Gene therapy aims at the introduction of gene(s) into somatic cells of humans for therapeutic purposes. The success of gene therapy is therefore dependent on the efficiency by which a therapeutic gene can be transferred to the patient's target tissues. In many cases, viruses are exploited for gene transfer purposes, and in particular gene transfer vectors derived from adenoviruses (adenoviral vectors) are often used to achieve this.

The fate of phenotypically defined human hematopoietic stem cells (hHSCs) in culture and the link between their surface marker expression profile and function are still controversial. We studied these aspects of hHSC biology by relating the expression of the early lineage markers (ELM) CD33, CD38, and CD71 on the surface of human umbilical cord blood (UCB) CD34(+) cells to their long-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse repopulation activity (LT-SRA). In uncultured UCB samples, LT-SRA was largely confined to the small CD34(+)ELM(-) cell fraction. CD34(+) cells expressing ELM markers at their surface usually lacked LT-SRA. After culturing UCB CD34(+) cells for 6 days in serum-free medium and on a feeder layer of Rat2 cells, the number of CD34(+)ELM(-) cells stayed roughly the same or showed a slight increase and the LT-SRA was preserved, suggesting a close association between LT-SRA and the CD34(+)ELM(-) phenotype. Indeed, transplantation of CD34(+)ELM(-) cells isolated from cultured UCB CD34(+) cells resulted in long-term hematopoietic reconstitution of conditioned NOD/SCID mice, whereas CD34(+)ELM(+) cells derived from the same cultures were devoid of LT-SRA. Remarkably, roughly 1% of the cells recovered from cultures initiated with isolated CD34(+)ELM(+) cells had lost ELM surface expression. Concurrently, the cultured CD34(+)ELM(+) cells acquired LT-SRA, suggesting that hematopoietic stem cells (HSCs) may arise by the dedifferentiation of early hematopoietic progenitor cells. The latter finding challenges the paradigm of unidirectional hematopoietic differentiation and opens new opportunities for HSC expansion prior to transplantation.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), making it amenable to gene- or cell-based therapies. Another possible treatment entails the combination of both principles by transplantation of autologous myogenic cells after their genetic complementation. This approach requires efficient and stable transduction of these cells with recombinant DMD. Recently, we generated a dual high-capacity (hc) adenovirus (Ad)–adeno-associated virus (AAV) hybrid vector (HV) that can deliver two full-length dystrophin-encoding modules into target cells. We showed that HV transduction of human cells containing AAV Rep proteins leads to the insertion of foreign DNA into the AAVS1 locus. Here, we improved HV entry into muscle cells from DMD patients. After having verified that these cells barely express the coxsackie B virus and Ad receptor (CAR), which constitutes the attachment molecule for Ad serotype 5 (Ad5) fibers, we equipped dual hcAd/AAV HV particles with Ad serotype 50 fiber domains to achieve CAR-independent uptake. These retargeted vectors complemented much more efficiently the genetic defect of dystrophin-defective myoblasts and myotubes than their isogenic counterparts with conventional Ad5 fibers. Importantly, the accumulation of β-dystroglycan along the membranes of vector-treated DMD myotubes indicated proper assembly of dystrophin-associated glycoprotein complexes.

ABSTRACT Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.

Bone marrow-derived human mesenchymal stem cells (hMSCs) lack the Coxsackie-adenovirus (Ad) receptor and thus are poorly transduced by vectors based on human Ad serotype 5 (Ad5). We investigated whether this problem could be overcome by using tropism-modified Ad5 vectors carrying fiber shaft domains and knobs of different human species B Ads (Ad5FBs). To allow quantitative analyses, these vectors coded for the enhanced green fluorescent protein (eGFP). Transgene expression analysis showed superior transduction of hMSCs by all Ad5FBs tested as compared with conventional Ad5 vectors. This was evident both by the frequency of eGFP-positive cells and by the eGFP level per cell. Highly efficient transduction of hMSCs, with limited variability between cells from different donors, was achieved with vectors displaying fiber domains of Ad serotypes 50, 35, and 16. These findings could not be reconciled with the very low levels of CD46, a recently identified receptor for species B Ads, on hMSCs, suggesting that AdFBs probably use receptors other than CD46 to enter these cells. We further observed that high eGFP levels were maintained in replication-restricted hMSCs for more than 30 days. In dividing hMSCs, foreign DNA delivered by Ad5FBs was expressed in a large fraction of the cells for approximately 3 weeks without compromising their replication capacity. Importantly, the transduced hMSCs retained their capacity to differentiate into adipocytes and osteoblasts when exposed to the appropriate stimuli.

Duchenne muscular dystrophy (DMD) is the most prevalent inheritable muscle disease. It is caused by mutations in the approximately 2.5-megabase dystrophin (Dys) encoding gene. Therapeutic attempts at DMD have relied on injection of allogeneic Dys-positive myoblasts. The immune rejection of these cells and their limited availability have prompted the search for alternative therapies and sources of myogenic cells. Stem cell-based gene therapy aims to restore tissue function by the transplantation of gene-corrected autologous cells. It depends on (i) the capacity of stem cells to participate in tissue regeneration and (ii) the efficient genetic correction of defective autologous stem cells. We explored the potential of bone marrow-derived human mesenchymal stem cells (hMSCs) genetically modified with the full-length Dys-coding sequence to engage in myogenesis. By tagging hMSCs with enhanced green fluorescent protein (EGFP) or the membrane dye PKH26, we demonstrated that they could participate in myotube formation when cultured together with differentiating human myoblasts. Experiments performed with EGFP-marked hMSCs and DsRed-labeled DMD myoblasts revealed that the EGFP-positive DMD myotubes were also DsRed-positive indicating that hMSCs participate in human myogenesis through cellular fusion. Finally, we showed that hMSCs transduced with a tropism-modified high-capacity hybrid viral vector encoding full-length Dys could complement the genetic defect of DMD myotubes.

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.

This chapter discusses the production systems of adenoviral vectors. Particular attention is paid to the generation and use of complementation cell lines that carry the El genes and emphasizes on the PER.C6 cell line, which was developed to prevent generation of replication-competent adenovirus (RCA) during propagation of El-deleted adenoviral vectors. Further, safety issues with respect to the use of the cell line for making clinical grade material are also addressed in this chapter. The PER.C6 cell line is the best substrate for the production of adenoviral vectors for gene therapy or vaccines. PER.C6 permits extensive analysis for adventitial agents and other safety concerns and thus is less hazardous than short-lived primary cell cultures for which testing must be repeated for each newly established culture.

ABSTRACT Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/ loxP -mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Gene therapy aims to complement or, ideally, correct defective genes. The broad clinical application of this emerging technology requires the development of safe high-capacity gene delivery vehicles that combine efficient transduction of dividing as well as quiescent cells with sustained transgene expression. Here we present a new hybrid vector system that unites favorable attributes of adenoassociated virus (AAV) and adenovirus (Ad) vectors in a single particle. This was achieved by inclusion of Ad packaging elements in different sized recombinant AAV genomes. In the presence of AAV replicative functions and a recombinant helper Ad, AAV/Ad hybrid particles were generated via encapsidation of AAV-dependent replicative intermediates into Ad capsids. In stringent in vitro models based on transduction of proliferating cells we show that AAV/Ad hybrid vectors are superior to Ad vectors in establishing prolonged transgene expression and can be used to deliver DNA fragments of at least 27 kb.

Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34+ CD33- CD38- CD71- cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34+ cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP+. Transduction by Ad5FBs of the less primitive fraction within UCB CD34+ cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34+ cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.

High doses of adenotk were injected into the cerebrospinal fluid of rats and nonhuman primates (Macaca mulatta). Vector administration was followed by ganciclovir administration for 14 days. Despite the absence of clinical symptoms, analysis of the cerebrospinal fluid (CSF) and histopathological examination of the central nervous system (CNS) of the monkeys (3 weeks after vector injection) were consistent with a viral meningitis. Immunohistochemical analysis of the inflammatory infiltrates in the monkeys revealed the presence of T and B lymphocytes, indicating a combined cellular and humoral immune response to the vector. This latter was supported by the finding of intrathecal anti-adenovirus antibody synthesis. Rats receiving high intrathecal adenotk doses showed a transient and dose-dependent clinical toxicity consisting of lethargy, hyperemic eyes and weight loss. Histopathological examination of the meninges showed a shift from polymorphonuclear infiltrates during the first post-injection days to clusters of mononuclear cells after 7 days. Acute toxicity is probably related to the early, innate immune response to the vector. In a separate experiment, high levels of IL-8 and IL-6, were measured during the first 2–3 post-injection days in the CSF of two monkeys which received intrathecal adenoLacZ. Therefore, these cytokines seem to play an important role in initiating the nonspecific immune response. In one monkey which received adenotk, recombinant adenovirus was cultured from serum samples obtained at the 7th post-injection day. At this time-point, no vector could be isolated from CSF samples. Based on these preclinical data, we recommend careful dose finding for clinical studies that aim to treat patients with leptomeningeal metastases.

Local intracoronary delivery of recombinant adenoviruses expressing anti-migratory or anti-proliferative proteins including human constitutive endothelial nitric oxide synthase (NOS3), plasminogen activator inhibitor 1 (PAI-1), or herpesvirus thymidine kinase (TK) (combined with ganciclovir) was used to prevent neointimal formation in porcine coronary arteries. After balloon injury of the left anterior descending (LAD) coronary artery, animals received an intramural injection of adenovirus (1.5 X 10(9) PFU) carrying either the NOS3 cDNA (AdCMVNOS3, n = 12), the PAI-1 cDNA (AdCMVPAI-1, n = 12), the TK cDNA (AdMLPItk, n = 12), or no cDNA (AdpL+, n = 12). After 28 days, morphometric analysis was performed on coronary sections from all segments demonstrating injury. The internal elastic lamina (IEL) fracture length normalized to the IEL perimeter (initial injury) and the neointimal area normalized to the vessel area (response to injury) were used to generate linear regression lines and calculate an index of stenosis for the respective treatment groups. The response to injury was significantly smaller in AdCMVNOS3- and AdMLPItk-infected animals than in AdpL+-infected animals (slopes = 0.86 +/- 0.05 and 0.69 +/- 0.07 versus 1.11 +/- 0.06, p < 0.005 and p < 0.0001, respectively) but not in AdCMVPAI-1-infected animals (slope = 1.26 +/- 0.04, p = 0.04). No viral shedding was observed and there was no acute systemic toxicity after gene transfer. An increase in neutralizing antibody titers against Ad vectors was observed without any detectable response to the transgene products (NOS3, PAI-1). Local gene transfer of NOS3 and TK may hold promise as a safe and effective adjunctive treatment to reduce neointimal formation after percutaneous coronary intervention in humans.

OBJECTIVE:To evaluate the potential of isolated limb perfusion (ILP) for efficient and tumor-specific adenovirus-mediated gene transfer in sarcoma-bearing rats. SUMMARY BACKGROUND DATA:A major concern in adenovirus-mediated gene therapy in cancer is the transfer of genes to organs other than the tumor, especially organs with a rapid cell turnover. Adjustment of the vector delivery route might be an option creating tumor specificity in therapeutic gene expression. METHODS:Rat hind limb sarcomas (5-10 mm) were transfected with recombinant adenoviruses. Intratumoral luciferase expression after ILP was compared with systemic administration, regional infusion, or intratumoral injection using a similar dose of adenoviruses carrying the luciferase marker gene. Localization studies using lacZ as a marker gene were performed to evaluate the intratumoral distribution of transfected cells after both ILP and intratumoral injection. RESULTS:Intratumoral luciferase activity after ILP or intratumoral administration was significantly higher compared with regional infusion or systemic administration. After ILP, luciferase gene expression was minimal in extratumoral organs, whether outside or inside the isolated circuit. Localization studies demonstrated that transfection was confined to tumor cells lying along the needle track after intratumoral injection, whereas after ILP, lacZ expression was found in viable tumor cells and in the tumor-associated vasculature. CONCLUSIONS:Using ILP, efficient and tumor-specific gene transfection can be achieved. The ILP technique might be useful for the delivery of recombinant adenoviruses carrying therapeutic gene constructs to enhance tumor control.

Gene transfer to synovial tissue by adenoviral vectors (Ad) was studied in vitro in cultured human synoviocytes and in vivo in seven primates with arthritis. Hyperplastic synovium was efficiently transduced with Ad.lacZ in vitro and in vivo in rhesus monkeys with collagen-induced arthritis, whereas chondrocytes were not transduced. Intraarticular injection of recombinant Ad harboring the luciferase gene showed the presence of reporter gene products only in Ad-injected joints. In addition, the feasibility of synovectomy by Ad harboring the herpes simplex virus thymidine kinase gene (tk) was studied. In vitro infection of synovium from rheumatoid arthritis patients with Ad.TK, followed by administration of ganciclovir, resulted in death of >90% of the synoviocytes. By mixing Ad.TK-infected with noninfected cells, it appeared that the presence of 10% infected synoviocytes resulted in the killing of more than 85% of the synoviocytes, demonstrating a substantial bystander effect. Intraarticular injection of Ad.TK in the knees of rhesus monkeys with arthritis, followed by treatment with ganciclovir for 14 days, resulted in increased apoptotic cell death in the synovium of Ad.TK-injected as compared with noninjected joints and ablation of the synovial lining layer. The procedure revealed no toxic side effects. These data suggest that nonsurgical synovectomy by tK gene therapy is feasible.