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The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4(+) and CD8(+) T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells.

Acetylcholine plays an important role in regulation of nervous system development and function. We are developing zebrafish (Danio rerio) as a model system to study the role of specific neuronal nicotinic acetylcholine receptor (nAChR) subtypes in development and the effects of nicotine on the developing vertebrate nervous system. We previously characterized the expression of several zebrafish nAChR subunits. To further develop the zebrafish model, here we report a study on the molecular characterization of two additional nAChR subunit genes, designated chrna6 and chrna4. Both zebrafish nAChRs have a high degree of sequence identity to nAChRs expressed in a variety of mammalian species. Reverse transcription polymerase chain reaction was used to show that both nAChR subunit RNAs were expressed early in zebrafish development, with the chrna4 transcript present at 3 hours postfertilization (hpf) and the chrna6 RNA present at 10 hpf. In situ hybridization was used to localize chrna6 and chrna4 RNA expression in 24, 48, 72, and 96 hpf zebrafish. The chrna6 and chrna4 RNAs were each expressed in a unique pattern, which changed during development. At various ages, chrna6 was expressed in Rohon-Beard sensory neurons, trigeminal ganglion, retina, and the pineal gland. Most notably, chrna6 was expressed in catecholaminergic neurons in the midbrain, but was also present in noncatecholaminergic cells in both midbrain and hindbrain. The expression of chrna6 RNA in catecholaminergic cells supports the use of zebrafish as a valid model system to better understand the molecular basis of cholinergic regulation of dopaminergic signaling and the role of alpha6-containing nAChRs in Parkinson's disease. The most notable chrna4 expression was in neural crest cells at 24 hpf and reticulospinal neurons in hindbrain at 48 hpf. chrna4 RNA exhibited a widespread and robust expression pattern in the midbrain in 72 hpf and 96 hpf zebrafish., (Copyright 2009 Wiley-Liss, Inc.)

Rationale: This review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure., Objectives: This review capitalizes on the authors' collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models., Results: Seven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses., Conclusions: The selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose-response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Cellular immune mechanisms that regulate viral gene expression within infected brain cells remain poorly understood. Previous work has shown that systemic immunization against adenovirus after vector delivery to the brain results in complete loss of brain cells infected by adenoviral vectors. Although T cells play an important role in this process, we demonstrate herein that B cells also significantly regulate transgene expression from the CNS. After the systemic immunization against adenovirus of animals injected via the brain with an adenoviral vector 30 days earlier, we uncovered substantial infiltration by CD19+ B cells of the area of the brain transduced by the virus. This suggests the involvement of B cells in the adaptive immune response-mediated loss of transduced cells from the brain. Confocal analysis of these brains demonstrated physical contacts between transduced brain cells and CD19+ cells. To test the hypothesis that B cells play a causal role in the loss of infected cells from the brain, we demonstrated that animals devoid of B cells were unable to eliminate transgene expression at early time points after immunization. This demonstrates that B cells play a necessary role in the loss of transgene expression at early, but not late, time points postimmunization. Thus, these data have important implications for our understanding of the role of B cells as immune effectors during the immune-mediated clearance of viral infections from the CNS, and also for understanding mechanisms operating in brain autoimmunity, as well as for the potential safety of clinical gene therapy for brain diseases.

The innate immune response, characterized by the rapid induction of proinflammatory genes, plays an important role in immune responses to viral vectors utilized in gene therapy. We demonstrate that several innate proinflammatory mRNAs, i.e., those coding for the interferon (IFN)-regulated proteins interferon regulatory factor 1, 2',5'-oligoadenylate synthetase, and double-stranded-RNA-dependent protein kinase as well as those coding for the chemokines RANTES, IFN-gamma-inducible protein 10, and monocyte chemoattractant protein 1, were all increased in a statistically significant manner in response to 1 x 10(8) IU, but not lower doses, of a first-generation adenovirus injected into the naïve brain. This indicates the presence of a threshold dosage of adenovirus needed to elicit an acute innate inflammatory response.

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

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.

Gene therapy aims to revert diseased phenotypes by the use of both viral and nonviral gene delivery systems. Substantial progress has been made in making gene transfer vehicles more efficient, less toxic, and nonimmunogenic and in allowing long-term transgene expression. One of the key issues in successfully implementing gene therapies in the clinical setting is to be able to regulate gene expression very tightly and consistently as and when it is needed. The regulation ought to be achievable using a compound that should be nontoxic, be able to penetrate into the desired target tissue or organ, and have a half-life of a few hours (as opposed to minutes or days) so that when withdrawn or added (depending on the regulatable system used) gene expression can be turned "on" or "off" quickly and effectively. Also, the genetic switches employed should ideally be nonimmunogenic in the host. The ability to switch transgenes on and off would be of paramount importance not only when the therapy is no longer needed, but also in the case of the development of adverse side effects to the therapy. Many regulatable systems are currently under development and some, i.e., the tetracycline-dependent transcriptional switch, have been used successfully for in vivo preclinical applications. Despite this, there are no examples of switches that have been employed in a human clinical trial. In this review, we aim to highlight the main regulatable systems currently under development, the gene transfer systems employed for their expression, and also the preclinical models in which they have been used successfully. We also discuss the substantial challenges that still remain before these regulatable switches can be employed in the clinical setting.

Glioblastoma multiforme is an intracranial tumor that has very poor prognosis. Patients usually succumb to their disease 6 to 12 months after they are diagnosed despite very aggressive treatment modalities. We tested the efficacy of a potent differentiation and proliferation factor for the professional antigen-presenting dendritic cells (DCs), i.e., Flt3L, for its potential role as a novel therapy for gliomas. We investigated the ability of recombinant adenoviral vectors encoding human soluble Flt3L (hsFlt3L) to improve the survival of Lewis rats bearing intracranial syngeneic CNS-1 gliomas. We show that RAdhsFlt3L can improve survival in a dose-dependent manner. Seventy percent of rats survive when treated with 8 x 10(7) pfu RAdhsFlt3L (P < 0.0005). In addition we demonstrate in both naive Lewis rats and C57BL/6 mice the presence of increased numbers of cells bearing DC markers (OX62 and MHCII, in rats, or CD11C, 33D1, MHCII, and F4/80, but not DEC205, in mice) in sites of brain delivery of RAdhsFlt3L. These results show that expression of hsFlt3L in the brain leads to the presence of cells displaying DC markers. We demonstrate that treatment with hsFlt3L leads to inhibition of tumor growth and significantly increased life span of animals implanted with syngeneic CNS-1 glioma cells. Animals that had survived for long periods, i.e., 6 months, had eliminated the implanted tumors after neuropathological analysis; on the other hand, some of the 3-month survivors still appeared to harbor brain tumors. Our results have profound implications for immune-mediated brain tumor therapy and also suggest the ability to recruit DC-like cells within the brain parenchyma in response to the local expression of Flt3L from adenoviral vectors.

We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.