Growth Factor Transgene Research Articles

684. Viral Gene Therapy in Tendon Healing: Adeno-Associated Virus-2 but Not Other Serotypes Effectively Transduces Intrasynovial Tenocytes with Persistent Expression of the Transgene

Purpose: Transfer of exogenous growth factor genes to injured tendons is a new and promising method for strengthening tendon repairs. Genes have been delivered to tenocytes through plasmid or adenoviral vectors, but gene transfer through adeno-associated viral (AAV) vectors has not been reported. It is not known whether AAVs can effectively transduce tenocytes and whether transduction rate is different for different AAV serotypes. We explored the efficiency of transduction of intrasynovial tenocytes with 7 serotypes of AAV and the persistency of its expression of a growth factor transgene.

Tf-lipoplex-mediated NGF gene transfer to the CNS: neuronal protection and recovery in an excitotoxic model of brain injury

The development of efficient systems for in vivo gene transfer to the central nervous system (CNS) may provide a useful therapeutic strategy for the alleviation of several neurological disorders. In this study, we evaluated the feasibility of nonviral gene therapy to the CNS mediated by cationic liposomes. We present evidence of the successful delivery and expression of both a reporter and a therapeutic gene in the rodent brain, as evaluated by immunohistochemical assays. Our results indicate that transferrin-associated cationic liposome/DNA complexes (Tf-lipoplexes) allow a significant enhancement of transfection activity as compared to plain complexes, and that 8/1 (+/-) Tf-lipoplexes constitute the best formulation to mediate in vivo gene transfer. We demonstrated that Tf-lipoplex-mediated nerve growth factor transgene expression attenuates the morphological damages of the kainic acid-induced lesion as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) vital staining. These findings suggest the usefulness of these lipid-based vectors in mediating the delivery of therapeutic genes to the CNS.

Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo

Endothelial cells (ECs) in normal vessels are poorly transducible by retroviral vectors, which require cell division for gene transduction. Among retroviruses, lentiviruses have the unique ability to integrate their genome into the chromatin of nondividing cells. Here we show that multiply attenuated, self-inactivating, lentiviral vectors transduce both proliferating and growth-arrested human umbilical vein ECs (HUVECs), human coronary artery ECs (HCAECs), and human coronary artery smooth muscle cells (HCASMCs), with high efficacy. Lentiviral vectors containing the enhanced green fluorescence protein (EGFP) transgene driven by either the cytomegalovirus or the elongation factor-1α promoter, but not the phosphoglycerate kinase promoter, directed high-level EGFP expression in endothelial and smooth muscle cells. The endothelium-specific Tie2 promoter also directed transgene expression in ECs. Re-insertion of cis-acting sequences from pol of human immunodeficiency virus type 1 (HIV-1) into the vectors improved transgene expression. A lentiviral vector containing the vascular endothelial growth factor transgene promoted EC proliferation and sprouting in vitro. In vivo gene transfer was studied by lumenal infusion of vector containing solutions into rat carotid arteries. Lentivirus-mediated EGFP gene transfer was observed in ~5% of ECs. Lentiviral vectors containing the LacZ transgene achieved detectable β-galactosidase activity in rat arteries, albeit at a lower level compared with adenoviral vectors. This difference was mainly due to the lower concentration of lentiviral vector preparations. Lentivirus-mediated gene transfer was associated with minimal neointimal hyperplasia and scant inflammatory cell infiltrates in the media and adventitia. These observations indicate that lentiviral vectors may be useful for genetic modifications of vascular cells in vitro and in vivo.

Vascular endothelial growth factor transgene expression in cell-transplanted hearts

ObjectiveWe evaluated the effect of transplanted cell type, time, and region of the heart on transgene expression to determine the potential of combined gene and cell delivery for myocardial repair. MethodsLewis rats underwent myocardial cryoinjury 3 weeks before transplantation with heart cells (a mixed culture of cardiomyocytes, smooth muscle cells, endothelial cells and fibroblasts, n = 13), vascular endothelial growth factor–transfected heart cells (n = 13), skeletal myoblasts (n = 13), vascular endothelial growth factor–transfected skeletal myoblasts (n = 13), or medium (control, n = 12). Vascular endothelial growth factor expression in the scar, border zone, and normal myocardium was evaluated at 3 days and at 1, 2, and 4 weeks by means of quantitative polymerase chain reaction. Transplanted cells and vascular endothelial growth factor protein were identified immunohistologically on myocardial sections. ResultsVascular endothelial growth factor levels were very low in control scars but increased transiently after medium injection. Transplantation with heart cells and skeletal myoblasts significantly increased vascular endothelial growth factor expression in the scar and border zone. Transplantation of vascular endothelial growth factor–transfected heart cells and vascular endothelial growth factor–transfected skeletal myoblasts further augmented vascular endothelial growth factor expression, resulting in 4- to 5-fold greater expression of vascular endothelial growth factor in the scar at 1 week. Peak vascular endothelial growth factor expression was greater and earlier in vascular endothelial growth factor–transfected heart cells than in vascular endothelial growth factor–transfected skeletal myoblasts. Vascular endothelial growth factor was primarily expressed by the transplanted cells. Some of the transplanted heart cells and vascular endothelial growth factor–transfected heart cells were identified in the endothelial layer of blood vessels in the scar. ConclusionsTransplantation of heart cells and skeletal myoblasts induces vascular endothelial growth factor expression in myocardial scars and is greatly augmented by prior transfection with a vascular endothelial growth factor transgene. Vascular endothelial growth factor expression is limited to the scar and border zone for 4 weeks. Both heart cells and skeletal myoblasts may be excellent delivery vehicles for cell-based myocardial gene therapy.

Transplants of cells genetically modified to express neurotrophin-3 rescue axotomized Clarke's nucleus neurons after spinal cord hemisection in adult rats.

To test the idea that genetically engineered cells can rescue axotomized neurons, we transplanted fibroblasts and immortalized neural stem cells (NSCs) modified to express neurotrophic factors into the injured spinal cord. The neurotrophin-3 (NT-3) or nerve growth factor (NGF) transgene was introduced into these cells using recombinant retroviral vectors containing an internal ribosome entry site (IRES) sequence and the beta-galactosidase or alkaline phosphatase reporter gene. Bioassay confirmed biological activity of the secreted neurotrophic factors. Clarke's nucleus (CN) axons, which project to the rostral spinal cord and cerebellum, were cut unilaterally in adult rats by T8 hemisection. Rats received transplants of fibroblasts or NSCs genetically modified to express NT-3 or NGF and a reporter gene, only a reporter gene, or no transplant. Two months postoperatively, grafted cells survived at the hemisection site. Grafted fibroblasts and NSCs expressed a reporter gene and immunoreactivity for the NGF or NT-3 transgene. Rats receiving no transplant or a transplant expressing only a reporter gene showed a 30% loss of CN neurons in the L1 segment on the lesioned side. NGF-expressing transplants produced partial rescue compared with hemisection alone. There was no significant neuron loss in rats receiving grafts of either fibroblasts or NSCs engineered to express NT-3. We postulate that NT-3 mediates survival of CN neurons through interaction with trkC receptors, which are expressed on CN neurons. These results support the idea that NT-3 contributes to long-term survival of axotomized CN neurons and show that genetically modified cells rescue axotomized neurons as efficiently as fetal CNS transplants.

Skin antigens in the steady state are trafficked to regional lymph nodes by transforming growth factor-beta1-dependent cells.

Antigen capturing in the skin and antigen trafficking into regional lymph nodes (LN) initiate immune responses. In this study, employing melanin granule (MG) as an easily traceable antigen in two mouse strains that carried steel factor or hepatocyte growth factor transgenes and had melanocytosis in the epidermis or in the dermis respectively, we investigated the mechanism of antigen trafficking from the skin. MG captured in the epidermis or dermis accumulated in the regional LN, but not other tissues. Only in alymphoplastic mice did MG-laden cells pass through the lymphatics and reached many tissues. Since inflammatory regions were not observed in the skin of either type of transgenic mouse, our developmental system enables us to investigate constitutive capturing and trafficking of insoluble antigens in the steady state. Both dendritic cells and macrophages were laden with MG in the regional LN. To determine which cells traffic antigens to the LN, we prepared double mutants that carried the transgenes and lacked transforming growth factor (TGF)-beta1, since mice lacking TGF-beta1 are reported to be deficient of Langerhans cells. Few MG were observed in the regional LN of these double-mutant mice. We also showed that signaling via macrophage colony stimulating factor receptor or Flt3/Flk2 is not essential for development of the cells for this antigen trafficking. These results indicate that antigens in the epidermis and dermis in the steady state are trafficked into regional LN only by TGF-beta1-dependent cells, which may be a dendritic cell lineage.

Th2 T cells Expressing Transgene PDGF-A Serve as Vectors for Gene Therapy in Autoimmune Demyelinating Disease

We hypothesized that T cells can be genetically modified to express growth factor transgene products capable of inducing oligodendrocyte progenitor proliferation. Autoreactive T cells isolated from SWXJ mice immunized with the p139–151 determinant of myelin proteolipid protein (PLP) were transfected with an antigen-inducible transgene for platelet-derived growth factor-A (PDGF), a growth factor important in regulating the development of oligodendrocytes. Isolated antigen-specific T cell clones expressed the PDGF transgene when stimulated with PLP 139–151 peptide and produced biologically active PDGF capable of inducing proliferation of oligodendrocyte progenitor cells. Furthermore, upon adoptive transfer, the PDGF transfected T cells migrated to the CNS and ameliorated ongoing disease. Our data indicate that autoreactive memory Th2 cells can be genetically modified so that upon engagement with self antigen they produce regenerative growth factors capable of mediating tissue repair during autoimmune disease.

Mice expressing a bovine basic fibroblast growth factor transgene in the brain show increased resistance to hypoxemic-ischemic cerebral damage.

Cerebral intraventricular infusion of acidic or basic fibroblast growth factor has been shown to attenuate ischemic damage to hippocampal CA1 neurons in the gerbil. The purpose of the present study was to determine if the basic fibroblast growth factor transgenic mouse has an enhanced ability to resist the effects of severe cerebral hypoxemia-oligemia. Mice that were transgenic for bovine basic fibroblast growth factor were exposed to right carotid artery ligation, hyperglycemia, and 20 minutes of 1% carbon monoxide. After 5 days' recovery, brains were examined for histological damage. Counts of CA1 neurons in the right hippocampus showed a significantly higher number of neurons per millimeter CA1 in hypoxic-ischemic transgenic mice compared with nontransgenic controls (transgenic, 260 +/- 33; nontransgenic, 151 +/- 37 neurons per millimeter CA1; P < .05). The results indicate that basic fibroblast growth factor transgenic mice, as judged by CA1 hippocampal neuronal survival, have an enhanced ability to resist the effects of a complex hypoxic-ischemic cerebral insult.

Altered expression of nerve growth factor in the skin of transgenic mice leads to changes in response to mechanical stimuli

It has recently become clear that the neurotrophic factor, nerve growth factor, interacts specifically with nociceptive sensory neurons during development and maturity. Indeed, it may serve as a critical link between inflammation and the hyperalgesia that ensues in adult animals. Nerve growth factor is normally expressed in limiting amounts in target tissues of sensory and postganglionic sympathetic neurons. In the present study we have altered the basal level of nerve growth factor expression in the skin by producing transgenic mice that express a fusion gene construct containing either a sense or antisense nerve growth factor complementary DNA linked to the K14 keratin promoter. The K14-nerve growth factor transgene (sense or antisense) is abundantly expressed in skin from approximately embryonic day 15 and is then constitutively expressed throughout the life of the animal. In light of the fact that systemic administration of nerve growth factor to neonatal or adult rats leads to hyperalgesia, we have asked whether mice expressing the sense K14-nerve growth factor transgene exhibit similar sensory abnormalities and whether mice expressing the antisense nerve growth factor complementary DNA were hypoalgesic. Here we show that mice over-expressing nerve growth factor in skin display a profound hyperalgesia to noxious mechanical stimulation. Additionally, K14-nerve growth factor antisense mice displayed a profound hypoalgesia to the same stimuli.