June R. Merwin

Targeted delivery of DNA for gene therapy via receptors

The general concept of targeted delivery of therapeutic agents was first recognized by Paul Ehrlich at the turn of the century I. Only recently has this strategy been applied to the delivery of DNA-protein complexes to cells 2. Most of the research on D N A delivery by this approach has been performed by targeting the liver-specific asialoglycoprotein receptor (ASGPr) (Refs 3-10) and the relatively ubiquitous transferrin receptor,1 19. DNA delivery to hepatocytes has also been effected using insulin-polylysine conjugates 2°, and to antigen-bearing cells using an antibody 21. The majority of these experiments use a covalently crosslinked receptor ligand-polycation conjugate to bind DNA in an electrostatic complex 5 (Fig. 1). Receptormediated endocytosis of the ligand carries the bound DNA into a cell, allowing subsequent expression of the foreign DNA (Fig. 2). This technique, which has been demonstrated in animals using the ASGPr (Refs 5-8), is promising for the delivery of therapeutic DNAs as well as antisense oligonucleotides2L

Interactions of Vascular Cells with Transforming Growth Factors-βa

The vascular system is lined by mitotically quiescent endothelial cells, which in addition to having a broad range of metabolic activities, provide a non-thrombogenic surface for blood flow. Beneath the endothelium, smooth muscle cells are found in the media of large vessels and pericytes are found in close association with the endothelial cells of various microvascular beds. These smooth muscle cells play major roles in the maintenance of the connective tissues of the vessel wall and in the control of vascular tone.’ Vascular cells (endothelial, pericyte, and smooth muscle) have been found to respond to injury in specific ways, depending upon the vascular bed and the cell type(s) injured. For example, following denudation injury evoked by angioplasty, endarterectomy, or autologous or synthetic grafting, large vessel endothelial cells bordering the affected area will exhibit rapid sheet migration over the exposed extracelMar matrix and proliferate in an attempt to reconstitute the normal continuous endothelial cell lining?.3 Medial smooth muscle cells of large and medium-sized vessels respond to vessel injury by migrating into the intima, where they proliferate and synthesize matrix components, which may result in the formation of a thickened intima that narrows the vessel lumen? In contrast, following soft tissue injury or in response to a variety of angiogenic factors, microvascular endothelial cells respond by freeing themselves from the constraints of their investing basement membranes and migrating and proliferating in the surrounding three-dimensional interstitial stroma and ultimately forming new microvessels? The role of pericytes (smooth muscle cell analogs found surrounding microvessels following new vessel formation) in the response to injury has been less well-studied and their origin(s) (endothelial, undifferentiated mesenchymalfibroblastic, or vascular smooth muscle cell) is still a matter of

In Vivo Gene Therapy via Receptor-Mediated DNA Delivery

Human gene therapy has advanced during the past ten years from a theoretical concept to a rapidly emerging technology. Tremendous technological strides in recombinant DNA methodologies have fostered molecular studies in such fields as regulation of gene expression, human genetics and disease states, and gene transfer techniques. The confluence of these fields has led to the emergence of gene therapy as a reality, albeit limited at present to a small number of clinical studies (Anderson, 1992; Miller, 1992). As practiced today most gene therapy protocols lie within the realm of high-cost, technology-intensive, individualized treatments, akin to such procedures as organ and bone marrow transplantation (Mulligan, 1991). This type of procedure, though beneficial, is limited in usefulness since it is not readily accessible to much of the population. Even for those to whom this type of gene therapy strategy would be available, the current inability to achieve permanent transgene expression necessitates periodic retreatments, making these procedures highly cost-ineffective over the long term.

The Interactions of Vascular Cells with Solid Phase (Matrix) and Soluble Factors

The vessel wall is composed of heterogeneous cell populations residing in a variety of vascular beds. Each cell type has different functions and morphologies but all of them have a role in the repair process following vascular injury. Responses to injury vary depending upon the type and extent of the injury and the vascular bed affected. The sheet migration and proliferation exhibited by large vessel endothelial cells is in striking contrast to the migration through soft tissues and tube formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells respond to injury by migrating into the intima, proliferating and synthesizing matrix, causing intimal thickening. The response to injury by vascular cells appears to be modulated, in part, by the composition and organization of the surrounding matrix and the various platelet factors and cytokines found at sites of injury. Furthermore, evidence has been accrued in culture, suggesting that solid phase (matrix) and soluble factors modulate each others effects on local vascular cell populations following injury.

Interactions of Matrix Components and Soluble Factors in Vascular Responses to Injury

The vascular system is lined by mitotically quiescent but metabolically active endothelial cells, which in addition to having a broad range of metabolic activities, provide a nonthrombogenic surface for blood flow. Beneath the endothelium, smooth muscle cells are found in the media of large vessels, and pericytes are found in close association with the endothelial cells of microvascular beds. The smooth muscle cells (pericytes) are thought to play major roles in maintaining vessel wall integrity, being responsible for the maintenance to the connective tissues of the vessel wall and in the control of vascular tone.8 Vascular cells (large and small vessel derived endothelial, pericyte, and smooth muscle cells) have been found to respond to injury in specific ways, depending upon the vascular bed and the cell type(s) injured. For example, following denudation injury evoked by angioplasty, endarterectomy or autologous or synthetic grafting, large vessel endothelial cells bordering the affected area will exhibit rapid sheet migration over the exposed extracellular matrix and proliferate in an attempt to reconstitute the normal continuous endothelial cell lining.15,20 The medial smooth muscle cells of large and medium-sized vessels respond to vessel injury by migrating into the intima, where they proliferate and synthesize matrix components, which results in the formation of a thickened intima which narrows the vessel lumen.34 In contrast, following soft tissue injury or in response to a variety of angiogenic factors, microvascular endothelial cells respond by freeing themselves from the constraints of their investing basement membranes. Following this, they migrate and proliferate in the surrounding three-dimensional interstitial stroma and ultimately form new microvessels.17

Cancer cell binding to E-selectin transfected human endothelia.

Human endothelial cells were transiently transfected with E-Selectin which enabled us to study tumor cell/endothelial interactions following engagement of E-Selectin without the added complications of metabolic stimulation, morphological changes, and/or up regulation of other adhesion molecules due to cytokine induction. Similar results were received from in vitro binding studies and FACS analyses on both Tumor Necrosis Factor-alpha activated and E-Selectin transfected endothelial cells. These data suggest that this methodology is appropriate for dissecting the individual activities of E-selectin while minimizing the participation of other adhesion molecules, thereby allowing us to develop a better understanding of the role of E-Selectin and endothelia in metastatic disease.

Vascular cell responses to a hybrid Transforming Growth Factor-Beta molecule

Functional biological assays were performed using a hybrid molecule of Transforming Growth Factor-Beta (TGF-5 beta) where nine amino acids near the cleavage site of TGF-beta 1 were substituted with nine amino acids located in the identical position of TGF-beta 2. Bovine aortic endothelial and smooth muscle cells as well as rat epididymal fat pad microvascular endothelia were studied in three distinct bioassays examining proliferation, migration and angiogenesis. The data suggested TGF-5 beta elicited results that do not differ significantly from the TGF-beta 1 isoform, while TGF-beta 2 expressed unique characteristics. We have also shown that these amino acid substitutions to TGF-beta 1 do not, in fact, alter the biological functions of the growth factor.

Acyclovir–Glycoprotein conjugates are potent inhibitors of hepatitis B virus replication

AbstractReceptor-mediated targeting to the liver using galactose-terminated ligands is a promising strategy for site-specific delivery of antiviral drugs for treatment of hepatitis B virus infection. Optimization of drug conjugates for receptor-mediated delivery depends, in part, on judicious selection of coupling chemistry between drug and receptor-specific ligand. We synthesized three chemically distinct conjugates: acyclovir linked through a γ-aminobutyryl ester to asialoorosomucoid; acyclovir linked through a succinyl ester to polylysine-asialoorosomucoid; and acyclovir linked to polylysine-asialoorosomucoid through a monophosphoryl linkage (ACV-MP-PL-ASOR). All conjugates were rapidly cleared by the liver when tail-vein injected in mice. The conjugates inhibited replication of hepatitis B virus (HBV) DNA in cultured cells at concentrations that were dependent on the structure of the cross-linker and the extent of modification that the protein endured during coupling with drug. ACV-MP-PL-ASOR displaye...