We explored the nature of the tumor-initiating cell in osteosarcoma, a bone malignancy that predominately occurs in children. Previously, we observed expression of Oct-4, an embryonal transcriptional regulator, in osteosarcoma cell cultures and tissues. To examine the relationship between Oct-4 and tumorigenesis, cells from an osteosarcoma biopsy (OS521) were stably transfected with a plasmid containing the human Oct-4 promoter driving a green fluorescent protein (GFP) reporter to generate the transgenic line OS521Oct-4p. In culture, only f24% of the OS521Oct-4p cells were capable of activating the transgenic Oct-4 promoter; yet, xenograft tumors generated in NOD/SCID mice contained f67% GFP + cells, which selectively expressed the mesenchymal stem cellassociated surface antigens CD105 and ICAM-1. Comparison of the tumor-forming capacity of GFP-enriched and GFPdepleted cell fractions revealed that the GFP-enriched fractions were at least 100-fold more tumorigenic, capable of forming tumors at doses of <300 cells, and formed metastases in the lung. Clonal populations derived from a single Oct-4/GFP + cell were capable of forming tumors heterogeneous for Oct-4/GFP expression. These data are consistent with the cancer stem cell model of tumorigenesis in osteosarcoma and implicate a functional link between the capacity to activate an exogenous Oct-4 promoter and tumor formation. This osteosarcoma tumor-initiating cell appears highly prolific and constitutes a majority of the cell population in a primary xenograft tumor, which may provide a biological basis for the particular virulence of this type of cancer.

The human b-globin gene locus is the subject of intense study, and over the past two decades a wealth of information has accumulated on how tissue-specific and stage-specific expression of its genes is achieved. The data are extensive and it would be difficult, if not impossible, to formulate a comprehensive model integrating every aspect of what is currently known. In this review, we introduce the fundamental characteristics of globin locus regulation as well as questions on which much of the current research is predicated. We then outline a hypothesis that encompasses more recent results, focusing on the modification of higher-order chromatin structure and recruitment of transcription complexes to the globin locus. The essence of this hypothesis is that the locus control region (LCR) is a genetic entity highly accessible to and capable of recruiting, with great efficiency, chromatinmodifying, coactivator, and transcription complexes. These complexes are used to establish accessible chromatin domains, allowing basal factors to be loaded on to specific globin gene promoters in a developmental stage-specific manner. We conceptually divide this process into four steps: (a) generation of a highly accessible LCR holocomplex; (b) recruitment of transcription and chromatin-modifying complexes to the LCR; (c) establishment of chromatin domains permissive for transcription; (d) transfer of transcription complexes to globin gene promoters.

Erythroid-specific, high level expression of the ␤-globin genes is regulated by the locus control region (LCR), composed of multiple DNase I-hypersensitive sites and located far upstream of the genes. Recent studies have shown that LCR core elements recruit RNA polymerase II (pol II). In the present study we demonstrate the following: 1) pol II and other basal transcription factors are recruited to LCR core hypersensitive elements; 2) pol II dissociates from and re-associates with the globin gene locus during replication; 3) pol II interacts with the LCR but not with the ␤-globin gene prior to erythroid differentiation in embryonic stem cells; and 4) the erythroid transcription factor NF-E2 facilitates the transfer of pol II from immobilized LCR constructs to a ␤-globin gene in vitro. The data are consistent with the hypothesis that the LCR serves as the primary attachment site for the recruitment of macromolecular complexes involved in chromatin structure alterations and transcription of the globin genes.The five genes of the human ␤-globin locus are expressed in erythroid cells in a tissue-and developmental stage-specific manner (1). Appropriate expression of the globin genes is regulated by many DNA elements that are located proximal and distal to the genes. The human ␤-globin locus control region (LCR) 1 is a powerful regulatory DNA element located far upstream of the genes and is required for high level expression of all the globin genes throughout development (1, 2). The LCR, unlike classical enhancer elements, operates in an orientationdependent manner (3). There is currently no consensus on how the LCR acts to stimulate globin gene transcription, but it is generally believed that it involves some form of communication between the LCR and the globin genes (4 -6). The LCR is composed of several regions that exhibit extremely high sensitivity to DNase I in erythroid cells (hypersensitive HS sites 1-5). The core HS sites contain clusters of transcription factor binding sites and are separated from each other by 2-4 kbp (2). The results from analyzing human LCR function at ectopic sites in the context of transgenic mice demonstrate that the HS sites synergistically enhance globin gene transcription (7-12), whereas studies in the endogenous murine locus show that the core HS sites function additively (13-15).Recent models view the LCR as a holocomplex in which the individual HS sites interact via extensive protein/DNA and protein/protein interactions (7,16,17). The LCR holocomplex may provide a highly accessible region for the efficient recruitment of macromolecular complexes involved in chromatin modification and transcription (18). Indeed, it has been shown that RNA polymerase II (pol II) is recruited to LCR HS sites in vitro and in vivo (19 -22), suggesting that transcription complexes are first recruited to the LCR and subsequently delivered to the globin genes (18). Sawado et al. (23) recently demonstrated that another important function of the LCR is to regulate transcription elongation at the adult ␤...

Background The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single-stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double-stranded form. To overcome this, we evaluated double-stranded self-complementary (sc) AAV as a vehicle for intra-articular gene delivery. Methods Conventional and scAAV vectors were used to infect lapine articular fibroblasts in culture to determine transduction efficiency, transgene expression levels, and nuclear trafficking. scAAV containing the cDNA for interleukin (IL)-1 receptor antagonist (Ra) was delivered to the joints of naïve rabbits and those with IL-1β-induced arthritis. From lavage of the joint space, levels of transgenic expression and persistence were measured by enzyme-linked immunosorbent assay. Infiltrating leukocytes were quantified using a hemocytometer. Results Transgene expression from scAAV had an earlier onset and was approximately 25-fold greater than conventional AAV despite the presence of similar numbers of viral genomes in the nuclei of infected cells. Fibroblasts transduced with scAAV produced amounts of IL1-Ra comparable to those transduced with adenoviral and lentiviral vectors. IL1-Ra was present in lavage fluid of most animals for 2 weeks in sufficient quantities to inhibit inflammation of the IL-1β-driven model. Once lost, neither subsequent inflammatory events, nor re-administration of the virus could re-establish transgene expression. Conclusions scAAV-mediated intra-articular gene transfer is robust and similarly efficient in both normal and inflamed joints; the resulting transgenic expression is sufficient to achieve biological relevance in joints of human proportion.

To understand the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver and overexpress transforming growth factor-beta 1 (TGF-β1) cDNA (Ad.TGF-β1) in the knee joints of immunocompromised rats. Following delivery, animals were killed periodically, and joint tissues were examined macroscopically and histologically. PCR-array was used to assay alterations in expression patterns of extracellular matrix (ECM)-associated genes. By days 5 and 10, TGF-β1 induced an increase in knee diameter and complete encasement of joints in dense scar-like tissue, locking joints at 90° of flexion. Histologically, massive proliferation of synovial fibroblasts was seen, followed by their differentiation into myofibroblasts. The fibrotic tissue displaced the normal architecture of the joint capsule and fused with articular cartilage. RNA expression profiles showed high levels of transcription of numerous MMPs, matricellular and ECM proteins. By day 30, the phenotype of the fibrotic tissue had undergone chondrometaplasia, indicated by cellular morphology, matrix composition and >100-fold increases in expression of collagen type II and cartilage link protein. Pre-labeling of articular cells by injection with recombinant lentivirus containing eGFP cDNA showed fibrotic/cartilaginous tissues appeared to arise almost entirely from local proliferation and differentiation of resident fibroblasts. Altogether, these results indicate that TGF-β1 is a potent inducer of arthrofibrosis, and illustrate the proliferative potential and plasticity of articular fibroblasts. They suggest the mechanisms causing arthrofibrosis share many aspects with tumorigenesis.

Toward the treatment of osteoarthritis (OA), the authors have been investigating self-complementary adeno-associated virus (scAAV) for intra-articular delivery of therapeutic gene products. As OA frequently affects weight-bearing joints, pharmacokinetic studies of scAAV gene delivery were performed in the joints of the equine forelimb to identify parameters relevant to clinical translation in humans. Using interleukin-1 receptor antagonist (IL-1Ra) as a secreted therapeutic reporter, scAAV vector plasmids containing codon-optimized cDNA for equine IL-1Ra (eqIL-1Ra) were generated, which produced eqIL-1Ra at levels 30- to 50-fold higher than the native sequence. The most efficient cDNA was packaged in AAV2.5 capsid, and following characterization in vitro, the virus was injected into the carpal and metacarpophalangeal joints of horses over a 100-fold dose range. A putative ceiling dose of 5 × 10 viral genomes was identified that elevated the steady-state eqIL-1Ra in the synovial fluids of injected joints by >40-fold over endogenous levels and was sustained for at least 6 months. No adverse effects were seen, and eqIL-1Ra in serum and urine remained at background levels throughout. Using the 5 × 10 viral genome dose of scAAV, and green fluorescent protein as a cytologic marker, the local and systemic distribution of vector and transduced cells following intra-articular injection scAAV.GFP were compared in healthy equine joints and in those with late-stage, naturally occurring OA. In both cases, 99.7% of the vector remained within the injected joint. Strikingly, the pathologies characteristic of OA (synovitis, osteophyte formation, and cartilage erosion) were associated with a substantial increase in transgenic expression relative to tissues in healthy joints. This was most notable in regions of articular cartilage with visible damage, where foci of brilliantly fluorescent chondrocytes were observed. Overall, these data suggest that AAV-mediated gene transfer can provide relatively safe, sustained protein drug delivery to joints of human proportions.

The human ␤-globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5 to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1-and KMnO 4 -sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.The human ␤-globin locus contains five genes (ε, G␥, A␥, ␦, and ␤), which are organized in sequential order on chromosome 11 (39). The gene order reflects the timing of expression during erythroid development, with the embryonic ε gene located at the 5Ј end and the adult ␤-globin gene at the 3Ј end of the locus. Developmental stage-specific expression is controlled mainly at the transcriptional level by a variety of geneproximal or -distal cis elements. The most prominent distal regulatory element is the ␤-globin locus control region (LCR), located from 8 to 22 kb upstream of the ε-globin gene and composed of several subregions that exhibit heightened sensitivity to digestion with exogenous DNase I (HS sites) in erythroid cells (14,18,41). Originally, five HS sites (HS1 to HS5) were associated with LCR function, but more recently additional sites were mapped even further 5Ј to the LCR (6). Many studies indicate that the human ␤-globin LCR is able to confer position-independent expression to linked globin genes in transgenic mice (18,19). It is generally believed that this activity is based on the ability of the LCR to provide an open accessible chromatin structure regardless of where the transgenic locus integrates in the mouse genome. Recent results suggest that the chromatin-opening function of the LCR may not be the primary activity in the endogenous mouse or human globin locus, because the LCR can be deleted without affecting general sensitivity to DNa...

The authors are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of interleukin-1 receptor antagonist (IL-1Ra), and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene transfer on a scale proportional to the human knee, a frequent site of OA incidence, studies were focused on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, a functional ceiling dose of ∼5 × 10 viral genomes was previously identified, which elevated the steady state levels of eqIL-1Ra in synovial fluids by >40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, the functional capacity of scAAV.IL-1Ra gene-delivery was examined in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by >400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and ∼25% improvement in total joint pathology by both magnetic resonance imaging and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively, these studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.