Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration.

Adeno-associated virus type 2 (AAV2) is the only virus known to integrate into a specific locus in the human genome. The locus, AAVS1, is on the q arm of chromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site-specific integration, that is, the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector that does insert the transgene into AAVS1. One component, rAAVSVAV2, contains the rep gene, driven by the simian virus 40 early promoter rather than the P5 promoter. Thus, the integration enhancer element (IEE) within P5, which greatly enhances site-specific integration, has been deleted. The other component, rAAVP5UF11, contains the P5 IEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS1. We have not detected any clones that have rep inserted anywhere. With the optimal multiplicity of infection and ratio of rAAVSVAV2 and rAAVP5UF11, the transgene integrated specifically at AAVS1 with high efficiency (>60%). Most importantly, the cloned cell lines with the AAVS1 site-specific integrated green fluorescent protein (GFP) were healthy and stably expressed GFP for 35 passages. An AAV vector that would integrate at a specific site with high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site-specific integration gene therapy could provide a novel approach for diseases that need long-term gene expression.

Comparison of air and ground transport of cardiac patients.

PURPOSE: To investigate the outcome of cardiac patients transported by helicopter versus ground ambulance SETTING: A hospital-based helicopter program in southeastern Minnesota METHODS: Retrospective chart review assessing an 18-month period (January 1998 to June 1999). Charts were reviewed for type of cardiac diagnosis, level of pain, treatments en route, time to intervention, and length of stay (LOS). Two-hundred-sixty-six cardiac patients came by helicopter. Of the 86 turndowns, 50 came by ground ambulance; 28 records were recovered in this group. These patients composed the comparison ground group. RESULTS: Prehospital time was less for patients transported by air than ground transports (P <.001). The amount of time from the call for transport until arrival at our hospital was less for helicopter transports (P =.002). Air transports had more patients with reduced chest pain on arrival. Difference in CCU LOS was not significant (P =.94). Air patients spent an average of 2 fewer days in the hospital than did ground patients (P =.036). DISCUSSION: Helicopter transport benefits the cardiac patient with decreased chest pain as a result of more treatments en route; decreased time from the call until arrival, resulting in decreased time to intervention; and shorter prehospital time and hospital stays. CONCLUSION: All of these improved variables relate to salvaged cardiac muscle.

Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors.

Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate-early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57Bl/6 mice. Potentially therapeutic serum levels of hAAT (600 microg/ml) were achieved after portal vein injection of doses of 4 x 10(9) infectious units (IU), a 10-fold lower dose than that required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3 x 10(10) IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.

Recombinant angiostatin prevents retinal neovascularization in a murine proliferative retinopathy model.

Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration.

Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle.

We report here that the DNA-dependent protein kinase (DNA-PK) affects the molecular fate of the recombinant adeno-associated virus (rAAV) genome in skeletal muscle. rAAV-human alpha1-antitrypsin (rAAV-hAAT) vectors were delivered by intramuscular injection to either C57BL/6 (DNA-PKcs(+)) or C57BL/6-SCID [severe combined immunodeficient (SCID), DNA-PKcs(-)] mice. In both strains, high levels of transgene expression were sustained for up to 1 year after a single injection. Southern blot analysis showed that rAAV genomes persisted as linear episomes for more than 1 year in SCID mice, whereas only circular episomal forms were observed in the C57BL/6 strain. These results indicate that DNA-PK is involved in the formation of circular rAAV episomes.

p27kip1-independent cell cycle regulation by MYC.

MYC transcription factors are potent stimulators of cell proliferation. It has been suggested that the CDK-inhibitor p27kip1 is a critical G1 phase cell cycle target of c-MYC. We show here that mouse embryo fibroblasts deficient for both p27kip1 and the related p21cip1 are still responsive to stimulation by c-MYC and can be arrested in G1 by a dominant negative mutant of c-MYC. This growth arrest can be overruled by ectopic expression of E2F or adenovirus E1A, but not by a mutant of E1A defective for binding to retinoblastoma family proteins. We show that fibroblasts with a genetic disruption of all three retinoblastoma family members (pRb, p107 and p130) are unresponsive to a dominant negative c-MYC mutant. These data indicate that p27kip1 is not the only rate limiting cell cycle target of c-MYC and suggest that regulation of E2F is also essential for c-MYC's mitogenic activity.

A genetic screen to identify genes that rescue the slow growth phenotype of c-myc null fibroblasts.

The c-myc gene is frequently over-expressed in human cancers and is involved in regulation of proliferation, differentiation and apoptosis. c-Myc is a transcription factor that acts primarily by regulating the expression of other genes. However, it has been very difficult to identify bona fide c-Myc target genes that explain its diverse biological activities. The recent generation of c-myc deficient Rat1A fibroblasts with a profound and stable growth defect provides a new system to search for genes that can substitute for c-myc in proliferation. In this study, we have attempted to identify genes that rescue the slow growth phenotype of c-myc null cells through introduction of a series of potent cell cycle regulatory genes and several retroviral cDNA expression libraries. None of the candidate genes tested, including SV40 T-antigen and adenovirus E1A, caused reversal of the c-myc null growth defect. Furthermore, extensive screens with high-complexity retroviral cDNA libraries from three different tissue sources revealed that only c-myc and N-myc rescued the c-myc null slow-growth phenotype. Our data support the notion that there are no functional equivalents of the myc family of proto-oncogenes and also suggest that there are no c-Myc-activated genes that alone can substitute for c-Myc in control of cell proliferation.

DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system.

The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288-11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966-7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.

Simplified production of a recombinant human angiostatin derivative that suppresses intracerebral glial tumor growth.

Angiostatin is an endogenous inhibitor of tumor neovascularization that inhibits the proliferation of endothelial cells. Production of sufficient quantities of biologically active angiostatin by the enzymatic cleavage of plasminogen has proven difficult in that it has delayed clinical testing. We have cloned, expressed, and purified a recombinant human angiostatin derivative (K1-3) using a mammalian expression system. Through the addition of a secretory signal and polyhistidine sequence tag, K1-3 can be purified from post-culture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability to bind to lysine-Sepharose. In vitro, recombinant K1-3 significantly suppressed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-competent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011). Growth suppression was accompanied by a 32% decrease (P = 0.01) in tumor neovascularization. This study demonstrates a simple method to produce a biologically active recombinant angiostatin derivative. The ability to suppress intracerebral tumor growth after systemic administration suggests that K1-3 is likely to have therapeutic value in the treatment of malignant glial tumors.

Adeno-associated virus (AAV) site-specific integration: formation of AAV-AAVS1 junctions in an in vitro system.

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.

Recombinant adeno-associated virus-mediated expression of O6-alkylguanine-DNA-alkyltransferase protects human epithelial and hematopoietic cells against chloroethylating agent toxicity.

Recombinant adeno-associated virus (rAAV) encoding the human O6-alkylguanine-DNA-alkyltransferase (hAT) protein and a selectable marker (Neo(r)) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeLa clones were isolated, 9 of which survived for 2-3 months before cell death ensued, presumably owing to the loss of G418 resistance. Northern blot analysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced from this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg protein was expressed relative to untransduced cells (800-900 fmol/mg protein). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold higher than background conferred significant resistance (p < 0.001) to the toxic effects of this drug. Two days following infection of K562 cells with the rAAV vector, immunoblot analysis showed that hAT protein was being produced. Three K562 clones, isolated using G418 selection, were studied in detail and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 cells contain <2 fmol of hAT/mg protein). As with HeLa cells, Northern blot analysis showed the production of an appropriately sized transcript and immunoblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Expression of hAT at levels 800- to 1500-fold higher than background conferred significant resistance (p < 0.001) to the toxic effects of mitozolomide. We have therefore successfully conferred a protective advantage against mitozolomide toxicity to cells by rAAV-mediated hAT expression.

Preventing the misuse of microorganisms: the role of the American Society for Microbiology in protecting against biological weapons.

The American Society for Microbiology's (ASM) involvement with issues surrounding biological weapons began during World War II and continues to the present time. The Public and Scientific Affairs Board (PSAB) of the ASM has played an important role in monitoring and responding to legislative and regulatory issues involving biological weapons. As this review makes apparent, there is no consensus of opinion among scientists on their role in biological defense research, or is it likely that there will ever be complete agreement. There is consensus that steps should be taken to prevent biological warfare and that openness of scientific research and global surveillance of disease outbreaks can significantly increase transparency for detecting development of biological weapons. The ASM recommends increased attention to and efforts directed toward global surveillance of disease outbreaks, not only to aid public health organizations in improving human health, but also to establish baseline data against which unusual disease outbreaks can be assessed. Issues of how best to increase global security and to achieve a scientifically based verification protocol of the Biological Weapons Convention are important and continue to be addressed by the ASM.

Blood usage in rotor-wing transport.

INTRODUCTION: Blood transfusion for hemorrhagic shock is standard therapy. The purpose of this study was to document the development of protocols for administering red blood cells and review the experience with inflight blood transfusions. SETTING: This study was conducted at a hospital-based rotor-wing (RW) program whose service area is a large rural area in southeastern Minnesota. A BK 117 with a flight nurse/flight nurse configuration was used. METHODS: The RW registry was accessed during a 3-year period (August 1993 to July 1996), and 2131 records were reviewed to retrospectively analyze blood use during transport. Blood bank records and protocols also were reviewed. RESULTS: This helicopter program maintains a refrigerator with 4 units of O-negative blood that is kept in the hangar and serviced by the blood bank. Blood was taken on all appropriate transports (91% interfacility, 9% scene), and 94 of 2131 patients (4%) were transfused. Criteria for blood transfusion included Hgb < 10, persistent hypotension after resuscitation, and clinical signs of shock. Medical conditions consisted of trauma (48% of patients), gastrointestinal (GI) bleed (25%), abdominal aortic aneurysm (AAA) (17%), and other (10%). The mean Hgb before and after transport was 8.9 and 10.2, respectively; 38% of the patients received an average of 3 units before transport. Overall, 2 units red blood cells were given in flight and another 12 units during hospitalization. No complications occurred with blood transfusions. CONCLUSIONS: Selected RW patients will benefit from in-flight blood transfusions. Proximity of blood storage to the helicopter is mandatory to avoid delays in transport. A close working relationship with blood bank personnel ensures ready availability of current O-negative blood.

Site-specific integration of adeno-associated virus into an episome with the target locus via a deletion-substitution mechanism.

Five site-specific adeno-associated virus integrants generated in a model system with an Epstein-Barr virus- based shuttle vector have been characterized. The results suggest a deletion-substitution mechanism of recombination.

Role of the adenovirus DNA-binding protein in in vitro adeno-associated virus DNA replication.

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences.

Repression of c-Myc responsive genes in cycling cells causes G1 arrest through reduction of cyclin E/CDK2 kinase activity.

The c-myc gene encodes a sequence-specific DNA binding protein involved in proliferation and oncogenesis. Activation of c-myc expression in quiescent cells is sufficient to mediate cell cycle entry, whereas inhibition of c-myc expression causes cycling cells to withdraw from the cell cycle. To search for components of the cell cycle machinery that are targets of c-Myc, we have made a mutant c-Myc protein, named MadMyc, that actively represses c-myc target genes. Expression of MadMyc in cycling NIH3T3 cells causes a significant accumulation of cells in G1. The MadMyc-induced G1 arrest is rescued by ectopic expression of cyclin E/CDK2 and cyclin D1/ CDK4, but not by Cdc25A, a known cell cycle target of c-Myc. The MadMyc G1 arrest does not require the presence of a functional retinoblastoma protein and is associated with a strong reduction in cyclin E/CDK2 kinase activity in arrested cells. MadMyc does not cause alterations in the expression levels of cyclin E, CDK2, p27kip1, cyclin D1 or CDK4 in G1-arrested cells. These data indicate that inhibition of c-Myc activity in exponentially growing cells leads to G1 arrest through loss of cyclin E-associated kinase activity.