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  1. Biologics Research Projects

Cell-specific And Gene-specific Targeting of Gene Therapy Vectors

Principal Investigator: Jakob Reiser, PhD
Office / Division / Lab: OTAT / DCGT / GTIB

General Overview

Gene therapy holds great potential for treating a variety of serious diseases, some of which are as yet incurable. One strategy for delivering therapeutic genes is to use a virus to carry them into cells. Such gene delivery vehicles are referred to as vectors. Viruses used as vectors are specially modified so they do not reproduce inside the target cells.

Over the past decade, a variety of researchers have developed gene therapy vectors based on human immunodeficiency virus (HIV). But while HIV-derived vectors are promising, they also pose a risk. These vectors might insert the therapeutic genes into chromosomes at sites that behave like "on switches" that activate cancer-causing genes. This problem occurred previously during a study in France, triggering leukemia in children being treated with gene therapy for an inherited disease of the immune system.

Our goal is to improve the safety of HIV-based vectors so they can be used in the future to treat patients without these risks. Therefore, we are trying to engineer such vectors so they insert genes directly into well-defined sites on human chromosomes that lack cancer-causing on-switches.

These studies will help researchers to design safer and more effective HIV-based gene therapy vectors. The knowledge gained from our research will also allow us to evaluate the risks associated with the use of HIV vectors and to provide timely advice on product development for FDA sponsors.

Scientific Overview

Lentiviruses are complex retroviruses that include human immunodeficiency viruses such as HIV-1. Gene therapy vectors based on HIV-1 are being developed to deliver therapeutic genes. While these vectors are promising, they pose risks in that they may activate oncogenes during random integration into the host genome. Our goal is to develop safer HIV-1-based lentiviral (LV) vectors by limiting their integration to well-defined sites in the human genome and by narrowing their tissue tropism.

Toward this goal, we have designed integrase-defective LV vectors capable of integrating at specific sites in the mouse genome through homologous recombination. To do this, we are pursuing a targeted trapping approach using a promoterless secretion trap vector containing genomic sequences corresponding to genomic loci to be targeted. We rely on the endogenous promoter of the gene to be targeted to drive the expression of a selectable marker gene including a drug resistance gene and a cell surface marker-encoding gene. In parallel, we are investigating the capacity of phage PhiC31 integrase to mediate region-specific vector integration.

Targeted gene delivery involves broadening the tropism of vectors to transduce previously non-permissive cells or replacing the viral tropism to transduce specific target cell exclusively. These approaches offer potential advantages of enhanced therapeutic effects and reduced side effects. We are in the process of designing improved targetable LV vectors that more efficient and more flexible than the currently available ones. These vectors will either bear a cell-binding domain linked to the vector's membrane plus a separate fusion domain derived from Sindbis virus or vesicular stomatitis virus or a molecular bridge consisting of soluble avian leukosis/sarcoma virus receptors fused to cell-specific ligands. Our initial focus will be interleukin 13 (IL13)-displaying vectors to target IL13 receptor-over-expressing cancer cells in vitro and in vivo. If the performance of the system (specificity, vector titers) turns out to be poor, we plan to improve it using directed evolution approaches.

Our research allows us to evaluate the technologies used to construct, manufacture and test novel LV vectors (purity) and to investigate the ability of these vectors to target specific cell types in animal models (potency and safety). The information gained from these studies will enable us to identify potential risks associated with making and evaluating LV vectors for clinical use.

We use a broad range of biological approaches, including cell culture, molecular biological and virological techniques, to construct, manufacture and test new HIV-1-based vectors. A special emphasis is on targetable vectors for cell-specific transduction and on vectors capable of site-specific integration.


DNA RNA Nanotechnol 2016 Jan;2(1):42-52
Aptamer guided delivery of nucleic acid-based nanoparticles.
Panigaj M, Reiser J

Gene Ther 2015 Mar;22(3):64-9
A scalable method to concentrate lentiviral vectors pseudotyped with measles virus glycoproteins.
Marino MP, Panigaj M, Ou W, Manirarora J, Wei CH, Reiser J

Nano Lett 2014 Oct 8;14(10):5662-71
Multifunctional RNA nanoparticles.
Afonin KA, Viard M, Koyfman AY, Martins AN, Kasprzak WK, Panigaj M, Desai R, Santhanam A, Grabow WW, Jaeger L, Heldman E, Reiser J, Chiu W, Freed EO, Shapiro BA

Hum Gene Ther Methods 2014 Apr;25(2):126-35
Analysis of partial recombinants in lentiviral vector preparations.
Kuate S, Marino MP, Reiser J

PLoS One 2013 Nov 29;8(11):e81131
Targeting of Herpes Simplex Virus 1 Thymidine Kinase Gene Sequences into the OCT4 Locus of Human Induced Pluripotent Stem Cells.
Ou W, Li P, Reiser J

J Biol Chem 2013 Jun 7;288(23):16895-904
Acquired substrate preference for GAB1 protein bestows transforming activity to ERBB2 kinase lung cancer mutants.
Fan YX, Wong L, Marino MP, Ou W, Shen Y, Wu WJ, Wong KK, Reiser J, Johnson GR

PLoS One 2013 May 22;8(5):e64389
Regulated expression of lentivirus-mediated GDNF in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro.
Yang WH, Yang C, Xue YQ, Lu T, Reiser J, Zhao LR, Duan WM

J Innate Immun 2013;5(3):219-30
Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils.
Zhou Y, Song K, Painter RG, Aiken M, Reiser J, Stanton BA, Nauseef WM, Wang G

Gene Ther 2013 Jan;20(1):43-50
Rapid titration of retroviral vectors using a beta-lactamase protein fragment complementation assay.
Ou W, Marino MP, Lu C, Reiser J

Gene Ther 2012 Dec;19(12):1133-40
Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells.
Goyvaerts C, De Groeve K, Dingemans J, Van Lint S, Robays L, Heirman C, Reiser J, Zhang XY, Thielemans K, De Baetselier P, Raes G, Breckpot K

Hum Gene Ther Methods 2012 Apr;23(2):137-47
Specific targeting of human interleukin (IL)-13 receptor alpha2-positive cells with lentiviral vectors displaying IL-13.
Ou W, Marino MP, Suzuki A, Joshi B, Husain SR, Maisner A, Galanis E, Puri RK, Reiser J

Mol Pain 2011 Aug 23;7:63
Lentiviral gene transfer into the dorsal root ganglion of adult rats.
Yu H, Fischer G, Jia G, Reiser J, Park F, Hogan QH

Retrovirology 2010 Jan 25;7:3
Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus.
Zhang XY, Kutner RH, Bialkowska A, Marino MP, Klimstra WB, Reiser J

J Virol Methods 2009 May;157(2):113-21
Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.
Kuroda H, Kutner RH, Bazan NG, Reiser J

Nat Protoc 2009;4(4):495-505
Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.
Kutner RH, Zhang XY, Reiser J

Cloning Stem Cells 2009 Mar;11(1):167-76
Generation of domestic transgenic cloned kittens using lentivirus vectors.
Gómez MC, Pope CE, Kutner RH, Ricks DM, Lyons LA, Ruhe MT, Dumas C, Lyons J, Dresser BL, Reiser J

BMC Biotechnol 2009 Feb 16;9:10
Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.
Kutner RH, Puthli S, Marino MP, Reiser J

J Gene Med 2008 Nov;10(11):1163-75
A comparative analysis of constitutive and cell-specific promoters in the adult mouse hippocampus using lentivirus vector-mediated gene transfer.
Kuroda H, Kutner RH, Bazan NG, Reiser J

J Immunol 2008 Sep 1;181(5):3049-56
TRIF and IRF-3 binding to the TNF promoter results in macrophage TNF dysregulation and steatosis induced by chronic ethanol.
Zhao XJ, Dong Q, Bindas J, Piganelli JD, Magill A, Reiser J, Kolls JK

Stem Cells Dev 2008 Jun;17(3):441-50
Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells.
Ricks DM, Kutner R, Zhang XY, Welsh DA, Reiser J

J Neurochem 2008 Feb;104(4):1055-64
Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain.
Zhang XY, Dinh A, Cronin J, Li SC, Reiser J

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