Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors.
(17/7311)
We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34(+) and CD34(+)38(-) clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34(+) and CD34(+)38(-) cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34(+), CD34(+)38(-) LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors. (+info)
Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine alpha-L-iduronidase deficiency.
(18/7311)
Canine alpha-L-iduronidase (alpha-ID) deficiency, a model of the human storage disorder mucopolysaccharidosis type I (MPS I), is an ideal system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells. We performed adoptive transfer of genetically corrected autologous hematopoietic cells in dogs with alpha-ID deficiency. Large volume marrow collections were performed on five alpha-ID-deficient dogs. Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks of culture to retroviral vectors bearing the normal canine alpha-ID cDNA. Transduced LTMC cells from deficient dogs expressed enzymatically active alpha-ID at 10 to 200 times the levels seen in normal dogs. An average of 32% of LTMC-derived clonogenic hematopoietic cells were provirus positive by polymerase chain reaction and about half of these expressed alpha-ID. Approximately 10(7) autologous gene-modified LTMC cells/kg were infused into nonmyeloablated recipients. Proviral DNA was detected in up to 10% of individual marrow-derived hematopoietic colonies and in 0.01% to 1% of blood and marrow leukocytes at up to 2 to 3 years postinfusion. Despite good evidence for engraftment of provirally marked cells, neither alpha-ID enzyme nor alpha-ID transcripts were detected in any dog. We evaluated immune responses against alpha-ID and transduced cells. Humoral responses to alpha-ID and serum components of the culture media (fetal bovine and horse sera and bovine serum albumin) were identified by enzyme-linked immunosorbent assay. Cellular immune responses to autologous alpha-ID but not neo(r) transduced cells were demonstrated by lymphocyte proliferation assays. To abrogate potential immune phenomena, four affected dogs received posttransplant cyclosporine A. Whereas immune responses were dampened in these dogs, alpha-ID activity remained undetectable. In none of the dogs engrafted with genetically corrected cells was there evidence for clinical improvement. Our data suggest that, whereas the alpha-ID cDNA may be transferred and maintained in approximately 5% of hematopoietic progenitors, the potential of this approach appears limited by the levels of provirally derived enzyme that are expressed in vivo and by the host's response to cultured and transduced hematopoietic cells expressing foreign proteins. (+info)
Mismatch repair and differential sensitivity of mouse and human cells to methylating agents.
(19/7311)
The long-patch mismatch repair pathway contributes to the cytotoxic effect of methylating agents and loss of this pathway confers tolerance to DNA methylation damage. Two methylation-tolerant mouse cell lines were identified and were shown to be defective in the MSH2 protein by in vitro mismatch repair assay. A normal copy of the human MSH2 gene, introduced by transfer of human chromosome 2, reversed the methylation tolerance. These mismatch repair defective mouse cells together with a fibroblast cell line derived from an MSH2-/- mouse, were all as resistant to N-methyl-N-nitrosourea as repair-defective human cells. Although long-patch mismatch repair-defective human cells were 50- to 100-fold more resistant to methylating agents than repair-proficient cells, loss of the same pathway from mouse cells conferred only a 3-fold increase. This discrepancy was accounted for by the intrinsic N-methyl-N-nitrosourea resistance of normal or transformed mouse cells compared with human cells. The >20-fold differential resistance between mouse and human cells could not be explained by the levels of either DNA methylation damage or the repair enzyme O6-methylguanine-DNA methyltransferase. The resistance of mouse cells to N-methyl-N-nitrosourea was selective and no cross-resistance to unrelated DNA damaging agents was observed. Pathways of apoptosis were apparently intact and functional after exposure to either N-methyl-N-nitrosourea or ultraviolet light. Extracts of mouse cells were found to perform 2-fold less long-patch mismatch repair. The reduced level of mismatch repair may contribute to their lack of sensitivity to DNA methylation damage. (+info)
Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors.
(20/7311)
PURPOSE: Adenovirus-mediated arterial gene transfer is a promising tool in the study of vascular biology and the development of vascular gene therapy. However, intraluminal delivery of adenoviral vectors causes vascular inflammation and neointimal formation. Whether these complications could be avoided and gene transfer efficiency maintained by means of delivering adenoviral vectors via the adventitia was studied. METHODS: Replication-defective adenoviral vectors encoding a beta-galactosidase (beta-gal) gene (AdRSVnLacZ) or without a recombinant gene (AdNull) were infused into the lumen or the adventitia of rabbit carotid arteries. Two days after infusion of either AdRSVnLacZ (n = 8 adventitial, n = 8 luminal) or AdNull (n = 4 luminal), recombinant gene expression was quantitated by histochemistry (performed on tissue sections) and with a beta-gal activity assay (performed on vessel extracts). Inflammation caused by adenovirus infusion was assessed 14 days after infusion of either AdNull (n = 6) or vehicle (n = 6) into the carotid adventitia. Inflammation was assessed by means of examination of histologic sections for the presence of neointimal formation and infiltrating T cells and for the expression of markers of vascular cell activation (ICAM-1 and VCAM-1). To measure the systemic immune response to adventitial infusion of adenovirus, plasma samples (n = 3) were drawn 14 days after infusion of AdNull and assayed for neutralizing antibodies. RESULTS: Two days after luminal infusion of AdRSVnLacZ, approximately 30% of luminal endothelial cells expressed beta-gal. Similarly, 2 days after infusion of AdRSVnLacZ to the adventitia, approximately 30% of adventitial cells expressed beta-gal. beta-gal expression was present in the carotid adventitia, the internal jugular vein adventitia, and the vagus nerve perineurium. Elevated beta-gal activity (50- to 80-fold more than background; P <.05) was detected in extracts made from all AdRSVnLacZ-transduced arteries. The amount of recombinant protein expression per vessel did not differ significantly between vessels transduced via the adventitia (17.1 mU/mg total protein [range, 8.1 to 71.5]) and those transduced via a luminal approach (10.0 mU/mg total protein [range, 3.9 to 42.6]). Notably, adventitial delivery of AdNull did not cause neointimal formation. In addition, vascular inflammation in arteries transduced via the adventitia (ie, T-cell infiltrates and ICAM-1 expression) was confined to the adventitia, sparing both the intima and media. Antiadenoviral neutralizing antibodies were present in all rabbits after adventitial delivery of AdNull. CONCLUSION: Infusion of adenoviral vectors into the carotid artery adventitia achieves recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Because adventitial gene transfer can be performed by means of direct application during open surgical procedures, this technically simple procedure may be more clinically applicable than intraluminal delivery. Moreover, despite the generation of a systemic immune response, adventitial infusion had no detectable pathologic effects on the vascular intima or media. For these reasons, adventitial gene delivery may be a particularly useful experimental and clinical tool. (+info)
Effect of in situ retroviral interleukin-4 transfer on established intracranial tumors.
(21/7311)
BACKGROUND: Current therapies for malignant gliomas remain largely ineffective. We have previously demonstrated that interleukin 4 (IL-4) exhibits antitumorigenic activity in athymic nude mice by promoting both eosinophil infiltration and inhibition of tumor angiogenesis (formation of new blood vessels). In this study, we investigated treatment of established rat C6 cell gliomas by retroviral delivery of IL-4 in situ. METHODS: Tumors grown subcutaneously in athymic nude mice or implanted intracranially in immunocompetent Wistar rats were implanted with ecotropic retrovirus (i.e., will replicate only in cells of closely related species) packaging cells (RPCs) that were transfected with a retroviral vector encoding mouse IL-4 (1C5 cells) or a control vector (SV cells). For the demonstration of the long-term effects of such treatment, C6 cells were also implanted into the contralateral hemisphere of the brains of rats previously treated with 1C5 RPCs. Tumor volume measurements and immunohistochemical analyses were performed. RESULTS: Implantation of 1C5 RPCs into subcutaneous C6 cell tumors resulted in tumor growth arrest that was associated with eosinophil infiltration and inhibition of angiogenesis. When 1C5 RPCs were stereotactically implanted into established intracranial tumors in rats, tumor volumes were dramatically smaller than in control animals (approximately 1.8 mm3 versus 70-80 mm3, respectively) 7 days after treatment. All 1C5 RPC-treated rats survived to 106 days after C6 cell implantation (99 days after treatment; an arbitrary end point), whereas control rats had to be killed 14 days after C6 cell implantation because of extensive tumor growth. Histologic analysis demonstrated that treated tumors were completely eradicated, and immunohistochemical analysis revealed an inhibition of tumor angiogenesis and infiltration by CD8+ cells and macrophages. C6 cells implanted contralaterally into the brains of long-term-surviving rats treated with 1C5 RPCs were also rapidly and completely rejected. CONCLUSIONS: Implantation of packaging cells producing IL-4 retrovirus leads to rapid eradication of rat C6 cell gliomas and provides sustained protection against further intracranial challenge. (+info)
Antitumor and immunotherapeutic effects of activated invasive T lymphoma cells that display short-term interleukin 1alpha expression.
(22/7311)
Expression of cytokines in malignant cells represents a novel approach for therapeutic treatment of tumors. Previously, we demonstrated the immunostimulatory effectiveness of interleukin 1alpha (IL-1alpha) gene transfer in experimental fibrosarcoma tumors. Here, we report the antitumor and immunotherapeutic effects of short-term expression of IL-1alpha by malignant T lymphoma cells. Activation in culture of T lymphoma cells with lipopolysaccharide-stimulated macrophages induces the expression of IL-1alpha. The short-term expression of IL-1alpha persists in the malignant T cells for a few days (approximately 3-6 days) after termination of the in vitro activation procedure and, thus, has the potential to stimulate antitumor immune responses in vivo. As an experimental tumor model, we used the RO1 invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the vertebral column and compress the spinal cord, resulting in hind leg paralysis and death of the mice. Activated RO1 cells, induced to express IL-1alpha in a short-term manner, manifested reduced tumorigenicity: approximately 75% of the mice injected with activated RO1 cells remained tumor free. IL-1 was shown to be essential for the eradication of activated T lymphoma cells because injection of activated RO1 cells together with IL-1-specific inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor, reversed reduced tumorigenicity patterns and led to progressive tumor growth and death of the mice. Furthermore, activated RO1 cells could serve as a treatment by intervening in the growth of violent RO1 cells after tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after the inoculation of violent cells, mortality was significantly reduced. IL-1alpha, in its unique membrane-associated form, in addition to its cytosolic and secreted forms, may represent a focused adjuvant for potentiating antitumor immune responses at low levels of expression, below those that are toxic to the host. Further assessment of the immunotherapeutic potential of short-term expression of IL-1alpha in activated tumor cells may allow its improved application in the treatment of malignancies. (+info)
IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells.
(23/7311)
Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer. (+info)
Selective regulation of cytokine induction by adenoviral gene transfer of IkappaBalpha into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-kappaB independent.
(24/7311)
Macrophages are the major cytokine producers in chronic inflammatory diseases, but the biochemical pathways regulating cytokine production are poorly understood. This is because genetic tools to dissect signaling pathways cannot be used in macrophages because of difficulties in transfection. We have developed an adenoviral technique to achieve high efficiency gene delivery into macrophages and recently showed that spontaneous TNF-alpha production in rheumatoid arthritis joint cells, chiefly from macrophages, is 75% blocked by adenoviral transfer of IkappaBalpha. In this report we use the same adenovirus to investigate whether the production of a number of proinflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, and IL-8) from human macrophages depends on NF-kappaB. While the cytokine response to certain inducers, such as LPS, PMA, and UV light, is blocked by overexpression of IkappaBalpha, the response to zymosan is not. In contrast, anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist) induced by LPS are only marginally inhibited by IkappaBalpha excess. These studies demonstrate several new points about macrophage cytokine production. First, there is heterogeneity of mechanisms regulating both the proinflammatory and anti-inflammatory cytokines within populations of a single cell type. In addition, the results confirm the utility of the adenoviral technique for functional analysis of cytokine induction. The results also confirm that there are autocrine and paracrine interactions regulating cytokine synthesis within a single cell type. The selectivity of NF-kappaB blockade for proinflammatory but not anti-inflammatory mediators indicates that in macrophages, NF-kappaB may be a good target for the treatment of chronic inflammatory diseases. (+info)