Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood-brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients.
Steroids destined for intracellular metabolic conversion or binding to nuclear receptors are believed to cross cell membranes by passive diffusion. According to this free hormone hypothesis, steroids bound to plasma carrier proteins are inactive because they cannot reach their intracellular targets (1). However, recent data show that carrier proteins may greatly facilitate steroid uptake by endocytosis of steroid-carrier complexes followed by intracellular release of the steroid (2, 3). Megalin, a member of the low density lipoprotein receptor family abundant in kidney proximal tubules, mediates endocytic uptake of complexes between the steroid 25 ( Megalin binds a large number of structurally unrelated ligands, and coreceptors may confer ligand specificity by sequestering and presenting their cargo to megalin (4). For example, intrinsic factorvitamin B 12 complex ) is taken up in the intestine by a tandem receptor-mediated mechanism; the complex is first bound to a receptor, cubilin, anchored to the outer leaflet of the plasma membrane possibly by an amphipathic helix (5), followed by endocytosis of cubilin and its cargo mediated by megalin (6, 7). The pivotal role of intestinal cubilin is underscored by the vitamin B 12 deficiency observed in patients with Imerslund-Gräsbeck disease characterized by defective cubilin incapable of binding IF-B 12 (8). These patients have low molecular weight proteinuria in addition to megaloblastic anemia, indicating dysfunction of cubilin coexpressed with megalin in kidney proximal tubules. However, whereas the role of cubilin in the intestine is well characterized, the physiological role in the kidney remains elusive.Here, we identify cubilin as an important coreceptor in the endocytic pathway for retrieval of 25(OH)D 3 -DBP complexes by megalin-mediated endocytosis in the kidney. We show that absence of cubilin or inhibition of its function markedly reduces cellular uptake of the steroid-carrier complex, and animals or patients lacking functional cubilin are characterized by abnormal vitamin D metabolism. This study identifies patients with mutations in an endocytic pathway that regulates steroid hormone metabolism. Materials and MethodsLigands, Receptors, and Antibodies. DBP was purified from human serum (2). Receptor-associated protein (RAP) was produced in Escherichia coli (9); 3 H-25(OH)D 3 was from Amersham Pharmacia, and 25(OH)D 3 was from Dr. A.-M. Kissmeyer (Leo Pharmaceutical Products, Ballerup, Denmark). Biotin-25(OH)D 3 was synthesized by coupling 25(OH)D 3 -3-(3Ј-aminopropyl)ether (10) with aminocaproic acid-biotin-4-nitrophenyl ester (Pierce) (11). Sterol-DBP complexes were prepared by incubating DBP with 10 to 100-fold excess labeled or unlabeled 25(OH)D 3 (2). Uncomplexed steroid was removed by gel filtration or dialysis. Human retinolbinding protein (RBP) was from Dr. G. Alexander (University of Oslo, Norway). Rabbit megalin and cubilin were purified as reported (5).The primary antibodies used were rabbit anti-human DBP and anti-human RBP (Dako), goat anti-h...
Introduction Cobalamin (vitamin B 12 ) is a coenzyme for the enzymes of intermediate metabolism, methionine synthase, and methylmalonyl-CoA mutase, and deficiency of the vitamin leads to potentially lethal manifestations such as megaloblastic anemia and severe combined degeneration of the central nervous system. Cobalamin deficiency, which is one of the most common vitamin-deficiency diseases, is most often due to failure at a step in the complicated and highly specific gastrointestinal uptake mechanisms for dietary cobalamin rather than an insufficient supply from food. 1 Intrinsic factor (IF) is a glycoprotein produced in the gastric epithelium. It tightly binds to cobalamin in the gastrointestestinal tract, and in the distal small intestine the IF-cobalamin complex is recognized by cubilin, a multiligand apical membrane protein that participates in endocytosis of the complex. 2,3 IF is subsequently degraded in enterocyte lysosomes, and cobalamin is secreted into plasma in complex with transcobalamin-II. 4 Cubilin is a large membrane protein (460 kDa) with a unique set of extracellular protein modules comprising 8 tandem epidermal growth factor domains followed by 27 tandem CUB domains (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) harboring the IF-cobalamin binding site (CUB domains 5-8). 5,6 Although cubilin has no apparent transmembrane segment or cytoplasmic tail, several studies have shown that binding of IF-cobalamin to cubilin leads to endocytosis of the ligand and recycling of the receptor. 2,3 Besides expression and function in the intestine, cubilin has many-fold higher expression in the apical membrane of kidney proximal tubule and rodent yolk sac epithelial cells. 2,3,7,8 Consistent with this pattern of expression, cubilin is involved in reabsorption of several specific nutrient-carrying proteins from renal glomerular filtrate, including albumin, 9 transferrin, 10 vitamin D-binding protein, 11 and apolipoprotein AI, 12,13 and cubilin has a crucial but not-yet-defined role in early embryonic development of rodents. 14 Evidence to date indicates that the mechanism of cubilin-mediated endocytosis is the same in the various cubilin-expressing epithelia. 2 Accordingly, IF-cobalamin is effectively endocytosed in a cubilin-dependent manner in the proximal tubule 15 and yolk sac, 16 and because these tissues have higher density of cubilin in apical membranes and less luminal proteolytic activity than intestine, they have been the preferred tissues for studying cubilin function. 2,8,[14][15][16] However, it should be noted that ligands other than IF-cobalamin are likely to be physiologically more important in the kidney and yolk sac because little or no gastric IF circulates in plasma. An Inside Blood analysis of this article appears in the front of this issue.Reprints: Søren K. Moestrup, Institute of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark; e-mail: skm@biobase.dk.The publication costs of this article were defrayed in part by page charge ...
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