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SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron, compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC - a difference which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.

BACKGROUNDCytomegalovirus (CMV) is the most common intrauterine infection, leading to infant brain damage. Prognostic assessment of CMV-infected fetuses has remained an ongoing challenge in prenatal care, in the absence of established prenatal biomarkers of congenital CMV (cCMV) infection severity. We aimed to identify prognostic biomarkers of cCMV-related fetal brain injury.METHODSWe performed global proteome analysis of mid-gestation amniotic fluid samples, comparing amniotic fluid of fetuses with severe cCMV with that of asymptomatic CMV-infected fetuses. The levels of selected differentially excreted proteins were further determined by specific immunoassays.RESULTSUsing unbiased proteome analysis in a discovery cohort, we identified amniotic fluid proteins related to inflammation and neurological disease pathways, which demonstrated distinct abundance in fetuses with severe cCMV. Amniotic fluid levels of 2 of these proteins - the immunomodulatory proteins retinoic acid receptor responder 2 (chemerin) and galectin-3-binding protein (Gal-3BP) - were highly predictive of the severity of cCMV in an independent validation cohort, differentiating between fetuses with severe (n = 17) and asymptomatic (n = 26) cCMV, with 100%-93.8% positive predictive value, and 92.9%-92.6% negative predictive value (for chemerin and Gal-3BP, respectively). CONCLUSIONAnalysis of chemerin and Gal-3BP levels in mid-gestation amniotic fluids could be used in the clinical setting to profoundly improve the prognostic assessment of CMV-infected fetuses.FUNDINGIsrael Science Foundation (530/18 and IPMP 3432/19); Research Fund - Hadassah Medical Organization.

The nasal mucosa constitutes the primary entry site for respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the imbalanced innate immune response of end-stage coronavirus disease 2019 (COVID-19) has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here, we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with a rapid increase in tissue-associated viral subgenomic mRNA and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon-stimulated genes, cytokines, and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tracts that are specific to SARS-CoV-2. The studies shed light on the role of the nasal mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCE In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here, we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues infected in parallel with SARS-CoV-2 and influenza virus, we found distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal mucosal infection model can be employed to assess the impact of viral evolutionary changes and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV. IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

Background Trastuzumab is a monoclonal antibody which demonstrates efficacy for HER2 positive breast cancer patients. Recently, an increased incidence of brain metastasis in trastuzumab-treated patients has been reported. The reason for this may be the effectiveness of systemic trastuzumab allowing patients to survive longer thus providing time for brain metastases to develop, along with the lack of penetration of systemic therapies through the blood brain barrier. In recent years, several administration routes to the brain have been evaluated. Albeit advances in the field, there is still a need for improved delivery of therapeutic antibodies to the brain. To address this challenge, we have developed two gene therapy-based methods enabling continuous secretion of active trastuzumab in the brain. Methods We have developed two gene therapy approaches for the delivery of the therapeutic anti-HER2 monoclonal antibody, trastuzumab, to the brain. We utilized the helper dependent adenovirus vector, containing trastuzumab light and heavy chains coding sequences (HDAd-trastuzumab). In the first approach, we used the Transduced Autologous Restorative Gene Therapy (TARGT) platform, in which dermal fibroblasts of human and mouse origin, are ex-vivo transduced with HDAd-trastuzumab vector, rendering continuous secretion of active trastuzumab from the cells locally. These genetically engineered cells were subsequently implanted intracranially to mice, contralateral to HER2 positive breast carcinoma cells inoculation site, enabling continuous secretion of trastuzumab in the brain. In the second approach, we used the same HDAd-trastuzumab viral vector, directly injected intracranially, contralateral to the HER2 positive breast carcinoma cells inoculation site. Both methods enabled therapeutic concentrations of local in-vivo production of active trastuzumab in a mouse model of brain metastatic breast cancer. Results Trastuzumab secreted from the TARGT platform demonstrated in-vitro affinity and immune recruitment activity (ADCC) similar to recombinant trastuzumab (Herceptin, Genentech). When implanted in the brain of HER2 positive tumor-bearing mice, both the TARGT platform of dermal fibroblasts engineered to secrete trastuzumab and direct injection of HDAd-trastuzumab demonstrated remarkable intracranial tumor growth inhibitory effect. Conclusions This work presents two gene therapy approaches for the administration of therapeutic antibodies to the brain. The TARGT platform of dermal fibroblasts engineered to secrete active trastuzumab, and the direct injection of HDAd-trastuzumab viral vector, both rendered continuous in-vivo secretion of active trastuzumab in the brain and demonstrated high efficacy. These two approaches present a proof of concept for promising gene therapy based administration methods for intracranial tumors as well as other brain diseases.

Nasal polyps are three-dimensional structures arising from the mucosa of the upper airway. Due to their complexity, the reliability of single-layer cell cultures and animal systems as research models is limited.To evaluate the feasibility of an ex vivo organ culture of human polyps, preserving tissue structure and function.Nasal polyps were excised during routine endoscopic sinus surgery for chronic rhinosinusitis and polyposis. Fresh tissue samples were used for pathological evaluation and for the preparation of 250-500 µm sections, which were incubated in culture media. Tissue viability was assessed by visualisation of cilia motility, measurement of glucose uptake, and an infectivity assay. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay and real-time polymerase chain reaction before and after the introduction of steroids.Polyp tissue viability was retained for 2-3 days as demonstrated by cilia motility, glucose uptake and preserved cellular composition. Tissue samples maintained their capacity to respond to infection by herpes simplex virus 1 and adenovirus. Introduction of dexamethasone to cultured tissue samples led to suppression of interferon-g production.The ex vivo nasal polyp organ culture reproduces the physiological, metabolic, and cellular features of nasal polyps. Furthermore, it shows a preserved capacity for viral infection and response to drugs. This system is a useful tool for the investigation nasal-polyps and for the development of novel therapies.

We have recently shown that the artemisinin derivative artemisone, which was screened against malaria in human clinical studies, is a potent inhibitor of human cytomegalovirus (HCMV). Here we evaluated the antiviral effect of artemisone when employed in 2-drug combinations with approved and experimental anti-HCMV agents. Using the Chou-Talalay method, we found that in-vitro combination of artemisone with cidofovir, brincidofovir, or with the HCMV UL97 inhibitor maribavir resulted in antiviral synergism and the combination of artemisone with ganciclovir or with the viral terminase inhibitors letermovir and BDCRB resulted in moderate synergism. Importantly, the combination of artemisone with maribavir demonstrated synergistic antiviral activity ex-vivo, in a clinically-relevant multicellular model of human placental tissues maintained in organ culture. Our findings provide the basis for the use of artemisone in synergistically acting drug combinations, to enhance viral control and reduce antiviral drug toxicities.

Human cytomegalovirus (HCMV) is a major cause of disease in immunocompromised individuals and the most common cause of congenital infection and neurosensorial disease. The expanding target populations for HCMV antiviral treatment along with the limitations of the currently available HCMV DNA polymerase inhibitors underscore the need for new antiviral agents with alternative modes of action. The antimalarial artemisinin derivative artesunate was shown to inhibit HCMV in vitro yet has demonstrated limited antiviral efficacy in vivo , prompting our search for more potent anti-HCMV artemisinin derivatives. Here we show that the innovative artemisinin derivative artemisone, which has been screened for its activity against malaria parasites in human clinical studies, is a potent and noncytotoxic inhibitor of HCMV. Artemisone exhibited an antiviral efficacy comparable to that of ganciclovir (50% effective concentration, 1.20 ± 0.46 μM) in human foreskin fibroblasts, with enhanced relative potency in lung fibroblasts and epithelial cells. Significantly, the antiviral efficacy of artemisone was consistently ≥10-fold superior to that of artesunate in all cells. Artemisone effectively inhibited both laboratory-adapted and low-passage-number clinical strains, as well as drug-resistant HCMV strains. By using quantitative viral kinetics and gene expression studies, we show that artemisone is a reversible inhibitor targeting an earlier phase of the viral replication cycle than ganciclovir. Importantly, artemisone most effectively inhibited HCMV infection ex vivo in a clinically relevant multicellular model of integral human placental tissues maintained in organ culture. Our promising findings encourage preclinical and clinical studies of artemisone as a new inhibitor against HCMV.

Protein drugs are currently delivered by bolus injection and although treatment frequently is successful, these methods also have major drawbacks, which call for the development of alternative technologies allowing prolonged delivery of these drugs. We developed a new ex vivo gene therapy platform called Transduced Autologous Restorative Gene Therapy (TARGT) for sustained long term production and secretion of autologous therapeutic proteins. A biopsy of dermal tissue taken from the patient is transduced ex vivo with a viral vector encoding the required gene under a constitutive promoter. Following measurement of protein secretion ex vivo, the transduced dermal tissue is implanted back into the patient, where it secretes the therapeutic protein into the circulation for several months or longer. A major hurdle to this approach is potential immunogenicity of the transduced tissue following implantation. In this paper we describe the preclinical and early clinical development of this technology, which allowed for overcoming these hurdles. To that end, we have used the helper dependent (HD) adenoviral vector with newly designed expression cassette containing genetic elements to optimize transgene expression. Moreover, we have developed procedures for TARGT tissue implantation, with measures to improve engraftment and reduce inflammation and rejection. Implantation of human TARGT to severe combined immune deficient (SCID) mice indicated long-term production of active proteins in the blood. Preliminary results of a clinical trial from two anemic end-stage renal disease patients, implanted with TARGTs expressing the human erythropoietin (EPO) gene, demonstrated prolonged secretion with physiologic blood level of the hormone and hemoglobin maintenance in the desired range, for a period of at least 5 months without exogenous EPO administration. We believe that the TARGT technology has the potential to become a platform for the sustained delivery of therapeutic proteins in various clinical indications.

The initial interplay between human cytomegalovirus (HCMV) and innate tissue response in the human maternal–fetal interface, though crucial for determining the outcome of congenital HCMV infection, has remained unknown. We studied the innate response to HCMV within the milieu of the human decidua, the maternal aspect of the maternal–fetal interface, maintained ex vivo as an integral tissue. HCMV infection triggered a rapid and robust decidual-tissue innate immune response predominated by interferon (IFN)γ and IP-10 induction, dysregulating the decidual cytokine/chemokine environment in a distinctive fashion. The decidual-tissue response was already elicited during viral-tissue contact, and was not affected by neutralizing HCMV antibodies. Of note, IFNγ induction, reflecting immune-cell activation, was distinctive to the maternal decidua, and was not observed in concomitantly-infected placental (fetal) villi. Our studies in a clinically-relevant surrogate human model, provide a novel insight into the first-line decidual tissue response which could affect the outcome of congenital infection.

Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of platelet-derived growth factor receptor alpha (PDGFRα) as a determinant of the developmentally acquired HCMV susceptibility.

NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection.

Human cytomegalovirus (HCMV) is the leading cause of congenital infection and is associated with a wide range of neurodevelopmental disabilities and intrauterine growth restriction. Yet our current understanding of the mechanisms modulating transplacental HCMV transmission is poor. The placenta, given its critical function in protecting the fetus, has evolved effective yet largely uncharacterized innate immune barriers against invading pathogens. Here we show that the intrinsic cellular restriction factor apo lipoprotein B e diting c atalytic subunit-like 3A (APOBEC3A [A3A]) is profoundly upregulated following ex vivo HCMV infection in human decidual tissues—constituting the maternal aspect of the placenta. We directly demonstrated that A3A severely restricted HCMV replication upon controlled overexpression in epithelial cells, acting by a cytidine deamination mechanism to introduce hypermutations into the viral genome. Importantly, we further found that A3 editing of HCMV DNA occurs both ex vivo in HCMV-infected decidual organ cultures and in vivo in amniotic fluid samples obtained during natural congenital infection. Our results reveal a previously unexplored role for A3A as an innate anti-HCMV effector, activated by HCMV infection in the maternal-fetal interface. These findings pave the way to new insights into the potential impact of APOBEC proteins on HCMV pathogenesis. IMPORTANCE In view of the grave outcomes associated with congenital HCMV infection, there is an urgent need to better understand the innate mechanisms acting to limit transplacental viral transmission. Toward this goal, our findings reveal the role of the intrinsic cellular restriction factor A3A (which has never before been studied in the context of HCMV infection and vertical viral transmission) as a potent anti-HCMV innate barrier, activated by HCMV infection in the authentic tissues of the maternal-fetal interface. The detection of naturally occurring hypermutations in clinical amniotic fluid samples of congenitally infected fetuses further supports the idea of the occurrence of A3 editing of the viral genome in the setting of congenital HCMV infection. Given the widely differential tissue distribution characteristics and biological functions of the members of the A3 protein family, our findings should pave the way to future studies examining the potential impact of A3A as well as of other A3s on HCMV pathogenesis.

Herpes simplex virus type 1 (HSV-1) initially infects the skin and subsequently spreads to the nervous system. To investigate and compare HSV-1 mode of propagation in the two clinically relevant tissues, we have established ex vivo infection models, using native tissues of mouse and human skin, as well as mouse brain, maintained in organ cultures. HSV-1, which is naturally restricted to the human, infects and spreads in the mouse and human skin tissues in a similar fashion, thus validating the mouse model. The spread of HSV-1 in the skin was concentric to form typical plaques of limited size, predominantly of cytopathic cells. By contrast, HSV-1 spread in the brain tissue was directed along specific neuronal networks with no apparent cytopathic effect. Two additional differences were noted following infection of the skin and brain tissues. First, only a negligible amount of extracellular progeny virus was produced of the infected brain tissues, while substantial quantity of infectious progeny virus was released to the media of the infected skin. Second, antibodies against HSV-1, added following the infection, effectively restricted viral spread in the skin but have no effect on viral spread in the brain tissue. Taken together, these results reveal that HSV-1 spread within the brain tissue mostly by direct transfer from cell to cell, while in the skin the progeny extracellular virus predominates, thus facilitating the infection to new individuals.

Zika virus (ZIKV) has emerged as a cause of congenital brain anomalies and a range of placenta-related abnormalities, highlighting the need to unveil the modes of maternal-fetal transmission. The most likely route of vertical ZIKV transmission is via the placenta. The earliest events of ZIKV transmission in the maternal decidua, representing the maternal uterine aspect of the chimeric placenta, have remained unexplored. Here, we show that ZIKV replicates in first-trimester human maternal-decidual tissues grown ex vivo as three-dimensional (3D) organ cultures. An efficient viral spread in the decidual tissues was demonstrated by the rapid upsurge and continued increase of tissue-associated ZIKV load and titers of infectious cell-free virus progeny, released from the infected tissues. Notably, maternal decidual tissues obtained at midgestation remained similarly susceptible to ZIKV, whereas fetus-derived chorionic villi demonstrated reduced ZIKV replication with increasing gestational age. A genome-wide transcriptome analysis revealed that ZIKV substantially upregulated the decidual tissue innate immune responses. Further comparison of the innate tissue response patterns following parallel infections with ZIKV and human cytomegalovirus (HCMV) revealed that unlike HCMV, ZIKV did not induce immune cell activation or trafficking responses in the maternal-fetal interface but rather upregulated placental apoptosis and cell death molecular functions. The data identify the maternal uterine aspect of the human placenta as a likely site of ZIKV transmission to the fetus and further reveal distinct patterns of innate tissue responses to ZIKV. Our unique experimental model and findings could further serve to study the initial stages of congenital ZIKV transmission and pathogenesis and evaluate the effect of new therapeutic interventions. IMPORTANCE In view of the rapid spread of the current ZIKV epidemic and the severe manifestations of congenital ZIKV infection, it is crucial to learn the fundamental mechanisms of viral transmission from the mother to the fetus. Our studies of ZIKV infection in the authentic tissues of the human maternal-fetal interface unveil a route of transmission whereby virus originating from the mother could reach the fetal compartment via efficient replication within the maternal decidual aspect of the placenta, coinhabited by maternal and fetal cells. The identified distinct placental tissue innate immune responses and damage pathways could provide a mechanistic basis for some of the placental developmental abnormalities associated with ZIKV infection. The findings in the unique model of the human decidua should pave the way to future studies examining the interaction of ZIKV with decidual immune cells and to evaluation of therapeutic interventions aimed at the earliest stages of transmission.

Herpes simplex virus type 1 (HSV-1) initiates productive infection in mucocutaneous tissues to cause cold sores and establishes latent infection in the trigeminal ganglia. Under certain circumstances, HSV-1 may cause encephalitis. Here, we compared host innate defenses against HSV-1 in the two clinically relevant tissues, skin and brain, using a unique ex vivo system of organ culture. Upon HSV-1 infection and spread, apoptosis induction was observed in the skin, but not in brain tissues. While the two tissues elicited interferon (IFN-β) response upon HSV1 infection, IFN induction was more robust in the skin compared to the brain. Moreover, antiviral response to exogenous IFNβ treatment was much stronger in the skin compared to brain tissues. This observation was not related to the availability of the IFN receptor on cells' surface. Taken together, our study demonstrates differential innate antiviral responses to HSV-1 infection that may be exploited in future development of selective and tissue-specific anti-viral treatments.

Human cytomegalovirus (HCMV) is the leading cause of congenital infection, associated with severe birth defects and intrauterine growth retardation. The mechanism of HCMV transmission via the maternal-fetal interface is largely unknown, and there are no animal models for HCMV. The initial stages of infection are believed to occur in the maternal decidua. Here we employed a novel decidual organ culture, using both clinically derived and laboratory-derived viral strains, for the ex vivo modeling of HCMV transmission in the maternal-fetal interface. Viral spread in the tissue was demonstrated by the progression of infected-cell foci, with a 1.3- to 2-log increase in HCMV DNA and RNA levels between days 2 and 9 postinfection, the expression of immediate-early and late proteins, the appearance of typical histopathological features of natural infection, and dose-dependent inhibition of infection by ganciclovir and acyclovir. HCMV infected a wide range of cells in the decidua, including invasive cytotrophoblasts, macrophages, and endothelial, decidual, and dendritic cells. Cell-to-cell viral spread was revealed by focal extension of infected-cell clusters, inability to recover infectious extracellular virus, and high relative proportions (88 to 93%) of cell-associated viral DNA. Intriguingly, neutralizing HCMV hyperimmune globulins exhibited inhibitory activity against viral spread in the decidua even when added at 24 h postinfection—providing a mechanistic basis for their clinical use in prenatal prevention. The ex vivo -infected decidual cultures offer unique insight into patterns of viral tropism and spread, defining initial stages of congenital HCMV transmission, and can facilitate evaluation of the effects of new antiviral interventions within the maternal-fetal interface milieu.

A novel approach for sustained production of therapeutic proteins is described, using genetic modification of intact autologous micro-organ tissue explants from the subject's own skin. The skin-derived micro-organ can be maintained viable ex vivo for extended periods and is transduced with a transgene encoding a desired therapeutic protein, resulting in protein-secreting micro-organ (biopump (BP)). The daily protein production from each BP is quantified, enabling drug dosing by subcutaneous implantation of the requisite number of BPs into the patient to provide continuous production to the circulation of a known amount of the therapeutic protein. Each implanted BP remains localized and is accessible, to enable removal or ablation if needed. Examples from preclinical and clinical studies are presented, including use of associated virus vector 1 and helper-dependent adenoviral vectors producing BPs to provide long-term sustained secretion of recombinant interferon-α and erythropoietin.

Summary Background Current treatments for nonmelanoma skin cancer include surgery, Mohs micrographic surgery, radiation, cryosurgery, photodynamic therapy, local chemotherapy and application of immunomodulators such as imiquimod. However, all have a 5-year recurrence rate of 1–40%. Gene therapy for the treatment of skin cancers is a promising new approach, as delivery of the vectors to the skin is simple and safety issues can be properly addressed. Objectives To develop an ex-vivo organ culture system for basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) tumours, and to test the feasibility of applying oncolytic viruses to these tumours. Methods We first optimized conditions for the maintenance of BCC and SCC tissues in organ culture, and demonstrated viability of the tissues ex vivo for 3–7 days. The tropism of two potential oncolytic viral vectors, herpes simplex virus type 1 (HSV-1) and adenovirus (AD), was next evaluated. Results Immunohistological analysis revealed that HSV-1 targeted tumour cells that expressed p63 and did not express keratin 15 or keratin 14 markers of keratinocytes. Further examination indicated that uninfected BCC and SCC tissues express two isoforms of p63 mRNA, and HSV-1 infection specifically enhanced expression of the TAp63 isoform. Furthermore, following infection, both HSV-1 and AD induced apoptosis in the BCC and SCC cells as indicated by the induction of activated caspase-3. Conclusions The results indicated a specific pattern of viral tropism to skin cancer cells that are critical for maintenance of the tumour. This new experimental system should aid in the analysis of new therapeutic modalities, such as oncolytic viruses, for future treatment of these skin tumours.

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.

Cancer of the rectum poses a complex therapeutic challenge because of its proximity to adjacent organs and anal sphincters. The addition of radiotherapy before surgical resection has been shown to confer good survival rates while preserving sphincter function. Nevertheless, radiation is associated with significant side effects. On the basis of our previous work showing that herpes simplex virus type-1 (HSV-1) preferentially infects human colon cancer, we set out to examine the oncolytic effect of HSV-1 on orthotopic rectal tumors in mice. Two vectors were compared for oncolytic activity, HSV-1(Gbeta) with wild-type replication and an attenuated HSV-1 vector (HSV-G47Delta). Intratumoral injection of HSV-1(Gbeta) and HSV-G47Delta resulted in a significant reduction or disappearance of the tumors and increased survival of mice. Although the use of HSV-1(Gbeta) was associated with systemic toxicity, HSV-G47Delta appears to possess a selective oncolytic activity. Moreover, infection with HSV-G47Delta resulted in the activation of the double-stranded RNA-dependent protein kinase (PKR) pathway. A significant improvement in viral replication and the antitumor effect was observed when the PKR inhibitor 2-aminopurine was coadministered with HSV-G47Delta to the tumor. In conclusion, the efficacy of local delivery of HSV-G47Delta combined with a specific chemical inhibitor of antiviral activity points to a novel therapeutic modality for rectal cancer and other solid tumors.

The skin is an attractive tissue for gene therapy applications to treat genetic disorders and to express systemically delivered transgenes encoding therapeutic proteins. Understanding the tissue tropism of vectors is a prerequisite for the design of gene therapy trials. Using an ex vivo system of organ culture, we studied factors that determined viral tropism to the epidermal and dermal cells in human and mouse skin. We applied in these studies a lentiviral vector pseudotyped with two glycoproteins that use different cell receptors (vesicular stomatitis virus glycoprotein [VSV-G] and amphotropic murine leukemia virus envelope). The extent of infection with the amphotropic pseudotype was much higher than that of VSV-G, especially at low multiplicities of infection. In contrast, the tropism of these two pseudotypes in skin tissues was similar; at low multiplicities the infection was limited to areas near the basal layer of the epidermis, whereas at high multiplicities the infection extended to the dermal layer. To overcome physical barriers in the skin, the epidermal and dermal layers were separated and infected. Whereas the human epidermis was readily infected, we could not detect infection of stem and early progenitor cells in their niche. In contrast, mouse epidermis was completely resistant to infection. Dermal cells of both species were readily infected with the two pseudotypes. Molecular analysis indicated that infection of mouse epidermal cells was restricted after proviral DNA synthesis and before integration. In conclusion, we show that lentiviral tropism in a solid tissue is dependent on several factors, extra- and intracellular, distinct of the cellular receptors.

Glucagon-like peptide-2 (GLP-2) is a naturally occurring 33 amino acid peptide, with t1/2 of 6-7 min. This peptide is produced by L cells of small intestine and has several important functions, such as increasing intestinal absorption, stimulating intestinal growth, reducing bone breakdown and impacting gastric and intestinal motility. It is well known that individuals with short bowel syndrome benefit from exogenous GLP-2 to improve intestinal absorption.Native peptides have been difficult to develop into drugs because of their short half-life, poor metabolic stability and rapid clearance. Manufactured peptides frequently require structural modification and, long-acting formulations. Moreover, the treatment with such peptides frequently uses high doses, which can lead to off-target effects such as nausea and vomiting in some patients. As an alternative, we developed a novel approach that allows our TARGT™ (Transduced Autologous Restorative Gene Therapy) technology to produce and secrete peptides such as GLP-2.The TARGT™ system is an ex-vivo gene therapy, which we have now demonstrated is capable of providing autologous, continuous peptide therapies, at physiological ranges. Our group previously demonstrated such findings for certain proteins. In one set of experiments described herein, the TARGT™ system consists of several 2 × 30 mm biopsies of dermal tissue (Micro-Organ, MO), extracted under local anesthetic followed by fibroblast transduction with a Helper-Dependent Adenoviral Vector (HDAd) containing a novel GLP-2 gene expression cassette. After culture, and measurement of peptide production, one or more transduced MOs (TARGTs™) are re-implanted into the patient as required to achieve the desired dose. The system allows dose flexibility and the TARGTs™ may be added or removed according to in-vivo secretion levels.Pre-clinical studies, using this novel technology, showed GLP-2 TARGT (TARGTGLP-2) in-vitro secretion levels of tens of micrograms per day. Preliminary data from in-vivo SCID mice experiments confirms that TARGT-secreted human GLP-2 analog reaches the mouse blood stream and exhibits a sustained secretion profile similar to tho se observed and reported with other proteins using the TARGT™ technology (e.g. EPO, IFNα). In addition, in-vivo SCID mice and rat experiments suggest that TARGT™ secreted GLP-2 analog is active, and increases intestinal villous height and intestinal crypt cell proliferation rate.Data obtained so far suggests that TARGTGLP-2 may provide a favorable PK profile and physiologic levels of continuous GLP-2 for extended periods of time, avoiding supra-physiological peak serum concentration and achieving continuous coverage with less exposure compared to exogenous GLP-2 injections. The current results suggest that the TARGT™ platform is a promising novel therapy for short bowel syndrome and potentially other diseases of endogenous protein/peptide deficiency.

The mechanism of herpes simplex virus type 1 (HSV-1) penetration into the brain and its predilection to infect certain neuronal regions is unknown. In order to study HSV-1 neurotropism, an ex vivo system of mice organotypic brain slices was established and the tissue was infected with HSV-1 vectors. Neonate tissues showed restricted infection confined to leptomeningeal, periventricular and cortical brain regions. The hippocampus was the primary parenchymatous structure that was also infected. Infection was localized to early progenitor and ependymal cells. Increasing viral inoculum increased the intensity and enlarged the infected territory, but the distinctive pattern of infection was maintained and differed from that observed with adenovirus and Vaccinia virus. Neonate brain tissues were much more permissive for HSV-1 infection than adult mouse brain tissues. Taken together, these results indicate a complex interaction of HSV-1 with different brain-cell types and provide a useful vehicle to elucidate the mechanisms of viral neurotropism.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role in many ligand/receptors interactions. In the present work, we describe a unique human monoclonal antibody, termed Y1-scFv, that is specific for a sulfated epitope in the platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) for binding to human platelets and thus effectively inhibits platelet aggregation. Limited proteolysis of GPIb molecule, using the endoproteases, mocarhagin and cathepsin G, revealed that a seven amino-acid epitope, Tyr-276 to Glu-282, contains the recognition site for Y1-scFv. This GPIb region contains three sulfated tyrosine residues. Binding studies of Y1-scFv to cells and to synthetic peptides in vitro indicated that of the seven residues comprising the epitope only sulfo-Tyr-276 and adjacent Asp-277 are critical for the interaction. To identify the reciprocal sequences in the antibody that recognize the sulfated epitope, we introduced mutations within the complementary-determining region of the heavy chain (CDR3H) of Y1-scFv (MRAPVI). Arginine residue in the second position was critical for the binding. Moreover, a mutant, containing two sequential arginine residues, in the second and third positions of the CDR3H (MRRPVI), showed a nine-fold increased binding to GPIb. This antibody mutant also demonstrated a significant increase in inhibition of vWF-dependent platelet aggregation and adhesion under flow. In conclusion, this unique antibody and mutants, that recognize a sulfated epitope in GP1b receptor, efficiently inhibited platelet adhesion and aggregation, making it a candidate for a new anti-thrombotic agent.

We undertook a Phase I/II trial in patients with apparent recurrent glioblastoma multiforme (GBM) based on imaging studies to determine the safety and tumor response of repetitive intravenous administration of NDV-HUJ, the oncolytic HUJ strain of Newcastle disease virus. The first part of the study utilized an accelerated intrapatient dose-escalation protocol with one-cycle dosage steps of 0.1, 0.32, 0.93, 5.9, and 11 billion infectious units (BIU) of NDV-HUJ (1 BIU = 1 x 10(9) EID(50) 50% egg infectious dose) followed by three cycles of 55 BIU. Virus was administered by intravenous infusion over 15 min. In the second part, patients received three cycles of 11 BIU. All patients without progressive disease were maintained with two doses of 11 BIU iv weekly. Eleven of the 14 enrolled patients (11-58 years, Karnofsky performance scale 50-90%) received treatment. Toxicity was minimal with Grade I/II constitutional fever being seen in 5 patients. Maximum tolerated dose was not achieved. Anti-NDV hemagglutinin antibodies appeared within 5-29 days. NDV-HUJ was recovered from blood, saliva, and urine samples and one tumor biopsy. One patient achieved a complete response. Intravenous NDV-HUJ is well tolerated. The findings of good tolerability and encouraging responses warrant the continued evaluation of NDV-HUJ in GBM, as well as other cancers.

Understanding the mechanisms of the apoptotic and anti apoptotic processes may lead to a better way to control these cascades. Here we demonstrated for the first time the feasibility to express a short functional peptide in mammalian cells that abrogates the apoptosis cascade through interference with the proteolytic activity of the initiator caspase 9 and the executing caspase 3 enzymes. The expression of a short peptide that includes the pseudo-substrate motif of the apoptosis inhibitor protein P35 (Asp-Gln-Met-Asp) leads to the abrogation of cell death induced through either the mitochondrial or the death receptors pathways. Short open reading frames have been detected in several mammalian mRNAs, primarily upstream of the main long reading frame (uORFs), however, direct evidence for de-novo peptides translation has not been provided. Utilizing biochemical and imaging techniques we demonstrate here that the functional recombinant peptide was localized to the cytpoplasmic fraction of the cell. In conclusion, this work demonstrates that ribosomes recognize short ORFs to translate stable short recombinant peptides in mammalian cells. Expression of these intracellular peptides results in the knock down of apoptotic processes to generate apoptosis resistant stable cells.

Systemic delivery of therapeutic proteins through gene transfer approaches has been carried out mostly by ex vivo transduction of single cells or by direct in vivo injection of an expression vector. In this work an intact miniature biopsy of human dermis (microdermis) is harvested and transduced ex vivo by a viral vector encoding a gene for the therapeutic protein. The microdermis preserves its three-dimensional structure and viability during the ex vivo manipulations. Furthermore, upon transduction with adenoviral and adeno-associated viral vectors the microdermis secretes recombinant human erythropoietin (hEPO). Biochemical analysis of the secreted hEPO showed similarity to the clinically approved recombinant hEPO. Subcutaneous implantation of microdermal hEPO into SCID mice exhibited hEPO secretion in the blood circulation and preserved elevated hematocrit for several months, demonstrating the technology's potential for sustained delivery of protein therapeutics.

Salivary glands are an accessible organ for gene therapy, enabling expression of recombinant proteins for both exocrine and endocrine secretion. Lentivirus-based vectors have many advantages for gene therapy, including their ability to infect nondividing cells and to stably integrate into the host genome, enabling long-term transgene expression without eliciting an inflammatory immune response. In the present study, murine salivary glands were inoculated with feline immunodeficiency virus (FIV)-based lentiviral vectors expressing various reporter genes. Luciferase expression was observed as early as 24 h posttransduction, peaked at 17–21 days, and remained stable for more than 80 days. Staining with X-gal suggested that mucous acinar cells were effectively transduced. FIV vector transduction with the secreted alkaline phosphatase gene increased serum levels in treated animals for up to 45 days, and the FIV vector harboring the interferon- γ (IFN- γ ) expression cassette induced an increase in IFN- γ serum levels as well as in the supernatant of salivary gland explant cultures. These results demonstrate that the transduction of salivary glands with nonprimate lentiviral vectors may provide a novel and highly effective vehicle for long-term endocrine transgene expression.

Although the V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) effectively elicits potent neutralizing antibody responses, the specificity of the antibody response is often restricted to T cell line adapted (TCLA) strains and a small subset of primary isolates, limiting its utility for an AIDS vaccine. In this study, we have compared Env immunogens with substituted V3 regions to combinations of strains from different clades and evaluated their ability to expand the breadth of the neutralizing antibody response. When the V3 region from HIV BaL was substituted for HIV HXB2, an effective neutralizing antibody response against several clade B primary isolates was elicited, but it remained restricted to neutralization of most clade B isolates. In an attempt to expand this response further, a linear epitope recognized by the broadly neutralizing 2F5 antibody was inserted into V3. A V3 2F5 epitope was identified that bound to 2F5 and elicited a potent 2F5 antibody response as an immunogen, but the antisera neutralized only a lab-adapted strain and not primary isolates. In contrast, combinations of Envs from clades A, B, and C, elicited neutralizing antibodies to a more diverse group of primary HIV-1 isolates. These studies suggest that combinations of Env immunogens, despite the limited reactivity of the V3 from each component, can be used to expand the breadth of the neutralizing antibody response.

Background Organ fragments can be cultured for weeks in vitro if they are prepared of microscopic thickness and if the basic organ structure is preserved. Such organ fragments, which we termed micro-organs (MOs), express in culture endogenous tissue-specific gene products. We have exploited this methodology to engineer MOs ex vivo by gene transfer. Methods MOs prepared from spleen, lung, colon and skin were infected using: herpes simplex type-1, adeno virus, vaccinia virus and murine leukemia virus (MuLV), carrying the reporter gene β-galactosidase. Results All four viral vectors infected MOs in culture, with adeno infection giving significantly higher values. After optimization, high levels of expression (>15% positive cells), comparable to those obtained with the adeno construct, were also obtained using the MuLV construct both in vitro and after implantation into syngeneic hosts. After implantation, the engineered tissue was found to remain localized, become vascularized, and to express the transduced gene for several months. Conclusions The system can be used to study interactions between viruses and tissues both ex vivo and in vivo. Furthermore, the approach proposes a novel platform for ex vivo gene therapy. Such engineered structures could be used as autologous biological pumps for continuous secretion in vivo of gene products of clinical importance. Copyright © 2005 John Wiley & Sons, Ltd.

The tropism of human immunodeficiency virus type 1 for chemokine receptors plays an important role in the transmission of AIDS. Although CXCR4-tropic virus is more cytopathic for T cells, CCR5-tropic strains are transmitted more frequently in humans for reasons that are not understood. Phenotypically immature myeloid dendritic cells (mDCs) are preferentially infected by CCR5-tropic virus, in contrast to mature mDCs, which are not susceptible to infection but instead internalize virus into a protected intracellular compartment and enhance the infection of T cells. Here, we define a mechanism to explain preferential transmission of CCR5-tropic viruses based on their interaction with mDCs and sensitivity to neutralizing antibodies. Infected immature mDCs differentiated normally and were found to enhance CCR5-tropic but not CXCR4-tropic virus infection of T cells even in the continuous presence of neutralizing antibodies. Infectious synapses also formed normally in the presence of such antibodies. Infection of immature mDCs by CCR5-tropic virus can therefore establish a pool of infected cells that can efficiently transfer virus at the same time that they protect virus from antibody neutralization. This property of DCs may enhance infection, contribute to immune evasion, and could provide a selective advantage for CCR5-tropic virus transmission.

Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.

The salivary gland has been suggested as an accessible organ for gene transfer to express recombinant proteins locally in the saliva, as well as for secretion to the blood circulation. The aim of this study was to evaluate the efficiency of gene transfer to salivary glands using different viral vectors: adenovirus, vaccinia, herpes simplex type 1 (HSV), and two retroviral vectors (murine leukemia virus (MuLV) and lentivirus). We show, by in situ staining and beta-galactosidase reporter activity assay, that the adenoviral and vaccinia vectors were able to deliver the reporter gene efficiently to acinar and duct cells. The HSV vector was less efficient and infected only the acinar cells. The lentiviral vector infected acinar and duct cells, but at a relatively low efficiency. The MuLV vector did not infect the salivary gland unless cell proliferation was induced. Host immune responses to viral infection, inflammation, apoptosis and lymphocyte infiltration, in the transduced glands, were assessed. The DNA viral vectors induced local lymphocyte infiltration and apoptosis. In contrast, the retroviral vectors did not induce an immune response. Our results describe the outcome of salivary gland infection with each of the five different viral vectors and indicate their advantages and limitations for transferring genes to the salivary glands.

During herpes simplex virus type 1 (HSV-1) lifelong latency in human peripheral sensory ganglia (PSG), only the viral latency-associated transcript (LAT) gene is expressed. This raises the possibility that this gene is involved in establishment of latency of the virus, its maintenance, or reactivation. Here we present data that indicate yet another independent function of the LAT gene: interference with HSV-1 superinfection of latently infected neurons. Neuronal cells stably expressing the LATs are protected from infection with HSV-1, but not from infection by other RNA and DNA viruses, including HSV-2. These unexpected findings may explain the observation that human PSG are latently infected with just a single strain of HSV-1, despite repeated exposure during life to multiple viral strains. Thus, the LATs may protect the latent HSV-1 reservoir from cytopathic superinfections, and at the same time the host is protected at the viral entry site from HSV-1 insults that may eventually cause viral encephalitis.

Background Rheumatoid arthritis (RA) is a symmetric inflammatory polyarthritis associated with high concentrations of pro-inflammatory, cytokines including tumor necrosis factor (TNF)-α. Adalimumab is a monoclonal antibody (mAb) that binds TNF-α, and is widely used to treat RA. Despite its proven clinical efficacy, adalimumab and other therapeutic mAbs have disadvantages, including the requirement for repeated bolus injections and the appearance of treatment limiting anti-drug antibodies. To address these issues, we have developed an innovative ex vivo gene therapy approach, termed transduced autologous restorative gene therapy (TARGT), to produce and secrete adalimumab for the treatment of RA. Methods Helper-dependent (HD) adenovirus vector containing adalimumab light and heavy chain coding sequences was used to transduce microdermal tissues and cells of human and mouse origin ex vivo, rendering sustained secretion of active adalimumab. The genetically engineered tissues were subsequently implanted in a mouse model of RA. Results Transduced human microdermal tissues implanted in SCID mice demonstrated 49 days of secretion of active adalimumab in the blood, at levels of tens of microgram per milliliter. In addition, transduced autologous dermal cells were implanted in the RA mouse model and demonstrated statistically significant amelioration in RA symptoms compared to naive cell implantation and were similar to recombinant adalimumab bolus injections. Conclusions The results of the present study report microdermal tissues engineered to secrete active adalimumab as a proof of concept for sustained secretion of antibody from the novel ex vivo gene therapy TARGT platform. This technology may now be applied to a range of antibodies for the therapy of other diseases.

Despite the considerable clinical impact of congenital human cytomegalovirus (HCMV) infection, the mechanisms of maternal-fetal transmission and the resultant placental and fetal damage are largely unknown. Here, we discuss animal models for the evaluation of CMV vaccines and virus-induced pathology and particularly explore surrogate human models for HCMV transmission and pathogenesis in the maternal-fetal interface. Studies in floating and anchoring placental villi and more recently, ex vivo modeling of HCMV infection in integral human decidual tissues, provide unique insights into patterns of viral tropism, spread, and injury, defining the outcome of congenital infection, and the effect of potential antiviral interventions.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Aims—To test the feasibility of gene transfer into hyaloid blood vessels and into preretinal neovascularisation in a rat model of retinopathy of prematurity (ROP), using diVerent viral vectors. Methods—Newborn rats were exposed to alternating hypoxic and hyperoxic conditions in order to induce ocular neovascularisation (ROP rats). Adenovirus, herpes simplex, vaccinia, and retroviral (MuLV based) vectors, all carrying the ‚ galactosidase (‚-gal) gene, were injected intravitreally on postnatal day 18 (P18). Two sets of controls were also examined: P18 ROP rats injected with saline and P18 rats that were raised in room air before the viral vectors or saline were injected. Two days after injection, the rats were killed, eyes enucleated, and ‚-gal expression was examined by X-gal staining in whole mounts and in histological sections. Results—Intravitreal injection of the adenovirus and vaccinia vectors yielded marked ‚-gal expression in hyaloid blood vessels in the rat ROP model. Retinal expression of ‚-gal with these vectors was limited almost exclusively to the vicinity of the injection site. Injection of herpes simplex yielded a punctuate pattern of ‚-gal expression in the retina but not in blood vessels. No significant ‚-gal expression occurred in rat eyes injected with the retroviral vector. Conclusions—Adenovirus is an eYcient vector for gene transfer into blood vessels in an animal model of ROP. This may be a first step towards utilising gene transfer as a tool for modulating ocular neovascularisation for experimental and therapeutic purposes. (Br J Ophthalmol 2001;85:991‐995)

Several years ago, a field strain retrovirus, avian hemangioma virus (AHV), was isolated from hemangioma tumors in layer hens. Sequence analysis indicated that the AHV genome contains the three prototypic retroviral genes, gag, pol, and env, and is devoid of an oncogene. In cultured endothelial cells, however, AHV induced a significant cytopathic effect through a typical apoptotic cascade. We now demonstrate that AHV also induces cell proliferation and anchorage-independent growth of BSC-1 epithelial cells and NIH-3T3 fibroblasts. This was shown by measurements of (1) cell viability, (2) DNA synthesis, (3) flow cytometry analysis of the cell DNA content, and (4) clonogenic efficiency of the infected cells. Anchorage-independent cell growth was demonstrated by colony formation in soft agar. Moreover, the AHV env gene was cloned into a MuLV-based retroviral vector, and infection of NIH-3T3 cells with this vector induced cell proliferation as well as clonogenic growth. These results suggest that AHV, which is devoid of an oncogene, is a pleiotropic activator capable of inducing either apoptosis or cellular proliferation, depending on the infected cell type.

The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy.

Transcription of herpes simplex virus type 1 (HSV-1) during latency produces two abundant latency-associated transcripts (LATs). We have recently shown, that during HSV-1 latency in mice trigeminal ganglia (TG) LATs are bound to polyribosomes (J Virol, 1997, 71, 2897-2904). In order to study the possible role of this binding in the latency process, we now extend the polyribosomal analysis to brainstem tissues of latently infected mice, that unlike TG do not support viral reactivation. We report here that the relative amounts of the LATs associated with polyribosomes in the brainstems of mice are significantly lower than those present in TG. We therefore show that binding of the 1.5 and 2.0 kilobases LATs to polyribosomes is tissue specific and hypothesize that this association may have a role in the reactivation function of HSV-1.

During herpes simplex virus type 1 (HSV-1) latency, limited viral transcription takes place. This transcription has been linked to the ability of the HSV-1 genome to reactivate and consists of abundant 2.0- and 1.5-kb collinear latency-associated transcripts (LATs), spanned by minor hybridizing RNA (mLAT). The 1.5-kb LAT is derived from the 2.0-kb LAT by splicing, and both transcripts contain two large overlapping open reading frames. The molecular action mechanisms of the latency-associated gene expression are unknown, and no HSV-1 latency-encoded proteins have been convincingly demonstrated. We have cloned the entire latency-associated transcriptionally active HSV-1 DNA fragment (10.4 kb) under control of a constitutive promoter and generated a neuronal cell line (NA4) stably transfected with the viral LAT's region. NA4 cells produced the 2.0- and the 1.5-kb LATs. Northern blotting and reverse transcription-PCR analysis of RNA from NA4 cells and from trigeminal ganglia of mice latently infected with HSV-1 revealed that the two abundant LAT species were present in the polyribosomal RNA fractions. After addition of EDTA, which causes dissociation of mRNA-ribosome complexes, both LATs could be detected only in subpolyribosomal, but not in polyribosomal fractions. These results show that (i) HSV-1 LATs are bound to polyribosomes during latency in vivo, as well as in neuronal cells in vitro, and therefore might be translated, and that (ii) the NA4 cell line is a suitable tool with which to look for HSV-1 latency-encoded gene products.