Development of novel cell surface CD34-targeted recombinant adenoassociated virus vectors for gene therapy

Recombinant adenoassociated virus (rAAV) type 2 vectors have been used to transduce a wide variety of cell types, including hematopoietic progenitor cells. For in vivo gene transfer, it is desirable to have an rAAV vector that specifically transduces selected target cells. As a first step toward generating an rAAV vector capable of targeting delivery in vivo, we have engineered a chimeric protein combining the AAV capsid protein and the variable region of a single-chain antibody against human CD34 molecules, a cell surface marker for hematopoietic stem/progenitor cells. Inclusion of the chimeric CD34 single-chain antibody-AAV capsid proteins within an rAAV virion significantly increased the preferential infectivity of rAAV for the CD34+ human myoleukemia cell line KG-1, which is normally refractory to rAAV transduction. Antibodies against the single-chain antibody and the CD34 protein blocked this transduction. This chimeric vector represents a significant improvement in the host range of rAAV and the first step toward specific gene delivery by rAAV vectors to cells of choice, in this case, hematopoietic progenitor cells, for the treatment of human disease.     

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.     

Transient inhibition of CD28 and CD40 ligand interactions prolongs adenovirus-mediated transgene expression in the lung and facilitates expression after secondary vector administration

Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced beta-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.     

Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.     

Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells

We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4+ T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4+ peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.     

Lipofectamine and related cationic lipids strongly improve adenoviral infection efficiency of primitive human hematopoietic cells

Adenoviral vectors have the potential to infect a large number of cell types including quiescent cells. Their use in hematopoietic cells is limited by the episomal form of their DNA, leading to transgene loss in the progeny cells. However, the use of this vector may be interesting for short-term in vitro modifications of primitive human hematopoietic cells. Therefore, we have investigated the ability of adenovirus to transduce cord blood CD34+ cells. Several promoters were tested using the lacZ reporter gene. The PGK and CMV promoters induced transgene expression in 18-25% of the cells, whereas the HTLV-I and especially the RSV promoter were almost inactive. To improve infection efficiency, adenovirus was complexed with cationic lipids. Lipofectamine, Cellfectin, and RPR120535b, but not Lipofectin, Lipofectace, or DOTAP, markedly improved transgene expression in CD34+ cells (from 19 to 35%). Lipofectamine strongly enhanced infection efficiency of the poorly infectable primitive CD34+CD38low cells (from 11 to 28%) whereas the more mature CD34+CD38+ cells were only slightly affected (from 24 to 31%). Lipofectamine tripled the infection of CFU-GMs and LTC-ICs derived from the CD34+CD38low cell fraction (from 4 to 12% and from 5 to 16%, respectively) and doubled that of BFU-Es (from 13 to 26%). We conclude that cationic lipids can markedly increase the efficiency of adenovirus-mediated gene transfer into primitive hematopoietic cells.     

Combinatorial blockade of calcineurin and CD28 signaling facilitates primary and secondary therapeutic gene transfer by adenovirus vectors in dystrophic (mdx) mouse muscles

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.     

Peripheral expansion of pre-existing mature T cells is an important means of CD4+ T-cell regeneration HIV-infected adults

The CD4+ T-cell pool in HIV-infected patients is in a constant state of flux as CD4+ T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+ T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+ T-cell population relative to the CD4/CD45RO+ population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+ T cells persist in vivo for weeks to months and that the CD4+ T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.     

Efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: establishment of a glial cell line with a functional CD4 receptor

Adeno associated virus (AAV) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. We have investigated the potential for using AAV as a vector for gene transfer into glial cells of the human fetal nervous system. Recombinant AAV vectors expression either the reporter gene beta-galactosidase or a human CD4 receptor were able to transduce both primary glial cells of the human fetal nervous system and an SV40 immortalized human fetal glial cell line (SVG). No difference in transduction efficiency was observed between the primary cells and the cell line which in both cases was as high as 95%. Stable transfectants of the glial cell line expressing the CD4 receptor were selected. An SVG/CD4 expressing line was then established. The presence of the CD4 receptor was confirmed by immunohistochemistry, Westerm immuno-blotting and flow cytometric analysis. The CD4 receptor was shown to be functional by infection of the SVG/CD4 cell line with the human immunodeficiency virus (HIV). Upon infection, the SVG/CD4 cells produced 20-fold higher levels of the HIV intracellular core antigen P24 than the CD4 negative parental cells and in addition formed syncytia. The use of AAV vectors should prove useful in biological investigations of human glial cells and offers promise as a means of ex vivo and in vivo gene delivery.     

Induction of specific T-cell tolerance by adenovirus-transfected, Fas ligand-producing antigen presenting cells

A major problem associated with adenovirus gene therapy is the T cell-mediated immune response, which is elicited by inoculation of the adenovirus vector and leads to rapid clearance of the virus and loss of transgene expression. In this study, the immune response to adenovirus was prevented by induction of specific T-cell tolerance by pretreatment with adenovirus-infected antigen-presenting cells (APC) that express Fas ligand. Compared with control-treated mice, the tolerized mice showed prolonged expression of lacZ upon administration of AdCMVlacZ 1 week after tolerance induction. In contrast to the control mice, the tolerized mice did not display proliferation of CD3+ T cells in the spleen in response to AdCMVlacZ. Tolerance induction also was indicated by the lower production of interferon-gamma and interleukin-2 by peripheral T cells isolated from AdCMVlacZ-challenged tolerized mice than by AdCMVlacZ-challenged control-treated mice. The T-cell tolerance was specific for the adenovirus as the T-cell responses to irrelative murine cytomegalovirus remained unimpaired. Our results indicate that adenovirus-specific T-cell tolerance can be induced by APCs that coexpress Fas ligand and adenovirus antigens. We propose that this new strategy can be used to induce tolerance to adenovirus vector gene therapy with resultant prolonged expression of the transgene.     

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.     

Use of bicistronic retroviral vectors encoding the LacZ gene together with a gene of interest: a method to select producer cells and follow transduced target cells

The coordinate expression of a marker gene and a therapeutic gene in one retroviral vector has considerable advantages. High-titer producer lines can potentially be selected on the basis of marker gene expression, and the expression of transduced genes in target cells can readily be followed. Moreover, target cells with stable high expression can be selected before use in therapeutic protocols or research questions. We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector. We used the LacZ gene as the marker gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of interest. Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, MFG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression. These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, and were capable of efficiently transducing Jurkat T cells and T-cell clones. When MFG-IL-7-IRES-LacZ-transduced Jurkat T cells were sorted on the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found. This demonstrates that selection of the LacZ marker gene also selects for cells that express the gene of interest at high levels. Moreover, T cells transduced with the dn tyrosine kinases and selected on the basis of LacZ expression showed functional alterations after T-cell receptor stimulation, demonstrating that retrovirally transduced signaling molecules can alter the function of T cells.     

Liver-directed gene transfer vectors

The ultimate goal of liver-directed gene therapy for genetic diseases is the stable expression of a therapeutic transgene in a significant proportion of hepatocytes. This article considers the various liver-directed gene transfer procedures studied so far. Performances and limitations of currently available vector systems are discussed with respect to their clinical relevance. Although some improvements have been reported, naked DNA and nonviral gene transfer vectors induce transient expression in only a limited number of cells. Clinical applications of retrovirus-mediated gene transfer are hampered by the need to induce hepatocyte division. First-generation adenovirus vectors are highly efficient; however, they induce an immune response leading to the rapid rejection of transduced cells. Promising new vector systems have emerged, including gutless adenovirus vectors, adeno-associated vectors, and lentivirus vectors. However, these systems are still poorly documented and their relevance to liver-directed gene therapy must be confirmed.     

Effect of dexamethasone and corticosterone on activity levels of ATPase, phosphomonoesterases and phosphodiesterase in liver, muscle and testis of post-hatched White Leghorn chicks

We have investigated the feasibility of using high-titer murine leukemia virus-based retroviral vectors to deliver exogenous genes to naive and chronically inflamed knee joints of rabbits in vivo. Intraarticular injection of retrovirus encoding beta-galactosidase (beta-gal or lacZ) was found to transduce synoviocytes in both naive and inflamed joints, but a significantly higher number of lacZ+ cells were found in inflamed knees. Using a retrovirus encoding a secretable marker, human growth hormone (hGH), quantitative comparison of ex vivo and in vivo gene delivery methods demonstrated that transgene expression following in vivo gene transfer was at least equivalent to that of the ex vivo method in inflamed knees. In addition, hGH transgene expression was maintained for at least 4 weeks. These experiments suggest that high-titer retroviral vector could be used for efficient in vivo gene transfer to inflamed joints in patients with rheumatoid arthritis (RA).     

Support of the diagnosis of pancreatitis by enzyme tests--old problems, new techniques

In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied, following direct injection in several regions of the central and peripheral nervous system. The distribution of the cells expressing the transgene appears to be determined by natural anatomical boundaries and possibly by the degree of myelinization of a particular brain region. In highly myelinated areas with a compact cellular structure (e.g. the cortex and olfactory bulb) the spread of the viral vector is limited to the region close to the injection needle, while in areas with a laminar structure (e.g. the hippocampus and the eye) more widespread transgene expression is observed. Retrograde transport of the viral vector may serve as an attractive alternative route of transgene delivery. A time course of expression of beta-galactosidase in neural cells in the facial nucleus revealed high expression during the first week after AdLacZ injection. However, a significant decline in transgene expression during the second and third week was observed. This may be caused by an immune response against the transduced cells or by silencing of the cytomegalovirus promoter used to drive transgene expression. Taken together, the data underscore that for each application of adenoviral vectors as gene transfer agents in the nervous system it is important to examine vector spread in and infectability of the neural structure that is subject to genetic modification.