The Section on Directed Gene Delivery assesses the ability of retroviral vectors to deliver efficiently deliver genes to appropriate target cells with the end goal of transferring therapeutic genes to cells of the central nervous system. We and others have determined that although Adeno-Associated Viral (AAV) vectors and lentiviral vectors target neurons gammaretroviral vectors preferentially target non-neuronal cells in the CNS. These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-deployment of gammaretroviral gene therapy to neurological disease. Over the past year we have developed a systematic series of tools to allow us to construct targeted viral vectors that can be purified and concentrated in a manner that will allow delivery to specific cell types in the rodent brain. Vesicular stomatitis virus (VSV) is the envelope of choice for most cell culture work because it imposes a broad host range unto viral vectors. However VSV viral enveloped particles are neurotoxic, which limits its application, especially in the brain. The envelope protein of gibbon ape leukemia virus (GALV) also imposes a broad host range on viral vectors and it is not neurotoxic. However methods for concentrating high titer GALV enveloped vectors has been limited by the absence of antibodies to GALV envelope proteins and an effective means of concentrating GALV vectors free from cell and serum components that have been demonstrated to have toxic effects on neurons in the brain. With this in mind we have now constructed two GALV envelope protein variants one can pseudotype gammaretroviral vectors and the second lentiviral vectors that contain a five residue epitope tag that is identified by a monoclonal antibody. Vectors bearing these envelopes can be identified in producer cells by flow cytometry or on Western blots (M. Liu and M. V. Eiden, Retrovirology 8:53, 2011). We have also constructed a GALV vectors that contains red fluorescent protein tagged internal proteins (M. Liu and M.V. Eiden Retrovirology 9:55, 2012). This reagent is important in proof of concept experiments that are being used to optimize GALV epitope antibody-conjugated sepharose columns for the purification of GALV lentiviral and gammaretroviral vectors. GALV enveloped vectors can infect a broad number of target cells. To specifically target astrocytic, neuronal and microglial compartments of the human brain we are employing different envelope proteins and cell specific promoters. We have also examined why some patients treated with gammaretroviral vectors in which the viral promoter is inactivated develop cancers as a consequence of the vector activating an adjacent oncogene. This represents a conundrum since activation of the oncogene appears to occur even when the vector employed is self-inactivated for promoter function (e.g., a SIN vector). We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the mRNA level and protein level. We found that viral promoter activity in currently employed gammaretroviral SIN vectors is not attenuated. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research and only SIN vectors that contain the removal of a larger segment of the viral promoter be used in future clinical gene therapy trials (W. Xu, J.L. Russ and M.V. Eiden, Molecular Therapy Vol.20, 2012). The pace of gene delivery to the brain in a clinical setting has been significantly limited by the lack of translation of advances in vector development. Simply put, CNS payloads have evolved but their delivery systems have not. The availability of optimized preclinical vectors for in vivo testing would be a valuable resource for the national neuroscience community where they would serve seamlessly as tools for hypothesis-driven basic research, and again for clinical research, without needing to be re-discovered and re-presented for the clinic by translational scientists. A repository of such vectors would significantly reduce the time from inception of proof-of-concept design to in vivo execution and would serve to reduce redundancy in the evaluation of vectors through highly interactive collaborations among investigators in neuroscience, molecular virology, and surgical neurology.

Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2012
Total Cost
$1,209,233
Indirect Cost
Name
U.S. National Institute of Mental Health
Department
Type
DUNS #
City
State
Country
Zip Code
Emery, Andrew C; Liu, Xiu-Huai; Xu, Wenqin et al. (2015) Cyclic Adenosine 3',5'-Monophosphate Elevation and Biological Signaling through a Secretin Family Gs-Coupled G Protein-Coupled Receptor Are Restricted to a Single Adenylate Cyclase Isoform. Mol Pharmacol 87:928-35
Siegal-Willott, Jessica L; Jensen, Nathaniel; Kimi, David et al. (2015) Evaluation of captive gibbons (Hylobates spp., Nomascus spp., Symphalangus spp.) in North American Zoological Institutions for Gibbon Ape Leukemia Virus (GALV). J Zoo Wildl Med 46:27-33
Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement et al. (2015) Genetic diversity of koala retroviral envelopes. Viruses 7:1258-70
Emery, Andrew C; Eiden, Maribeth V; Eiden, Lee E (2014) Separate cyclic AMP sensors for neuritogenesis, growth arrest, and survival of neuroendocrine cells. J Biol Chem 289:10126-39
Emery, Andrew C; Eiden, Maribeth V; Mustafa, Tomris et al. (2013) Rapgef2 connects GPCR-mediated cAMP signals to ERK activation in neuronal and endocrine cells. Sci Signal 6:ra51
Emery, Andrew C; Eiden, Maribeth V; Eiden, Lee E (2013) A new site and mechanism of action for the widely used adenylate cyclase inhibitor SQ22,536. Mol Pharmacol 83:95-105
Xu, Wenqin; Stadler, Cynthia K; Gorman, Kristen et al. (2013) An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo. Proc Natl Acad Sci U S A 110:11547-52
Ozturk, Ferhat; Park, Paul J; Tellez, Joseph et al. (2012) Expression levels of the PiT-2 receptor explain, in part, the gestational age-dependent alterations in transduction efficiency after in utero retroviral-mediated gene transfer. J Gene Med 14:169-81
Liu, Meihong; Eiden, Maribeth V (2012) A mutant retroviral receptor restricts virus superinfection interference and productive infection. Retrovirology 9:51
Xu, Wenqin; Russ, Jill L; Eiden, Maribeth V (2012) Evaluation of residual promoter activity in γ-retroviral self-inactivating (SIN) vectors. Mol Ther 20:84-90

Showing the most recent 10 out of 17 publications