Interleukin-24 enhancing antitumor activity of chimeric oncolytic adenovirus for treating acute promyelocytic leukemia cell.

BACKGROUND: Acute promyelocytic leukaemia (APL) is a clonal disease arising by hematopoietic stem cell (HSC), which characterized by inappropriate proliferation/differentiation or survival of immature myeloid progenitors. Oncolytic adenoviruses have been under widespread investigation as anticancer agents. Recently, our data suggested that tumor cells were cured by AdCN205-IL-24, an adenovirus serotype 5-based conditionally replicating adenovirus expressing IL-24 after infection. METHODS: In this study, we created a novel fiber chimeric oncolytic adenovirus AdCN306-IL-24 that has Ad11 tropism and approved CAR (coxsackie adenovirus receptor, CAR)-independent cell entry, which could allow development of selective cytopathic effects (CPE) in APL cells in vitro. RESULTS: Formidable cytotoxic effect was specifically implemented in APL cells after infection with AdCN306-IL-24. The expression of IL-24 was up-regulated upon treated with accepted tumors. And the vector also induced superior cytolytic effects activity in APL cells by activation of programmed cell death. CONCLUSIONS: Taken together, our data suggested that chimeric oncolytic adenovirus AdCN306-IL-24 could express IL-24 gene, representing a potential therapeutics for acute promyelocytic leukemia.

Temporal dynamics of adenovirus 5 gene expression in normal human cells.

Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR.

Adenovirus Early Proteins and Host Sumoylation.

The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system.

Clinical Alarms in intensive care: implications of alarm fatigue for the safety of patients / Alarmes clínicos em terapia intensiva: implicações da fadiga de alarmes para a segurança do paciente / Alarmas clínicas en terapia intensiva: implicaciones de la fatiga de alarmas para la seguridad del paciente

OBJECTIVES: to identify the number of electro-medical pieces of equipment in a coronary care unit, characterize their types, and analyze implications for the safety of patients from the perspective of alarm fatigue. METHOD: this quantitative, observational, descriptive, non-participatory study was conducted in a coronary care unit of a cardiology hospital with 170 beds. RESULTS: a total of 426 alarms were recorded in 40 hours of observation: 227 were triggered by multi-parametric monitors and 199 were triggered by other equipment (infusion pumps, dialysis pumps, mechanical ventilators, and intra-aortic balloons); that is an average of 10.6 alarms per hour. CONCLUSION: the results reinforce the importance of properly configuring physiological variables, the volume and parameters of alarms of multi-parametric monitors within the routine of intensive care units. The alarms of equipment intended to protect patients have increased noise within the unit, the level of distraction and interruptions in the workflow, leading to a false sense of security. .

OBJETIVOS: identificar o número de alarmes dos equipamentos eletromédicos numa unidade coronariana, caracterizar o tipo e analisar as implicações para a segurança do paciente na perspectiva da fadiga de alarmes. MÉTODO: trata-se de estudo quantitativo observacional descritivo, não participante, desenvolvido numa unidade coronariana de um hospital de cardiologia, com capacidade para 170 leitos. RESULTADOS: registrou-se o total de 426 sinais de alarmes, sendo 227 disparados por monitores multiparamétricos e 199 alarmes disparados por outros equipamentos (bombas infusoras, hemodiálise, ventiladores mecânicos e balão intra-aórtico), nas 40h, numa média total de 10,6 alarmes/hora. CONCLUSÃO: os resultados encontrados reforçam a importância da configuração de variáveis fisiológicas, do volume e dos parâmetros de alarmes dos monitores multiparamétricos à rotina das unidades de terapia intensiva. Os alarmes dos equipamentos destinados a proteger os pacientes têm conduzido ao aumento do ruído na unidade, à fadiga de alarmes, a distrações e interrupções no fluxo de trabalho e à falsa sensação de segurança. .

OBJETIVOS: identificar el número de alarmas de los equipamientos electromédicos en una unidad coronariana, caracterizar el tipo y analizar las implicaciones para la seguridad del paciente en la perspectiva de fatiga de alarmas. MÉTODO: se trata de un estudio cuantitativo, observacional, descriptivo, no participante, desarrollado en una unidad coronariana de un hospital de cardiología, con capacidad de 170 camas. RESULTADOS: se registró un total de 426 señales de alarmas, siendo 227 disparadas por monitores multiparamétricos y 199 disparadas por otros equipamientos (bombas de infusión, hemodiálisis, ventiladores mecánicos y balón intraaórtico), durante 40h, con un promedio total de 10,6 alarmas/hora. CONCLUSIÓN: los resultados encontrados refuerzan la importancia de la configuración de las variables fisiológicas, del volumen y de los parámetros de alarma de los monitores multiparamétricos, a la rutina de las unidades de terapia intensiva. Las alarmas de los equipamientos destinados a proteger a los pacientes, han llevado al aumento del ruido en la unidad, a la fatiga de alarmas, a las distracciones e interrupciones en el flujo de trabajo y a una falsa sensación de seguridad. .

CRM1-dependent transport supports cytoplasmic accumulation of adenoviral early transcripts.

The life cycle of adenoviruses is divided by convention into early and late phases, separated by the onset of viral genome replication. Early events include virus adsorption, transport of the genome into the nucleus, and the expression of early genes. After the onset of viral DNA replication, transcription of the major late transcription unit (MLTU) and thereby synthesis of late proteins is induced. These steps are controlled by an orchestra of regulatory processes and require import of the genome and numerous viral proteins into the nucleus, as well as active transport of viral transcripts and proteins from the nucleus to the cytoplasm. The latter is achieved by exploiting the shuttling functions of cellular transport receptors, which normally stimulate the nuclear export of cellular mRNA and protein cargos. A set of adenoviral early and late proteins contains a leucine-rich nuclear export signal of the HIV-1 Rev type, known to be recognized by the cellular export receptor CRM1. However, a role for CRM1-dependent export in supporting adenoviral replication has not been established. To address this issue in detail, we investigated the impact of two different CRM1 inhibitors on several steps of the adenoviral life cycle. Inhibition of CRM1 led to a reduction in viral early and late gene expression, viral genome replication, and progeny virus production. For the first time, our findings indicate that CRM1-dependent shuttling is required for the efficient export of adenoviral early mRNA.

Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue.

Treatment of invasive adenovirus (Ad) disease in hematopoietic stem cell transplant (SCT) recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs) targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977) and late protein hexon (H-892) were compared in peripheral blood (PB) and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules.

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.

Lentivectors encoding immunosuppressive proteins genetically engineer pancreatic beta-cells to correct diabetes in allogeneic mice.

The effectiveness of genetic engineering with lentivectors to protect transplanted cells from allogeneic rejection was examined using, as a model, type 1 diabetes treatment with beta-cell transplantation, whose widespread use has been limited by the requirement for sustained immunosuppressive treatment to prevent graft rejection. We examined whether lentivectors expressing select immunosuppressive proteins encoded by the adenoviral genome early region 3 (AdE3) would protect transplanted beta-cells from an alloimmune attack. The insulin-producing beta-cell line beta TC-tet (C3HeB/FeJ-derived) was transduced with lentiviruses encoding the AdE3 proteins gp19K and RID alpha/beta. The efficiency of lentiviral transduction of beta TC-tet cells exceeded 85%. Lentivector expression of gp19K decreased surface class I major histocompatibility complex expression by over 90%, whereas RID alpha/beta expression inhibited cytokine-induced Fas upregulation by over 75%. beta TC-tet cells transduced with gp19K and RID alpha/beta lentivectors, but not with a control lentivector, provided prolonged correction of hyperglycemia after transplantation into diabetic BALB/c severe combined immunodeficient mice reconstituted with allogeneic immune effector cells or into diabetic allogeneic BALB/c mice. Thus, genetic engineering of beta-cells using gp19K- and RID alpha/beta-expressing lentiviral vectors may provide an alternative that has the potential to eliminate or reduce treatment with the potent immunosuppressive agents necessary at present for prolonged engraftment with transplanted islets.

Construction of adenovirus type 5 early region 1 and 4 virus mutants.

This chapter describes a novel strategy that simplifies the generation and production of adenovirus type 5 (Ad5) mutants carrying defined mutations in early transcription units 1 (E1) and 4 (E4). The strategy involves three recombinant plasmids containing E1 (pE1-1235), E4 (pE4-1155), or the wild-type genome that lacks a portion of E3 (pH5pg4100). To generate recombinant viruses, mutations are first introduced into pE1- and/or pE4-transfer plasmids by site-directed mutagenesis. The mutagenized constructs are then ligated into plasmid pH5pg4100 containing the Ad backbone by direct cloning. Infectious viral DNAs are released from the recombinant plasmids by PacI-digestion and transfected into the complementing cell lines 293 or W162, and viral progeny are isolated and amplified. The advantages of this strategy are multiple: all cloning steps are carried out in Escherichia coli, and any genetic region of the viral E1 and/or E4 transcription units can be specifically modified or deleted. Moreover, foreign genes can be introduced into the E1 and/or E4 regions, and expression of viral or therapeutic genes can be controlled by cell-type specific and/or inducible promoters.

Positive and negative effects of adenovirus type 5 helper functions on adeno-associated virus type 5 (AAV5) protein accumulation govern AAV5 virus production.

Full replication of adeno-associated virus type 5 (AAV5) is sustained by adenovirus type 5 (Ad5) helper functions E1a, E1b, E2a, E4Orf6, and virus-associated (VA) RNA; however, their combined net enhancement of AAV5 replication was comprised of both positive and negative individual effects. Although Ad5 E4Orf6 was required for AAV5 genomic DNA replication, it also functioned together with E1b to degrade de novo-expressed, preassembled AAV5 capsid proteins and Rep52 in a proteosome-dependent manner. VA RNA enhanced accumulation of AAV5 protein, overcoming the degradative effects of E4Orf6, and was thus required to restore adequate amounts of AAV5 proteins necessary to achieve efficient virus production.

Complex effect of adenovirus early region proteins on innate immune system.

Adenoviruses (Ads) cause acute and persistent infections. The genome of Ads has five early transcription units that are the first viral genes expressed during an active infection. The Early Region 1A (E1A) gene of the adenovirus genome is crucial for adenovirus transformation of the host cell. Ads E1A block some aspects of the innate immune system to enable viruses to invade the host cell. E1A suppresses nitric oxide (NO) production through transcriptional control of the inducible NO synthase (iNOS) gene. This inhibition of NO production may enable the virus to persist in human tissue because NO is an antiviral effector of the innate immune system. E1A also blocks secretory leukoprotease inhibitor (SLPI) and elafin/skin-derived antileukoproteinase (SKALP) secretion by alveolar epithelial cells. Recent scientific evidence suggests that SLPI and elafin/SKALP have broad-spectrum antibiotic activities that include bactericidal and antifungal properties. The inhibition of inflammation by Ad early region proteins is complex, as certain early region proteins can promote as well as inhibit inflammation depending on the genetic context of the virus. E1A DNA and protein are frequently detected in the lungs of chronic obstructive pulmonary disease (COPD) patients and it is associated with an increased inflammatory response. E1A enhances intercellular adhesion molecule-1 and interleukin-8 mRNA expression with lipopolysaccharide stimulation. Understanding the roles of the Ad gene products in the induction and inhibition of innate inflammatory functions will help us to clarify the pathogenesis of the chronic respiratory illness including COPD.

Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery.

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

Complete sequence and organization of the human adenovirus serotype 46 genome.

Out of 51 human adenoviral serotypes recognized to date, 32 of them belong to species D. Members of species D adenoviruses are commonly isolated from immune suppressed patients (organ transplant) and patients suffering from AIDS. The role of species D adenoviruses in pathogenesis is currently unclear. To derive new insights into the genetic content and evolution of species D adenoviruses and as a first step towards development of human adenovirus serotype 46 (Ad46) as vector, the complete nucleotide sequence of the virus was determined. The size of the genome is 35,178 bp in length with a G+C content of 56.9%. All the early and late region genes are present in the expected locations of the genome. The deduced amino acid sequences of all late region genes, with the exception of fiber, exhibited high degree of homology with the corresponding proteins of other adenoviruses. The deduced amino acid sequences of early regions E1, E3 and E4 showed a high degree of homology with the corresponding proteins of adenoviruses belonging to species D and less homology with the corresponding proteins of adenoviruses of other species. The homologues of Ad5 E3 region genes encoding 12.5K, gp19K, 10.4K, 14.5K and 14.7K are conserved in the genome of Ad46. However, the E3 region of Ad46 lacks genes encoding 6.7K and adenovirus death protein (ADP) but contains two additional open reading frames with a coding capacity of 433 and 281 amino acids. The fiber protein of Ad46 is 200 amino acids smaller than the fiber protein of Ad5 and contains only 10 pseudo-repeats in the shaft region. To facilitate the manipulation of the genome, the complete genome of Ad46 was cloned into a single bacterial plasmid. Following transfection into E1 complementing cell lines, the virus was recovered demonstrating the feasibility of viral genome manipulation for generation of recombinant viruses.

Adenovirus E1A orchestrates the urokinase-plasminogen activator system and upregulates PAI-2 expression, supporting a tumor suppressor effect.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.

Viral and cellular oncogenes induce rapid mitochondrial translocation of p53 in primary epithelial and endothelial cells early in apoptosis.

In p53-dependent apoptosis in response to genotoxic and hypoxic stress, a fraction of induced wild-type p53 rapidly translocates to mitochondria, triggering a rapid first wave of mitochondrial membrane permeabilization and apoptosis that is later fortified by the transcriptional program of p53. However, whether this direct mitochondrial program also occurs upon oncogenic stress is unknown. In normal cells, oncogenic signals can induce a p53-dependent fail-safe mechanism to counter uncontrolled proliferation by engaging p53-dependent apoptosis. To address whether mitochondrial p53 contributes to oncogene-induced fail-safe apoptosis, p53 translocation was determined in primary human epithelial and endothelial cells overexpressing c-Myc, E1A or E2F1. Serum starvation of these cells, but not of control cells, triggered rapid p53 accumulation at mitochondria, accompanied by cytochrome c and SMAC release and followed by apoptosis. Our data establishes the contribution of the transcription-independent mitochondrial p53 pathway to apoptosis of primary cells in response to deregulated oncogenes.

Induction and inhibition of innate inflammatory responses by adenovirus early region proteins.

First-generation adenovirus (Ad) gene therapy vectors deleted for the E1A, E1B, and E3 regions and carrying foreign genes under the control of strong foreign promoters induce high-level innate inflammatory responses within the first 24 hrs after transduction. Both uptake of the capsid and expression of gene products encoded by the vector contribute to the innate inflammatory response. Natural infections by Ad are frequently asymptomatic, suggesting that Ad has potent methods of inhibiting inflammation. The inability of Ad vectors to counter inflammatory responses suggests that the products of the Ad genes deleted in vector construction play critical roles in inhibiting these responses. Genetic analysis of the roles of Ad early region gene functions in vivo demonstrated that a virus made replication-incompetent by deletion of the preterminal protein gene and deleted for the transcriptional activation function of E1A effectively inhibits the innate inflammatory processes induced by Ad vectors. The mechanism(s) by which the Ad early region proteins inhibit inflammation is complex, as certain early region proteins can promote as well as inhibit inflammation, depending on the genetic context of the virus. Understanding of the roles of the Ad gene products in the induction and inhibition of innate inflammatory functions offers potential for the development of non-inflammatory vectors as well as for understanding of the mechanisms by which inflammation is regulated.

Complete nucleotide sequences and genome organization of four chimpanzee adenoviruses.

The complete nucleotide sequences for four adenoviruses-Simian Adenoviruses 21, 22, 23, and 24, originally isolated from chimpanzees, were determined. The genome organization of the chimpanzee adenoviruses was found to be similar to that of other adenoviruses. The viral gene products of the adenoviruses Simian Adenoviruses 22, 23, and 24 are very closely related to those of the (previously sequenced) chimpanzee adenovirus Simian Adenovirus 25. Simian Adenovirus 21 is most similar to human subgroup B adenoviruses HAdV-3, HAdV-7, and HAdV-35. Analysis of the capsid proteins hexon and fiber of the chimpanzee adenoviruses also supports the placement of Simian Adenovirus 21 in subgroup B and Simian Adenoviruses 22, 23, and 24 in subgroup E.

Porcine adenovirus type 3 E1 transcriptional control region contains a bifunctional regulatory element.

We identified a bifunctional regulatory element located between nt 374 and 431 upstream of TATA box of porcine adenovirus (PAV) 3 E1A promoter. Deletion of the element dramatically reduced the steady-state level of E1A mRNA, but increased that of E1B, which lies immediately downstream of E1A. The mutant virus displayed defective replication at early times of infection, but replicated nearly as efficiently as wild-type PAV-3 at late times of infection. This defect was complemented with coinfecting wild-type virus in a mixed infection. The results indicated that the upstream activation sequences (UAS) of E1A overlap the upstream repression sequences (URS) of E1B, although both transcription units are transcribed from different promoters.

Delivery of GDNF by an E1,E3/E4 deleted adenoviral vector and driven by a GFAP promoter prevents dopaminergic neuron degeneration in a rat model of Parkinson's disease.

A new adenoviral vector (Ad-GFAP-GDNF) (Ad=adenovirus, GFAP=glial fibrillary acidic protein, GDNF=glial cell line-derived neurotrophic factor) was constructed in which (i) the E1,E3/E4 regions of Ad5 were deleted and (ii) the GDNF transgene is driven by the GFAP promoter. We verified, in vitro, that the recombinant GDNF was expressed in primary cultures of astrocytes. In vivo, the Ad-GFAP-GDNF was injected into the striatum of rats 1 week before provoking striatal 6-OHDA lesion. After 1 month, the striatal GDNF levels were 37 pg/microg total protein. This quantity was at least 120-fold higher than in nontransduced striatum or after injection of the empty adenoviral vector. At 3 months after viral injection, GDNF expression decreased, whereas the viral DNA remained unchanged. Furthermore, around 70% of the dopaminergic (DA) neurons were protected from degeneration up to 3 months as compared to about 45% in the control groups. In addition, the amphetamine-induced rotational behavior was decreased. The results obtained in this study on DA neuron protection and rotational behavior are similar to those previously reported using vectors with viral promoters. In addition to these results, we established that a high level of GDNF was present in the striatum and that the period of GDNF expression was prolonged after injection of our adenoviral vector.