Gene transfer into vascular cells

AbstractThe goal of gene therapy is to introduce foreign deoxyribonucleic acid (DNA) into somatic cells to correct or prevent disorders caused by the malfunction of genes within a diseased individual. Overexpression of recombinant genes at specific sites within the vasculature can provide insights into vascular biology and potential treatments for various cardiovascular disorders such as restenosis. Methods for the introduction of foreign DNA into endothelial and vascular smooth muscle cells have been developed recently. These include the genetic modification of endothelium in vitro and implantation in vivo on arterial segments, direct infection of the arterial wall in vivo with a replication-defective retroviral vector expressing a recombinant gene and direct transfer of genes into vascular cells in vivo with use of liposomes. Although still in its formative stages, gene transfer into the vasculature holds promise as a potential treatment for vascular diseases, including atherosclerosis and restenosis. This approach may also provide insight into the role of specific gene products in the development of pathologic lesions

Gene transfer and cardiovascular disease

Through the introduction of foreign DNA into somatic cells, the aim of gene therapy is to correct or prevent disorders caused by the absence or malfunction of genes within a diseased individual. Expression of recombinant genes at specific sites within the vasculature can provide insights into vascular biology and potential treatments for various cardiovascular disorders. In our studies, we have developed methods for the transfer of recombinant genes into the endothelium and vascular smooth muscle cells by using retroviral vectors and liposomal transfection. Although these techniques are still in the formative stages, gene transfer into the endothelium and other vascular cells is a new approach to the treatment of vascular diseases, including atherosclerosis and restenosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29517/1/0000604.pd

Safety and Toxicity of Catheter Gene Delivery to the Pulmonary Vasculature in a Patient with Metastatic Melanoma

Overview summary Transcatheter delivery of HLA-B7 DNA and cationic liposomes into a segment of a pulmonary artery was safely performed in 1 patient with tumor nodules in the lung. No immunologic or organ toxicities were observed. Percutaneous catheter gene delivery has been performed in humans. Further refinements of this approach may lead to useful treatments for a variety of human diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63196/1/hum.1994.5.9-1089.pd

A New Cationic Liposome DNA Complex Enhances the Efficiency of Arterial Gene Transfer In Vivo

Overview summary GAP-DLRIE/DOPE, a new cationic liposome preparation, is an efficient liposomal vector that increases gene expression in arteries compared to naked DNA or previously described cationic DNA–liposome complexes by more than 15-fold. Although less efficient than adenoviral gene transfer, these levels of gene expression represent a significant improvement in liposome transfection in vivo and approach levels observed with clinically acceptable doses of adenoviral vectors. The improvement in gene expression, together with the relative safety associated with liposomal gene transfer, suggests that such nonviral vectors may be appropriate for human gene therapy protocols which utilize catheter-based gene delivery.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63105/1/hum.1996.7.15-1803.pd

Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells

Dendritic cells (DCs) capture and internalize human immunodeficiency virus (HIV)-1 through C-type lectins, including DC-SIGN. These cells mediate efficient infection of T cells by concentrating the delivery of virus through the infectious synapse, a process dependent on the cytoplasmic domain of DC-SIGN. Here, we identify a cellular protein that binds specifically to the cytoplasmic region of DC-SIGN and directs internalized virus to the proteasome. This cellular protein, leukocyte-specific protein 1 (LSP1), was defined biochemically by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane. LSP1 interacted specifically with DC-SIGN and other C-type lectins, but not the inactive mutant DC-SIGNΔ35, which lacks a cytoplasmic domain and shows altered virus transport in DCs. LSP1 diverts HIV-1 to the proteasome. Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1−/− mice also showed an increase in transfer of HIV-1BaL to a human T cell line. Proteasome inhibitors increased retention of viral proteins in lsp1+/+ DCs, and substantial colocalization of virus to the proteasome was observed in wild-type compared with LSP1-deficient cells. Collectively, these data suggest that LSP1 protein facilitates virus transport into the proteasome after its interaction with DC-SIGN through its interaction with cytoskeletal proteins

Envelope Exchange for the Generation of Live-Attenuated Arenavirus Vaccines

Arenaviruses such as Lassa fever virus cause significant mortality in endemic areas and represent potential bioterrorist weapons. The occurrence of arenaviral hemorrhagic fevers is largely confined to Third World countries with a limited medical infrastructure, and therefore live-attenuated vaccines have long been sought as a method of choice for prevention. Yet their rational design and engineering have been thwarted by technical limitations. In addition, viral genes had not been identified that are needed to cause disease but can be deleted or substituted to generate live-attenuated vaccine strains. Lymphocytic choriomeningitis virus, the prototype arenavirus, induces cell-mediated immunity against Lassa fever virus, but its safety for humans is unclear and untested. Using this virus model, we have developed the necessary methodology to efficiently modify arenavirus genomes and have exploited these techniques to identify an arenaviral Achilles' heel suitable for targeting in vaccine design. Reverse genetic exchange of the viral glycoprotein for foreign glycoproteins created attenuated vaccine strains that remained viable although unable to cause disease in infected mice. This phenotype remained stable even after extensive propagation in immunodeficient hosts. Nevertheless, the engineered viruses induced T cell–mediated immunity protecting against overwhelming systemic infection and severe liver disease upon wild-type virus challenge. Protection was established within 3 to 7 d after immunization and lasted for approximately 300 d. The identification of an arenaviral Achilles' heel demonstrates that the reverse genetic engineering of live-attenuated arenavirus vaccines is feasible. Moreover, our findings offer lymphocytic choriomeningitis virus or other arenaviruses expressing foreign glycoproteins as promising live-attenuated arenavirus vaccine candidates

Genetic Modification of Human Peripheral Blood Lymphocytes with a Transdominant Negative Form of Rev: Safety and Toxicity

Overview summary Expression of Rev M10, a transdominant mutant form of the Rev gene, in T cell lines confers resistance to HIV in vitro. Isertion of this Rev M10 gene into PBL appears to be nontoxic and well-tolerated by SCID mice. These results demonstrate that genetic modification of T cells by an antiviral gene can be performed safely and without overt toxicity. This finding encourages the development of therapeutic strategies to genetically protect T cells to prolong their survival in HIV-infected individuals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63234/1/hum.1995.6.8-997.pd

CONFORMATIONALLY STABILIZED HIV ENVELOPE IMMUNOGENS

Isolated immunogens including a HIV-1 gp120 polypeptide or immunogenic fragment thereof stabilized in a CD4 bound confirmation by crosslinked cysteines, and methods of their use are disclosed. The immunogens are useful, for example, for generating an immune response to HIV-1 gp120 in a Subject