RESUMO
Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted. We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.
Assuntos
Fibroblastos/enzimologia , Fibroblastos/transplante , Terapia Genética/métodos , Regiões Promotoras Genéticas/genética , Transfecção/métodos , Adenosina Desaminase/genética , Animais , Células Cultivadas , DNA , Fibronectinas/genética , Expressão Gênica , Vetores Genéticos , Humanos , Ratos , Ratos Endogâmicos F344 , Sequências Reguladoras de Ácido Nucleico , Retroviridae/genética , Fatores de TempoRESUMO
To approach the goal of consistent long-term erythropoietin (Epo) expression in vivo, we developed an implantation procedure in which transduced autologous vascular smooth muscle was introduced into rats in a chamber created from a polytetrafluoroethylene (PTFE) ring placed under the serosa of the stomach. The implant became vascularized and permitted the long-term survival of smooth muscle cells expressing Epo. Hematocrits of treated animals increased rapidly and monitored over 12 months gave a mean value of 56.0 +/- 4. 0% (P < .001; n = 9), increased from a presurgery mean of 42.3 +/- 1. 6%. Hemoglobin levels rose from a presurgery mean of 15.2 +/- 0.4 g/dL and for 12 months were significantly elevated with a mean value of 19.5 +/- 1.3 g/dL (P < .001; n = 9). The hematocrit and hemoglobin levels of control animals receiving human adenosine deaminase (ADA)-expressing cells were not significantly different from baseline (P > .05; n = 5). In response to tissue oxygenation, kidney, and (to a lesser extent) liver are specific organs that synthesize Epo. Treated animals showed downregulation of endogenous Epo mRNA in kidney over a 12-month period. The PTFE implant provides sustained gene delivery, is safe, and is minimally invasive. It allows easy engraftment of transduced cells and may be applied generally to the systemic delivery of therapeutic proteins such as hormones and clotting factors.
Assuntos
Implantes de Medicamento , Eritropoetina/biossíntese , Próteses e Implantes , Estômago , Adenosina Desaminase/genética , Animais , Células Cultivadas/transplante , DNA Complementar/genética , Desenho de Equipamento , Eritropoese , Eritropoetina/genética , Regulação da Expressão Gênica , Genes Reporter , Hematócrito , Hemoglobinas/biossíntese , Humanos , Canamicina Quinase/biossíntese , Rim/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/transplante , Especificidade de Órgãos , Politetrafluoretileno , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/biossíntese , Transfecção , Transplante HeterotópicoRESUMO
Granulocyte colony-stimulating factor (G-CSF) regulates granulocyte precursor cell proliferation, neutrophil survival, and activation. Cyclic hematopoiesis, a disease that occurs both in humans and grey collie dogs is characterized by cyclical variations in blood neutrophils. Although the underlying molecular defect is not known, long-term daily administration of recombinant G-CSF eliminates the severe recurrent neutropenia, indicating that expression of G-CSF by gene therapy would be beneficial. As a prelude to preclinical studies in affected collie dogs, we monitored hematopoiesis in rats receiving vascular smooth muscle cells transduced to express G-CSF. Cells transduced with LrGSN, a retrovirus expressing rat G-CSF, were implanted in the carotid artery and control animals received cells transduced with LASN, a retrovirus expressing human adenosine deaminase (ADA). Test animals showed significant increases in neutrophil counts for at least 7 weeks, with mean values of 3,670 +/- 740 cells/microliter in comparison to 1,870 +/- 460 cells/microliter in controls (p < 0.001). Thus, in rats G-CSF gene transfer targeted at vascular smooth muscle cells initiated sustained production of 1,800 neutrophils/microliter, a cell number that would provide clinical benefit to patients. Lymphocytes, red cells and platelets were not different between control and test animals (p > 0.05). These studies indicate that retrovirally transduced vascular smooth muscle cells can provide sustained clinically useful levels of neutrophils in vivo.
