vector, or untreated, for at least 18 days after vector administration (the longest survival time analyzed). A smaller, but significant, protective effect was also seen 9 days after intravitreal injection of recombinant CNTF. Because apoptotic cell death has been shown to be the common terminal fate of photoreceptors in the rd mouse and many other animal models of retinitis pigmentosa, these results suggest the possibility that administration of neurotrophic factors may prove beneficial in reducing photoreceptor loss in multiple forms of retinitis pigmentosa.

Rapid gene transfer into cultured hippocampal neurons and acute hippocampal slices using adenoviral vectors.

Primary cultures of hippocampal neurons were infected with an adenovirus coding for beta-galactosidase. Expression could be detected as early as 4 h after infection and steadily increased to high levels at 24 h without evidence for a functional impairment of the infected neurons. Similarly, adenovirus-mediated gene transfer into acute hippocampal slices was detectable 4 h after infection and could be localized to discrete areas of the CA1 region by microinjection of the virus stock solution. Infected slices were still suitable for electrophysiological experiments.

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Adenovirus-mediated gene transfer to retinal ganglion cells.

PURPOSE: To evaluate the capacity of a replication-defective adenoviral vector to transport retrogradely from the superior colliculus to the nuclei of retinal ganglion cells and to express in these cells vector-encoded transgene. METHODS: A replication-deficient adenovirus encoding an expression cassette for the Escherichia coli gene lacZ was injected into the right superior colliculus of mice. Brain sections and both eyes were tested histochemically and/or immunohistochemically for E. coli beta-galactosidase (LacZ) activity at 3, 7, 14, and 30 days after injection. RESULTS: lacZ expression was detected in the retinal ganglion cells of the contralateral eye at 7, 14, and 30 days after injection, but no expression was observed in the retina at 3 days after injection. No signs of retinal pathology was observed histologically. LacZ-positive cells also were found in other afferent systems to the superior colliculus, such as the reticular formation, layer V of the ipsilateral visual cortex, and pars reticulata of the ipsilateral substantia nigra. CONCLUSIONS: Injection of an adenovirus vector into the superior colliculus is an effective means to transfer and express a gene in retinal ganglion cells while avoiding damage to the eye tissues; thus, it represents a potentially useful tool to manipulate gene expression selectively in the retina.

Prevention of immune reactions triggered by first-generation adenoviral vectors by monoclonal antibodies and CTLA4Ig.

The therapeutic potential of adenovirus-mediated gene transfer using first-generation vectors is severely limited by the fact that only transient expression is achievable in immunocompetent animals. The loss in transgene expression can be attributed at least in part to the appearance of detrimental immune responses directed toward vector and/or transgene-encoded determinants. FK506 and cyclosporin A both reduced these immune responses. These immunosuppressants, however, may induce many severe side effects during prolonged use. An alternative strategy has been developed to overcome these problems following in vivo transfection of muscles of adult immunocompetent mice with a delta E1/E3a adenoviral vector encoding a beta-galactosidase (beta-Gal) expression cassette. YTS 177 (an anti-CD4 monoclonal antibody) as well as CTLA4Ig, a recombinant protein, partially controlled the immune responses. They were indeed able to reduce the muscle infiltration by CD4+ and CD8+ cells but they failed to repress the humoral response. Co-administration of YTS 191 (an anti-CD4), YTS 169 (an anti-CD8), and TIB 213 (an anti-CD11a) was, however, very efficient in blocking both cellular and humoral immune reactions. This combination of monoclonal antibodies allowed strong and stable transgene expression over 1 month. These data underline the potential of monoclonal antibodies as immunosuppressive adjunct treatment for adenovirus-mediated gene transfer.

Myoblast transplantation in monkeys: control of immune response by FK506.

Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-gal) gene. These myoblasts were then transplanted to 14 different monkeys, 6 of which were immunosuppressed with FK506. Without immunosuppression, only a few myoblasts and myotubes expressing beta-gal were observed 1 week after the transplantation, but no cells expressing beta-gal were observed after 4 weeks. This result was attributed to immune responses since infiltration by CD4+ or CD8+ lymphocytes was abundant 1 week after transplantation but not after 4 weeks. The expression of interleukin 6 (IL-6), interleukin 2 (IL-2), granulocyte/macrophage colony stimulating factor (GM-CSF), transforming growth factor-beta (TGF-beta) and granzyme B mRNAs was increased in the myoblast-injected muscle indicating that the infiltrating lymphocytes were activated. Moreover, antibodies against the donor myoblasts were detected in 3 out of 6 cases. When the monkeys were immunosuppressed with FK506, muscle fibers expressing beta-galactosidase (beta-gal) were present 1, 4 and 12 weeks after the transplantation. There was neither significant infiltration by CD4 or CD8 lymphocytes, nor antibodies detected. The mRNA expression of most cytokines was significantly reduced as compared to the nonimmunosuppressed monkeys. These results indicate that FK506 is effective in controlling short-term immune reactions following myoblast transplantation in monkeys and suggest that it may prove useful for myoblast transplantation in Duchenne Muscular Dystrophy patients.