Molecular therapy : the journal of the American Society of Gene Therapy
-
Neuropathic pain is a difficult clinical problem that is often refractory to medical management. Glial-derived neurotrophic factor (GDNF) administered intrathecally has been shown to prevent or reduce pain in an animal model of neuropathic pain, but cannot be delivered in the required doses to treat human pain. We have previously demonstrated that peripheral subcutaneous inoculation of a replication-incompetent herpes simplex virus (HSV)-based vector can be used to transduce neurons of the dorsal root ganglion. ⋯ Reinoculation of the vector reestablished the antiallodynic effect with a magnitude that was at least equivalent to the initial effect. Vector-mediated GDNF expression blocked the nonnoxious touch-induced increase in c-fos expression in dorsal horn characteristic of the painful state. Gene transfer to produce a trophic factor offers a novel approach to the treatment of neuropathic pain that may be appropriate for human therapy.
-
Dystrophin-based gene therapy treatments aimed at correcting the Duchenne muscular dystrophy phenotype require stable expression of normal dystrophin (DYST) protein in myocytes without immune responses, which would compromise long-term expression. To predict cytotoxic T-cell-mediated responses elicited by transgenes, we used here H-2-negative HLA-A*0201 transgenic mice and identified human DYST epitopes, which elicit HLA-A*0201-restricted cytotoxic T cell activities. Among a series of eight peptides predicted from the human DYST sequence, not shared with the endogenous mouse DYST sequence, four of them were able to bind to HLA-A*0201 molecules and to induce cytotoxic T lymphocyte (CTL) responses. ⋯ Our results demonstrate that muscle injection with human DYST DNA systematically triggers CTL responses against this HLA-A*0201-restricted human DYST1281 peptide, which is present in long human DYST isoforms. Identification of such immunodominant human DYST epitopes and use of peptide/HLA tetramers will allow the immunomonitoring of CTL responses in HLA-phenotyped Duchenne muscular dystrophy patients undergoing gene therapy. Finally, the knowledge of HLA-A*0201-restricted human DYST peptides will be of importance to test, in mouse models, new immunomodulatory interventions allowing long-term engraftment of human dystrophin.
-
Comparative Study
Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice.
Lentiviral-mediated gene delivery holds significant promise for sustained gene expression within living systems. Vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 1-based lentiviral vectors can be used to introduce transgenes in a broad spectrum of dividing as well as nondividing cells. In the current study, we construct a lentiviral vector carrying two reporter genes separated by an internal ribosomal entry site and utilize that virus in delivering both genes into neuroblastoma cells in cell culture and into cells implanted in living mice. ⋯ Imaging of both reporter genes using microPET and optical CCD camera in living mice is feasible, with the optical approach being more sensitive, and a high correlation (R(2) = 0.86) between gene expressions is again observed in lentiviral-infected N2a tumor xenografts. Indirect imaging of HSV1-sr39tk suicide gene therapy utilizing Fluc is also feasible and can be detected with increased sensitivity by using the optical CCD. These preliminary results validate the use of lentiviral vectors carrying reporter genes for multimodality imaging of gene expression and should have many applications, including imaging of xenografts, metastasis, and cell trafficking as well as noninvasive monitoring of lentiviral-mediated gene delivery and expression.
-
X-linked adrenoleukodystrophy (ALD), an inherited demyelinating disorder of the central nervous system, can be corrected by allogeneic bone marrow transplantation, likely due to the turnover of brain macrophages that are bone marrow derived. ALD is characterized by an accumulation of very long chain fatty acids (VLCFA) due to the deficiency of an ATP binding cassette transporter that imports these fatty acids in peroxisomes. Murine retroviral transduction results in metabolic correction of ALD CD34(+) cells in vitro but reinfusion of these cells into ALD patients would not provide clinical benefit owing to the absence of selective advantage conferred by transgene expression. ⋯ Efficient expression of the ALD gene was demonstrated in monocytes/macrophages derived from cultures of transduced ALD CD34(+) cells and in long-term culture initiating cells. VLCFA metabolism was corrected in transduced CD34(+), CFU-derived, and LTC-derived cells, indicating that the vector-encoded ALD protein was fully functional. Transplantation of transduced ALD CD34(+) cells into NOD/SCID mice resulted in long-term expression of ALD protein in monocytes/macrophages derived from engrafted stem cells.
-
Adenoviral vector-mediated gene transfer to skeletal muscle is a promising potential treatment for Duchenne muscular dystrophy. However, the immunological response to viral antigens and the therapeutic protein expressed by the delivered gene could prevent effective treatment. In this study, we investigated the immune response induced by adenoviral and dystrophin antigens presented by high-capacity adenoviral vector-mediated dystrophin and beta-galactosidase delivery to skeletal muscle of a mouse model that is both dystrophin-deficient and lacZ transgenic. ⋯ The development of an anti-dystrophin antibody response in mice treated with the high-capacity adenoviral vector as neonates suggested that dystrophin antigens were presented to the immune system at a time remote from the gene delivery, when the immune system was mature. Interestingly, an antibody response against beta-galactosidase developed late in the course of mice treated with the high-capacity adenoviral vector as neonates, suggesting a loss of tolerance to beta-galactosidase, a self-antigen in these transgenic mice. Our results suggest that future human trials of dystrophin gene delivery will need to address the potential for immunity induced by ongoing segmental degeneration of partially treated muscle fibers and presentation of recombinant dystrophin antigens in the context of a Duchenne muscular dystrophy patient.