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.
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.
|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 22:567-74|
|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|
|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|
|Titus, Mark A; Zeithaml, Brian; Kantor, Boris et al. (2012) Dominant-negative androgen receptor inhibition of intracrine androgen-dependent growth of castration-recurrent prostate cancer. PLoS One 7:e30192|
|Cockrell, Adam S; van Praag, Henriette; Santistevan, Nicholas et al. (2011) The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles. Retrovirology 8:51|
|Kantor, Boris; Bayer, Matthew; Ma, Hong et al. (2011) Notable reduction in illegitimate integration mediated by a PPT-deleted, nonintegrating lentiviral vector. Mol Ther 19:547-56|
|Johnson, Jarrod S; Gentzsch, Martina; Zhang, Liqun et al. (2011) AAV exploits subcellular stress associated with inflammation, endoplasmic reticulum expansion, and misfolded proteins in models of cystic fibrosis. PLoS Pathog 7:e1002053|
|Kantor, Boris; Ma, Hong; Webster-Cyriaque, Jennifer et al. (2009) Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection. Proc Natl Acad Sci U S A 106:18786-91|
|Bayer, Matthew; Kantor, Boris; Cockrell, Adam et al. (2008) A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector. Mol Ther 16:1968-76|
|Li, Chengwen; Hirsch, Matthew; Asokan, Aravind et al. (2007) Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo. J Virol 81:7540-7|
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