Inhibition of pathological brain angiogenesis through systemic delivery of AAV vector expressing soluble FLT1.

The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, for example, age-related macular degeneration. We hypothesized that adeno-associated viral vector (AAV)-mediated sFLT1 expression could be used to inhibit abnormal brain angiogenesis. We tested the anti-angiogenic effect of sFLT1 and the feasibility of using AAV serotype 9 to deliver sFLT1 through intravenous injection (IV) to the brain angiogenic region. AAVs were packaged in AAV serotypes 1 and 2 (stereotactic injection) and 9 (IV injection). Brain angiogenesis was induced in adult mice through stereotactic injection of AAV1-VEGF. AAV2-sFLT02 containing sFLT1 VEGF-binding domain (domain 2) was injected into the brain angiogenic region, and AAV9-sFLT1 was injected into the jugular vein at the time of or 4 weeks after AAV1-VEGF injection. We showed that AAV2-sFLT02 inhibited brain angiogenesis at both time points. IV injection of AAV9-sFLT1 inhibited angiogenesis only when the vector was injected 4 weeks after angiogenic induction. Neither lymphocyte infiltration nor neuron loss was observed in AAV9-sFLT1-treated mice. Our data show that systemically delivered AAV9-sFLT1 inhibits angiogenesis in the mouse brain, which could be utilized to treat brain angiogenic diseases such as brain arteriovenous malformation.

Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress.

OBJECTIVE: To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). METHODS: Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by ß-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. RESULTS: Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H(2)O(2) and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. CONCLUSIONS: AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model.

Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization.

Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae.

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.

Influence of electronic resonances on mode selective excitation with tailored laser pulses.

Femtosecond time-resolved coherent anti-Stokes Raman scattering (fs-CARS) gives access to ultrafast molecular dynamics. However, the gain of the temporal resolution entails a poor spectral resolution due to the inherent spectral width of the femtosecond excitation pulses. Modifications of the phase shape of one of the exciting pulses results in dramatic changes of the mode distribution reflected in coherent anti-Stokes Raman spectra. A feedback-controlled optimization of specific modes making use of phase and/or amplitude modulation of the pump laser pulse is applied to selectively influence the anti-Stokes signal spectrum. The optimization experiments are performed under electronically nonresonant and resonant conditions. The results are compared and the role of electronic resonances is analyzed. It can be clearly demonstrated that these resonances are of importance for a selective excitation by means of phase and amplitude modulation. The mode selective excitation under nonresonant conditions is determined mainly by the variation of the spectral phase of the laser pulse. Here, the modulation of the spectral amplitudes only has little influence on the mode ratios. In contrast to this, the phase as well as amplitude modulation contributes considerably to the control process under resonant conditions. A careful analysis of the experimental results reveals information about the mechanisms of the mode control, which partially involve molecular dynamics in the electronic states.

Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.

Fas ligand/Fas-mediated apoptosis in human coronary artery smooth muscle cells: therapeutic implications of fratricidal mode of action.

OBJECTIVE: This study aimed to determine the mode of action of Fas ligand (FasL)/Fas at mediating apoptosis so as to evaluate the potential of FasL in gene therapy for restenosis. METHODS: Passaged human coronary artery smooth muscle (HCASM) cells were infected with recombinant adenoviral vectors expressing murine FasL. Various parameters of FasL expression and apoptosis were measured using FACS, immunofluorescence, calorimetric, and cytotoxicity assays. RESULTS: Most HCASM cells under normal growth conditions expressed Fas and were shown to be susceptible to membrane bound but not soluble FasL. However, some FasL expressing cells survived for up to 7 days. These surviving cells were observed to be spatially distributed and were not in direct physical contact with each other. Upon examination, it was determined that although the majority of the surviving cells expressed FasL, only 30% expressed both Fas and FasL. These cells were capable of inducing apoptosis of target cells and some were also susceptible to FasL expressing cells, provided that the effector and target cells were in close physical contact. FasL/Fas-mediated apoptosis was inhibited by p35, a baculovirus gene that inhibits caspases. Additionally, in contrast to HCASM cells, neither membrane-bound nor soluble FasL induced apoptosis in coronary artery endothelial cells. CONCLUSIONS: FasL expressing HCASM cells do not undergo FasL/Fas mediated "suicide" but kill neighboring cells bearing Fas in a "fratricidal" manner. A small population of HCASM cells expresses no surface Fas. These results imply that HCASM cells transduced in vivo with FasL may serve as "scavengers" and exert a bystander effect on surrounding cells that may be enhanced by co-expression of p35. As FasL-mediated apoptosis occurs in coronary arterial smooth muscle but not endothelial cells, FasL may also offer an advantage over other genes for use in restenosis since the latter may indiscriminately delay re-endothelialization at the sites of gene.

Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35.

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.

Preirradiation of host (monkey) cells mitigates the effects of UV upon simian virus 40 DNA replication.

We are examining the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences (5 or 10 J/m2) of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV 40 DNA synthesis (incorporation of thymidine into viral DNA) in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication.

Preirradiation of host cells does not alter blockage of simian virus 40 replication forks by pyrimidine dimers.

Do damage-inducible responses in mammalian cells alter the interaction of lesions with replication forks? We have previously demonstrated that preirradiation of the host cell mitigates UV inhibition of SV40 DNA replication; this mitigation can be detected within the first 30 min after the test irradiation. Here we test the hypotheses that this mitigation involves either (1) rapid dimer removal, (2) rapid synthesis of daughter strands past lesions (trans-dimer synthesis), or (3) continued progression of the replication fork beyond a dimer. Cells preirradiated with UV were infected with undamaged SV40, and the effects of UV upon viral DNA synthesis were measured within the first hour after a subsequent test irradiation. In preirradiated cells, as well as in non-preirradiated cells, pyrimidine dimers block elongation of daughter strands; daughter strands grow only to a size equal to the interdimer distance along the parental strands. There is, within this first hour after UV, no evidence for trans-dimer synthesis, nor for more rapid dimer removal either in the bulk of the parental DNA or in molecules in the replication pool. Progression of the replication forks was analyzed by electron microscopy of replicating SV40 molecules. Dimers block replication-fork progression in preirradiated cells to the same extent as in non-preirradiated cells. These experiments argue strongly against the hypotheses that preirradiation of host cells results in either the rapid removal of dimers, trans-dimer synthesis, or continued replication-fork progression beyond dimers.

Ultrafast vibrational dynamics observed in higher electronic excited states of iodine using pump-UV DFWM spectroscopy.

By using a combination of an initial pump pulse and a degenerate four-wave mixing process, we show that an interrogation of the vibrational dynamics occurring in different electronic states of molecules is possible. The technique is applied to iodine. The initial pump pulse is used to populate the B((3)Pi) state of molecular iodine in the gas phase. Now, by using an internal time delay in the DFWM process, which is resonant with the transition between the B state and a higher lying ion-pair state, the vibrational dynamics of the B state and the ion-pair state could be observed. States of even symmetry are investigated, which are accessed by a one photon transition from the B state. By a proper choice of the wavelengths used for the pump and DFWM beams, the dynamics of ion-pair states belonging to two different tiers are monitored.

Vibrational dynamics of excited electronic states of molecular iodine.

Femtosecond degenerate four-wave-mixing spectroscopy following an initial pump laser pulse was used to observe the wave packet dynamics in excited electronic states of gas phase iodine. The focus of the investigation was on the ion pair states belonging to the first tier dissociating into the two ions I-(1S) + I+(3P2). By a proper choice of the wavelengths of the initial pump and degenerate four-wave-mixing pulses, we were able to observe the vibrational dynamics of the B (3)Pi(u) (+) state of molecular iodine as well as the ion pair states accessible from there by a one-photon transition. The method proves to be a valuable tool for exploring higher lying states that cannot be directly accessed from the ground state due to selection rule exclusion or unfavorable Franck-Condon overlap.

Fine-mapping of sequences that suppress splicing in the E3 complex transcription unit of adenovirus.

Complex transcription units encode multiple mRNAs that arise by alternative processing of a common pre-mRNA. Some mechanism must exist that maintains the balance among the various mRNAs. The E3 complex transcription unit of adenovirus encodes four major mRNAs (termed a, c, f, and h) and several minor mRNAs (d, e, i). mRNAs a and c account for about one-half of E3 mRNAs. Using virus deletion mutants we have identified a region (termed Region I) which, when deleted, leads to the formation of doubly spliced mRNAs f and h at the expense of singly spliced mRNAs a and c. Region I consists of at most 129 nucleotides. Deletions as small as 24 nucleotides markedly abolished the splice-suppressing function of Region I. We propose that Region I normally functions to suppress splicing in the E3 pre-mRNA and thereby allow mRNA a and c to be formed in abundance. We have also identified another region, designated as Region IA, that appears to specifically suppress splicing to form the doubly-spliced minor mRNAs d and e. Neither Region I nor Region IA include a conventional splice site or cleavage/polyadenylation site. We speculate that Region I and Region IA probably suppress splicing by binding a trans-acting factor.

Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus-polylysine-DNA complexes.

The E1 deleted adenoviral vectors are efficient at gene transfer to cells in culture or in animals. However, their use is limited because of an immune-mediated loss of transduced cells. This immune response is believed to result from low-level production of viral antigens from these vectors after gene transfer. The early region 4 (E4) of adenovirus produces a number of proteins that play an important role in adenoviral and host gene regulation during infection of mammalian cells. There is interest in developing E4 deficient adenovirus for gene therapy, especially in the context of developing a combined E1/E4 deleted vector. Towards this goal, a method by which to complement and propagate an E4 deficient adenovirus (dl 1014) in the E1 complementing 293 cell line, using a novel and simple rescue technique, has been developed. Purified adenovirus deficient in E4 gene expression (dl 1014) was conjugated to expression plasmids containing the E4-open reading frame 6 gene or complete E4 region to produce adenovirus-polylysine-DNA complexes that were used to transfect 293 cells. The derived virus obtained from this transfection did not replicate on 293 cells but did replicate on W162 cells (E4+) confirming that the virus was indeed deleted for E4. Viral yield was high ranging from 3 x 10(8) to 9 x 10(8) plaque forming units per 10(6) 293 cells. This method has general application to the production of new adenoviral mutants that will be useful for developing second generation adenoviral vectors.

Map of cis-acting sequences that determine alternative pre-mRNA processing in the E3 complex transcription unit of adenovirus.

The E3 complex transcription unit of adenovirus encodes four major mRNAs (a, c, f, and h) and two minor (d and e) mRNAs with overlapping exons, alternative splice sites, and two polyadenylation sites, termed E3A (upstream) and E3B (downstream). mRNAs a and d use the E3A polyadenylation site, and mRNAs c, e, f, and h use the E3B site. We have analyzed virus mutants with deletions throughout the E3 region in order to identify cis-acting sequences that function in E3 pre-mRNA processing. The results presented in this report as well as previous results are summarized as follows. (i) Deletions in the first (5') intron at nucleotides (nt) 372 to 768 in E3 had no effect unless they removed the consensus sequence for the nt 372 5' splice site; however, the overall pattern of E3 mRNAs did not change significantly. (ii) Deletions in region I (nt 1441 to 2044) eliminated mRNAs a and c and resulted in corresponding increases in mRNAs f and h; we propose that region I contains sequences that suppress splicing. (iii) Mutations in region II (nt 2161 to 2243) resulted in nearly exclusive synthesis of mRNA f; this phenotype is understood and is discussed. (iv) Changing the AUUAAA component of the E3A poly(A) addition signal to AAUAAA resulted in increased mRNA a levels, suggesting that the E3A poly(A) addition signal is intrinsically inefficient. (v) Deletions in region III (nt 2488 to 3002) decreased mRNA a levels about two- to threefold and specifically increased mRNA f levels; we suggest that region III facilitates use of the E3A polyadenylation site. (vi) Deletions in region IV (nt 2904 to 3251) increased mRNA a levels about two- to threefold; we suggest that region IV may contain sequences that facilitate use of the E3B polyadenylation site. A map of sequences that determine alternative pre-mRNA processing in region E3 is now nearly complete.

The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.

We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit.

The E3-11.6K protein of adenovirus is an Asn-glycosylated integral membrane protein that localizes to the nuclear membrane.

The 11,600 MW (101 amino acids; 11.6K) protein of adenovirus 2 (Ad2) is a protein of unknown function which is synthesized in low amounts during early stages of infection but in very high amounts at late stages. The 11.6K protein migrates as three major groupings of diffuse bands of ca. 14K, 21K, and 31K on SDS-PAGE, indicating that 11.6K undergoes post-translational modification. We show here that 11.6K is Asn-glycosylated with complex (endo H-resistant) oligosaccharides and that 11.6K is an integral membrane protein. Immunofluorescence indicated that 11.6K initially is associated with the endoplasmic reticulum and Golgi apparatus and that it ultimately localizes to the nuclear membrane. The 11.6K protein is predicted to have a single signal-anchor sequence at residues 41-62 and only one potential Asn-linked glycosylation site at residue 14; thus, 11.6K must be oriented in the membranes with its NH2-terminus in the lumen and its COOH-terminus in the cytoplasm. The signal-anchor and glycosylation features of 11.6K are preserved in Ad2 and Ad5 (group C), and in Ad3 and Ad7 (group B), but the sequence of 11.6K is more diverged among these serotypes than is the sequence of most other adenovirus proteins.

The E3-11.6-kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants.

We have reported that an 11,600-Da nuclear membrane glycoprotein named adenovirus death protein (ADP), encoded by the E3 region, is required for the efficient death (lysis) of adenovirus (Ad)-infected cells. We postulated that ADP mediates the release of virions from cells at the conclusion of replication. Here we provide further characterization of cells infected by adp+ and adp- Ads. Using virus mutants with deletions in the individual E3 genes, we show that only mutants that lack ADP have small plaques that are slow to develop. Mutants in the adp gene replicated as well as wild-type Ad, but the cells lysed much more slowly. Cell lysis and viability were determined by plaque size, cell morphology, trypan blue exclusion, the release of lactate dehydrogenase, and the MTT assay for mitochondrial activity. ADP is required for efficient lysis of human A549, KB, 293, and MCF-7 cells. A549 cells infected with adp+ Ads began to die at 2-3 days postinfection and were dead by 6 days. With adp mutants, > 80% of cells remained viable for 5-6 days; when the medium was changed, > 80% of cells were viable after 7 days and 10-20% after 14 days. When the MTT assay was used, there was an increase in mitochondrial activity, suggesting that Ad infection stimulates respiratory metabolism. Nearly all nuclei from wild-type Adinfected cells lacked DAPI-stained DNA by 7 days, whereas with an adp mutant nearly all nuclei stained brightly after 15 days. Nuclei from adp mutant-infected cells were extremely swollen and full of virus, and appeared to have an intact nuclear membrane. Cells infected with wild-type Ad had many vacuoles and perhaps a disrupted nuclear membrane; they did not display features typical of apoptosis.

Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient inbred strains.

High efficiency gene transfer and gene expression in hepatocytes in vivo can be achieved using recombinant adenoviral vectors. However, the persistence of gene expression in different experimental animal models has been variable. To determine if similar differences could be observed in a single species, persistence of gene expression was studied in inbred strains of mice using a recombinant adenoviral vector that expresses human alpha 1-antitrypsin. Marked variability in the persistence of gene expression ranging from several weeks (C3H/HeJ and Balb/c) to more than 3 months [C57Bl/6, B10.A(2R) and B10.BR] was observed when this vector was transduced in different strains of inbred mice. This variability did not correlate with H-2 type. To evaluate the role of T and B cell immunity in the persistence of gene expression, congenic C3H-scid and Balb/c-scid mice were studied and found to have indefinite gene expression from transduced hepatocytes. These animals unlike their immunocompetent counter-parts were able to undergo secondary transduction of hepatocytes with a different recombinant adenoviral vector. These findings suggest that as yet unidentified genetic loci influence the persistence of adenovirus-mediated hepatic gene expression in vivo, and these effects are mediated at least in part, by the antigen specific immune system.