Laboratory for Experimental Orthopaedics
Madry, H., Cucchiarini, M., Stein, U., Remberger, K. Menger, M.D., Kohn, D., Trippel, S.B. Sustained transgene expression in cartilage defects in vivo after transplantation of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system. Journal of Gene Medicine, 2003, 5 (6): 502-9.
BACKGROUND: Genetically modified chondrocytes may be able to modulate articular cartilage repair. To date, transplantation of modified chondrocytes into cartilage defects has been restricted to viral vectors. We tested the hypothesis that a recombinant gene can be delivered to sites of cartilage damage in vivo using chondrocytes transfected by a lipid-mediated gene transfer method. METHODS: Isolated lapine articular chondrocytes were transfected with an expression plasmid vector carrying the P. pyralis luciferase gene using the reagent FuGENE 6. Transfected chondrocytes were encapsulated in alginate spheres and implanted into osteochondral defects in the knee joints of rabbits. RESULTS: In vitro, luciferase activity in pCMVLuc-transfected spheres showed an early peak at day 2 post-transfection and remained elevated at day 32, the longest time point evaluated. The number of viable chondrocytes in non-transfected and transfected spheres increased over the period of cultivation. In vivo, luciferase activity was maximal at day 5 post-transfection, declined by day 16, but was still present at day 32. On histological analysis, the alginate-chondrocyte spheres filled the cartilage defects and were surrounded by a fibrous repair tissue composed of spindle-shaped cells. CONCLUSIONS: These data demonstrate the successful introduction of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system into osteochondral defects and the sustained expression of the transgene in vivo. This method may be used to define the effects of genes involved in cartilage repair and may provide alternative treatments for articular cartilage defects.
Cucchiarini, M., Ren, X.L., Perides, G., Terwilliger, E.F. Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and 5 vectors. Gene Therapy, 2003, 10 (8): 657-67.
Microglia represent a crucial cell population in the central nervous system, participating in the regulation and surveillance of physiological processes as well as playing key roles in the etiologies of several major brain disorders. The ability to target gene transfer vehicles selectively to microglia would provide a powerful new approach to investigations of mechanisms regulating brain pathologies, as well as enable the development of novel therapeutic strategies. In this study, we evaluate the feasibility of specifically and efficiently targeting microglia relative to other brain cells, using vectors based on two different serotypes of adeno-associated virus (AAV) carrying cell-type-specific transcriptional elements to regulate gene expression. Among a set of promoter choices examined, an element derived from the gene for the murine macrophage marker F4/80 was the most discriminating for microglia. Gene expression from vectors controlled by this element was highly selective for microglia, both in vitro and in vivo. To our knowledge, this is the first demonstration of selective expression of transferred genes in microglia using AAV-derived vectors, as well as the first utilization of recombinant AAV-5 vectors in any macrophage lineage. These results provide strong encouragement for the application of these vectors and this approach for delivering therapeutic and other genes selectively to microglia.
Madry, H., Cucchiarini, M., Terwilliger, E.F., Trippel, S.B. Recombinant adeno-associated virus vectors efficiently and persistently transduce chondrocytes in normal and osteoarthritic human articular cartilage. Human Gene Therapy, 2003, 14 (4): 393-402.
Successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. In the present study we tested the hypothesis that recombinant adeno-associated virus (rAAV) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. Using an rAAV vector carrying the Escherichia coli beta-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter/enhancer (rAAV-lacZ), transduction efficiency exceeded 70% for isolated normal human adult articular chondrocytes, and osteoarthritic human articular chondrocytes. These were comparable to the transduction efficiency obtained with neonatal bovine articular chondrocytes. Transduction of explant cultures of articular cartilage resulted in reporter gene expression within the tissue of all three cartilage types to a depth exceeding 450 mm, which remained present until 150 days. When rAAV-lacZ vectors were applied to femoral chondral defects and osteochondral defects in vivo in a rat knee model, reporter gene expression was achieved for at least 10 days after transduction. These data suggest that AAV-based vectors can efficiently transduce and stably express foreign genes in articular chondrocytes, including chondrocytes of normal and osteoarthritic human articular cartilage. The data further suggest that the same rAAV vectors are capable of transducing chondrocytes in situ within their native matrix to a depth sufficient to be of potential clinical significance. Finally, the data demonstrate that these rAAV vectors are capable of effectively delivering recombinant genes to chondral and osteochondral defects in vivo.
Madry H. Gene transfer in anterior cruciate ligament surgery - basic science and clinical applications. Der Orthopäde, 2002, 31(8): 799-809.
Current challenges in anterior cruciate ligament surgery include graft remodeling and tendon-to-bone healing. The development over the past few decades of methods for delivering genes to musculoskeletal tissues has stimulated interest in its application for orthopedic problems, including anterior cruciate ligament surgery. Despite substantial progress, a number of technical issues need to be addressed before gene transfer might be considered as an approach to improve the structural and functional properties of anterior cruciate ligament grafts. The aim of this review is to illustrate the principles of somatic gene transfer and to apply them to the cells that constitute the anterior cruciate ligament. Special characteristics that dictate the experimental strategies will be outlined.
Madry, H., Padera, Seidel, J., B. Freed, L., Langer, R., Trippel S.B., Vunjak-Novakovic, G. Gene transfer of a human insulin-like growth factor-I cDNA enhances tissue engineering of cartilage. Human Gene Therapy 2002, 13 (13): 1621-30.
The repair of articular cartilage lesions remains a clinical problem. Two novel approaches to cartilage formation, gene transfer and tissue engineering, have been limited by short-term transgene expression in transplanted chondrocytes and inability to deliver regulatory signals to engineered tissues according to specific temporal and spatial patterns. We tested the hypothesis that the transfer of a cDNA encoding the human insulin-like growth factor I (IGF-I) can provide sustained gene expression in cell-polymer constructs in vitro and in vivo and enhance the structural and functional properties of tissue-engineered cartilage. Bovine articular chondrocytes genetically modified to overexpress human IGF-I were seeded into polymer scaffolds, cultured in bioreactors in serum-free medium, and implanted subcutaneously in nude mice; constructs based on nontransfected or lacZ-transfected chondrocytes served as controls. Transgene expression was maintained throughout the duration of the study, more than 4 weeks in vitro followed by an additional 10 days either in vitro or in vivo. Chondrogenesis progressed toward the formation of cartilaginous tissue that was characterized by the presence of glycosaminoglycans, aggrecan, and type II collagen, and the absence of type I collagen. IGF-I constructs contained increased amounts of glycosaminoglycans and collagen and confined-compression equilibrium moduli as compared with controls; all groups had subnormal cellularity. The amounts of glycosaminoglycans and collagen per unit DNA in IGF-I constructs were markedly higher than in constructs cultured in serum-supplemented medium or native cartilage. This enhancement of chondrogenesis by spatially defined overexpression of human IGF-I suggests that cartilage tissue engineering based on genetically modified chondrocytes may be advantageous as compared with either gene transfer or tissue engineering alone.
Madry, H., Zurakowski, D., Trippel, S.B. Overexpression of human insulin-like growth factor-I promotes new tissue formation in an ex vivo model of articular chondrocyte transplantation. Gene Therapy 2001, 8(19): 1443-9.
Articular cartilage, the tissue that forms the gliding surface of joints, has a poor regenerative capacity. Insulin-like growth factor-I (IGF-I) is a polypeptide that is anabolic and mitogenic for cartilage. Transfection of articular chondrocytes with an expression plasmid vector containing the cDNA for human IGF-I under the control of the cytomegalovirus promoter/enhancer led to expression of the transgene and synthesis of biologically relevant amounts of IGF-I protein. Transplantation of transfected articular chondrocytes on to the surface of articular cartilage explants led to the formation of a new tissue layer on the cartilage explant surface. The new tissue was characterized by the presence of type II collagen and proteoglycan and by the absence of type I collagen, consistent with hyaline-like cartilage. The tissue formed by the chondrocytes expressing IGF-I was thicker and contained more cells than controls transfected with an expression plasmid vector containing the Escherichia coli (E. coli) beta-galactosidase (lacZ) gene. Transplantation of articular chondrocytes that overexpress human IGF-I also increased DNA synthesis and the synthesis of glycosaminoglycans by the underlying explant cartilage chondrocytes. These results identify a mechanism by which IGF-I may simultaneously promote chondrogenesis and shift cartilage homeostasis in an anabolic direction. The data further suggest that therapeutic growth factor gene transfer may be applicable to articular cartilage.
Madry, H., Reszka, R., Bohlender, J., Wagner, J. Efficacy of cationic-liposome mediated gene transfer to mesangial cells in vitro and in vivo. Journal of Molecular Medicine 2001, 79(4), 184-9.
Mesangial cells represent a major target for gene transfer approaches to the kidney. To establish a liposome-based system for transfection of mesangial cells we analyzed the efficacy and toxicity of different cationic liposomes and other nonviral transfection methods in primary cultures of rat and human mesangial cells using the Escherichia coli beta-galactosidase (lacZ) gene as a marker. In addition, an expression vector containing a human renin cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer was generated, introduced into mesangial cells, and assayed in a system of transient gene expression. In vivo, gene transfer was studied after infusion of liposome/DNA complexes in the kidney of rats via the renal artery. Transfection efficiency ranged from 5.5% with DMRIE Liposomes in rat mesangial cells to 1.1% with LipofectAmine liposomes in human mesangial cells. Cytotoxicity following transfection was dependent on the transfection method. Transfection with the human renin expression vector led to the secretion of 11 pg/104 cells/48 h human renin in rat mesangial cells, 3,600 pg/104 cells/48 h in 293 cells, and 113 pg/104 cells/48 h human renin in opossum kidney cells. In vivo, infusion of liposomes was accompanied by nephrotoxicity and did not result in marker gene expression. Together the data demonstrate that cationic liposomes are useful tools for transferring genes into mesangial cells, including human mesangial cells. Cationic liposomes provide a functional system for the synthesis and secretion of human renin in mesangial cells and other mammalian kidney cells. The current limitation of the evaluated liposomes for an efficient in vivo gene transfer to mesangial cells is the toxicity upon intrarenal arterial administration.
Cucchiarini, M., Kamer, A.R., Grabscheid, B., Diepolder, H.M., Gerlach, T.J., Gruner, N., Santantonio, T., Reichen, J., Pape, G.R., Cernay, A. Vigorous peripheral blood cytotoxic T cell response during the acute phase of hepatitis C infection. Cellular Immunology 2000, 203(2): 111-23.
After infection by hepatitis C virus (HCV), a minority of patients develop acute symptomatic disease and some of them are able to clear the virus. In this study, we analyzed peripheral blood mononuclear cells from nine patients with acute symptomatic disease with respect to their cytotoxic T lymphocyte (CTL) response using a panel of HCV-derived peptides in a semiquantitative secondary in vitro culture system. We could detect early CTL responses in 67% of these patients. The CTL responses were directed against multiple viral epitopes, in particular within the structural (core 2-9, core 35-44, core 131-140, and core 178-187) and nonstructural regions of the virus (NS3 1073-1081, NS3 1406-1415, NS4 1807-1816, NS5 2252-2260, and NS5B 2794-2802). We compared the CTL responses displayed by recently and chronically infected HLA-A2-positive patients. Virus-specific CTLs were detectable in chronic carriers but the percentage of positive peptide-specific CTL responses was significantly higher in recently infected patients (P = 0.002). Follow-up of recently infected patients during subsequent disease development showed a significant decrease in the values and proportions of positive peptide-specific CTL responses (P = 0.002 and 0.013, respectively). Patients with limited viral replication exhibited significantly more vigorous early responses (P = 0.024). These data suggest a protective role for the early antiviral CTL response in HCV infection.
Madry, H., Trippel, S.B. Efficient lipid-mediated gene transfer to articular chondrocytes. Gene Therapy 2000, 7(4): 286-91.
We examined nonviral, lipid-mediated gene transfer methods as potential tools for efficient transfection of articular chondrocytes. Transfection conditions were determined for primary cultures of normal human articular, osteoarthritic human articular and normal bovine articular chondrocytes using a lacZ reporter gene construct with the commercially available cationic liposomes Cellfectin, DMRIE-C, LipofectAmine, Lipofectin, LipoTaxi, TransFast and the lipid-based reagent FuGENE 6. Optimized conditions were then evaluated in an ex vivo model of chondrocyte transplantation. FuGENE 6 transfection produced the maximum levels of transgene expression. Transfection efficiency was cell type specific and affected by DNA concentration, lipid/DNA ratio and the presence of hyaluronidase, a matrix-degrading enzyme. Analysis of X-gal staining demonstrated an efficiency of 41.0% in normal bovine articular chondrocytes, 20.7% in normal human articular chondrocytes and 7.8% in osteoarthritic human chondrocytes. Transfected chondrocytes were found to successfully populate the articular cartilage surface in explant cultures. Transplanted genetically modified chondrocytes adhered to the articular cartilage and continued to produce beta-galactosidase for 2 weeks. This evaluation and optimization of lipid-based gene transfer into articular chondrocytes may serve as a useful tool in studies of genes involved in articular cartilage damage and repair and as a potential delivery method for therapeutic genes.
Cucchiarini, M., Barcellini-Couget, S., Lefebvre, J.-C., Doglio, A. T-cells chronically infected with HIV do not contain enough Nef to promote CD4 down-modulation in the absence of envelope-mediated effects. Journal of AIDS and Human Retrovirology 1998, 17(2):112-9.
Among HIV viral proteins, envelope glycoproteins and Nef have been both suggested to participate in CD4 downregulation during the course of HIV infection. In a previous study, we provided evidence that a mutant form of CD4 that does not bind gp120 was never downregulated in chronically HIV-1- and HIV-2-infected CEM cells. To further investigate the relative effects of Nef or glycoproteins in CD4 downregulation, recombinant vaccinia virus (VV) vectors were used to express high levels of HIV-1 viral proteins in cells expressing both wild-type and mutant CD4. It was demonstrated that during HIV infection, overexpression of Nef, achieved through the VV expression system, was necessary to induce CD4 downregulation in the mutant CD4-expressing cell model. These results are consistent with the hypothesis that Nef-mediated CD4 downregulation depends on the cellular levels of Nef expression. We concluded that during the late stage of viral replication, CD4 downregulation is mostly due to gp120 and not to Nef because of a low level of Nef expression.
Wagner, J., Madry, H., Reszka, R. Gene transfer - Focus on the Kidney. Nephrology Dialysis Transplantation 1995, 10(10): 1801-7.
Renal gene transfer techniques are being developed as a novel experimental approach to understand the pathogenesis of renal disease and to potentially develop new therapeutic tools. We review the currently available technology to introduce foreign genetic material into renal tissue, i.e., retroviral, adenoviral, and liposomal transfer systems with their respective advantages and caveats. Today, the transfer efficiency of these methods appears to be sufficiently high to study the effects of transduced genes on renal function and morphology in rat kidney. This will allow (i) the elucidation of the function of genes on the course of renal disease in experimental animal models and (ii) the modulation of local expression of endogenous genes which presumptively contribute to renal pathology in these models. One strategy to accomplish this aim is the use of recombinant DNA technology to design antisense DNA constructs or oligonucleotides, which interfere with the renal expression of target genes. We will also discuss some of the shortcomings of the currently used techniques with respect to potential therapeutic use of gene transfer systems and gene modulation.
Cagnon, L., Cucchiarini, M., Lefebvre, J.-C., Doglio, A. Protection of a T-cell line from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule. Journal of AIDS and Human Retrovirology 1995, 9(4):349-58.
Adenovirus VA1 gene is efficiently transcribed by RNA polymerase III and gives rise to a small highly ordered RNA. To inhibit replication of human immunodeficiency virus (HIV), a chimeric VA1 RNA molecule was designed that contained a short antisense RNA sequence complementary to a conserved region of the HIV-1 rev encoding mRNA (28 nucleotides). This sequence, which was inserted into a projecting loop of the VA1 RNA central domain, was mainly single stranded and available for binding with its complementary sequence. The chimeric VA1 antisense was abundantly expressed in human cells constituting 3% of mRNA and promoted strong and specific inhibition of HIV-1 gene replication. The stable expression of antisense RNA in human T cells (CEM) protected these cells from HIV-1 multiplication for at least 3 months. No side effects were detected because of the lack of antisense effect upon replication of the closely related HIV-2. The VA1 gene may provide a suitably compact gene cassette for the intracellular expression of short antisense RNA directed against HIV.
Cucchiarini, M., Cagnon, L., Giordanengo, V., Doglio, A., Lefebvre, J.-C. Induction by human immunodeficiency virus types 1 and 2 of degradation of CD4 but not of a CD4 mutant unable to bind viral glycoproteins. Journal of AIDS and Human Retrovirology 1995, 8(5):427-36.
HIV-1 appears to use a multiple gene strategy to regulate CD4 receptor expression, which emphasizes the importance of this regulation in the viral life cycle. The cytoplasmic interaction between gp160 and CD4 is probably the major event governing CD4 down-regulation, although other viral proteins, such as Nef (CD4 cell surface localization) and Vpu (CD4 degradation), are thought to participate as well. Because of the lack of vpu in HIV-2, we investigated the effects of two HIV-2 isolates (ROD 10 and EHO) on CD4 expression in the CEM T-cell line. We found that these HIV-2 strains induce CD4 degradation to a similar extent as that induced by an HIV-1 isolate (BRU). To assess the role of each viral protein involved in CD4 regulation (gp, Nef and Vpu), we developed cell lines expressing a mutated form of CD4 unable to efficiently bind gp160, in addition to their endogenous CD4. Using this system, we provide evidence that the mutated CD4 is always expressed in HIV-1-, and HIV-2-infected cells, independent of the presence of Nef, while the endogenous CD4 is completely lost. These results highlight the key role of intracytoplasmic gp-CD4 interaction, explaining in vitro the CD4 down-regulation in T-cell lines.
Lefebvre, J.-C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., Peyron, J.-F. Altered glycosylation of leukosialin, CD43, in HIV-1-infected cells of the CEM line. Journal of Experimental Medicine 1994, 180(5):1609-17.
CD43 (leukosialin, gpL115, sialophorin) is a major sialoglycoprotein widely expressed on hematopoietic cells that is defective in the congenital immunodeficiency Wiskott-Aldrich syndrome. It is thought to play an important role in cell-cell interactions and to be a costimulatory molecule for T lymphocyte activation. Using a metabolic 35SO42- radiolabeling assay or biotinylation of cell surface proteins, we describe here that CD43 are sulfated molecules the glycosylation of which is altered in human immunodeficiency virus type 1 (HIV-1)-infected leukemic T cells of the CEM line. Hyposialylation of O-glycans and changed substitution on N-acetylgalactosamine residues are observed. The glycosylation defect is associated with an impairment of CD43-mediated homotypic aggregation which can be restored by resialylation. The hyposialylation of CD43 on HIV-1+ cells may explain the high prevalence of autoantibodies directed against nonsialylated CD43 that have been detected in HIV-1-infected individuals. A defect in glycosylation of important molecules such as CD43 or, as we recently described, CD45 may explain alterations of T cell functions and viability in HIV-1-infected individuals. In addition, a possible implication of hyposialylation in the HIV-1-infected cells entrapment in lymph nodes could be envisioned.
Madry, H., Emkey, G., Zurakowski, D., Trippel, S. B. Overexpression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation. Journal of Gene Medecine, 2004, 6 (2): 238-45.
BACKGROUND: Genetically engineered chondrocytes could be used to enhance cartilage repair. Fibroblast growth factor 2 (FGF-2) is a mitogen for chondrocytes and may be a candidate for gene transfer approaches to stimulate chondrocyte proliferation. In the present study, we tested the hypothesis that human FGF-2 (hFGF-2) gene transfer into articular chondrocytes modulates cell proliferation in an ex vivo model of chondrocyte transplantation. METHODS: Transfection of articular chondrocytes with an expression plasmid vector carrying the cDNA for hFGF-2 under the control of the cytomegalovirus promoter/enhancer mediated transgene expression and synthesis of biologically relevant amounts of the recombinant hFGF-2 protein. Articular chondrocytes transfected with the Escherichia coli beta-galactosidase (lacZ) gene or a hFGF-2 cDNA were transplanted onto the surface of articular cartilage explants. RESULTS: The tissue formed by the chondrocytes expressing hFGF-2 was thicker and contained more cells than control cultures. Quantitative analysis of [(3)H]thymidine and [(35)S]sulfate incorporation in composite cultures revealed that hFGF-2 transfection stimulated mitogenic activity in the new tissue but did not augment matrix glycosaminoglycan synthesis. CONCLUSIONS: These data support the concept that chondrocytes overexpressing a hFGF-2 cDNA selectively modulate cell proliferation in an ex vivo model of chondrocyte transplantation. These results suggest that therapeutic hFGF-2 gene transfer may be applicable for the treatment of articular cartilage disorders, such as traumatic defects in which cellular repopulation is a therapeutic goal.
Lennerz, C., Madry, H., Ehlhardt, S., Venzke, T., Zang, K. D., Mehraein, Y. Parvovirus B19-related chronic monoarthritis: immunohistochemical detection of virus-positive lymphocytes within the synovial tissue compartment: two reported cases. Clinical Rheumatology, 2004, 23 (1): 59-62.
Apart from systemic symptoms of viral infection parvovirus B19, the infectious agent in erythema infectiosum, can lead to mainly self-limited acute and chronic arthropathy. Because mild subclinical features of the disease can be easily overlooked, joint affections might appear as isolated symptoms. We here report two cases of chronic monoarthritic symptoms of unknown origin, where immunohistochemical detection of B19-positive lymphocytic cells in the synovial tissue led to the diagnosis of B19 arthropathy. In conclusion, respective virus diagnostics should be considered even in chronic monosymptomatic arthritic lesions. The pathology of B19 arthropathy seems to be due to direct virus infection of cells within the synovia.