Nonintegrating lentiviral viral vectors (NILVs) present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling the short-term expression of potentially hazardous gene products. In recent studies, NILVs proved efficient at inducing an antigen specific immune response in vivo and are currently being used as a platform to deliver zinc finger nucleases to mediate site-specific gene editing. However, additional improvements in the NILV system are required to render this promising gene delivery system suitable for human clinical trials. These improvements include: a) increasing episomal gene expression, b) reducing illegitimate vector integration, c) developing an efficient and nonimmunogenic gene regulation system, and d) establishing an efficient vector production system. To achieve these improvements, we outline here a research proposal consisting of four aims.
In aim 1 we propose to develop a novel NILV deleted of the cis inhibitory elements recognized by the host LSF-YY1 and AP-4 transcriptional repressor complexes. This proposed modification will alleviate the transcriptional silencing typical of NILVs and will improve their efficacy.
Aim 2 will focus on the development of a novel inducible NILV system premised on alternative splicing. In contrast to currently used inducible systems, the novel splicing-regulated NILVs do not contain a synthetic transactivator, which could potentially induce a cell-mediated immune response.
In aim 3, we will focus on characterization and in vivo testing of a novel polypurine tract (PPT) deleted NILV, which exhibits reduced illegitimate integration. In the last aim, we will establish the first integrase-deficient stable packaging cell line, which will produce high-titer NILVs bearing the improvements described in aims 1-3. All new vectors containing the different modifications will carry an improved human factor IX (hFIX) cDNA. The vectors will be produced either by transient transfection or by the novel stable packaging cell line, and their ability to cure FIX deficiency will be tested in a hemophilia B mouse model.

Public Health Relevance

Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, several limitations inherent to this promising system limit its utilization in human clinical trials. The goal of the proposed research is to advance the NILV system to the point at which it will be considered suitable for human clinical trials. To this end, we propose a four-aim research plan. In aim 1, we will improve the NILVs'gene expression. Aim 2 will focus on the development of a nonimunogenic inducible lentiviral vector system. In aim 3, we will test the ability of a novel vector with reduced illegitimate integration to support therapeutic levels of transgene expression in vivo. In the last aim, to facilitate large-scale NILV production, we will establish the first stable packaging cell line for NILV.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Mckeon, Catherine T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
Zip Code
Simmons, Aaron B; Bretz, Colin A; Wang, Haibo et al. (2018) Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy. Angiogenesis 21:751-764
Becker, Silke; Wang, Haibo; Simmons, Aaron B et al. (2018) Targeted Knockdown of Overexpressed VEGFA or VEGF164 in Müller cells maintains retinal function by triggering different signaling mechanisms. Sci Rep 8:2003
Suwanmanee, Thipparat; Ferris, Martin T; Hu, Peirong et al. (2017) Toward Personalized Gene Therapy: Characterizing the Host Genetic Control of Lentiviral-Vector-Mediated Hepatic Gene Delivery. Mol Ther Methods Clin Dev 5:83-92
Hu, Peirong; Li, Yedda; Nikolaishvili-Feinberg, Nana et al. (2016) Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects. J Neurosci Res 94:1152-68
Hu, Peirong; Li, Yedda; Sands, Mark S et al. (2015) Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors. Mol Ther Methods Clin Dev 2:15025
Monahan, Paul E; Sun, Junjiang; Gui, Tong et al. (2015) Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial. Hum Gene Ther 26:69-81
Shen, Shen; Berry, Garrett E; Castellanos Rivera, Ruth M et al. (2015) Functional analysis of the putative integrin recognition motif on adeno-associated virus 9. J Biol Chem 290:1496-504
Wang, Haibo; Yang, Zhihong; Jiang, Yanchao et al. (2014) Quantitative analyses of retinal vascular area and density after different methods to reduce VEGF in a rat model of retinopathy of prematurity. Invest Ophthalmol Vis Sci 55:737-44
Jiang, Yanchao; Wang, Haibo; Culp, David et al. (2014) Targeting Müller cell-derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity. Invest Ophthalmol Vis Sci 55:824-31
Suwanmanee, Thipparat; Hu, Genlin; Gui, Tong et al. (2014) Integration-deficient lentiviral vectors expressing codon-optimized R338L human FIX restore normal hemostasis in Hemophilia B mice. Mol Ther 22:567-574

Showing the most recent 10 out of 23 publications