Cell-specific And Gene-specific Targeting of Gene Therapy Vectors
Jakob Reiser, Ph.D.
Office of Tissues and Advanced Therapies
Division of Cellular and Gene Therapies
Gene Transfer and Immunogenicity Branch
Biosketch
I received a Ph.D. in biochemistry from the University of Basel in Switzerland, after which I obtained training in virology and protein chemistry as an American Cancer Society Fellow in the Department of Biochemistry, Stanford University School of Medicine. While at Stanford I studied proteins encoded by Simian virus 40—work that supported development of the original Western blot technique. I also developed a novel chromatin immunoprecipitation assay that maps binding sites of specific DNA binding proteins on viral chromatin.
In 1994, I joined the NIH/NINDS to work on viral vectors for gene therapy, focusing primarily on the design of novel vector systems for transgene delivery into nondividing cells. I was among the first to design lentiviral vectors based on HIV-1. My paper was one of the three original reports that emerged on the subject in 1996.
In 1999 I joined the LSU Health Sciences Center in New Orleans as an associate professor of medicine and to serve as the director of the LSU Gene Therapy Vector Core. My goal was to develop improved gene and protein transfer strategies for the CNS. My work dealing with lentiviral vectors and transgene delivery to the CNS was supported by R01 and R21 grants from the NIH, on which I was the PI.
My research at CBER has focused on three of the major limitations of lentiviral vectors for clinical gene therapy, including the potential of such vectors to activate oncogenes during random integration into the host genome and to transduce cells in off-target tissues in vivo. Additionally, we are working on issues related to scaled-up lentiviral vector manufacturing.
To address these shortcomings, I am developing safer HIV-1-based lentiviral vectors by, 1) limiting their integration to well-defined sites in the human genome, and 2) narrowing their tissue tropism through targeted transduction. In addition, I am working on improved manufacturing strategies for lentiviral vectors.
General Overview
Gene therapy holds great potential for treating a variety of serious diseases, some of which are 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 two and a half decades, 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 studies in Europe, 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 do not pose these risks when used to treat patients. 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 ex vivo and in vivo. While these vectors are promising, they pose the risk of activating oncogenes or disrupting critical cellular genes during random integration into the host genome.
Our goal is to develop safer HIV-1-based lentiviral 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 lentiviral vectors that integrate through homology directed repair (HDR) at the AAVS1 "safe harbor" locus that is present on chromosome 19. HDR events are augmented via nuclease-induced DNA damage, mediated by zinc finger nucleases (ZFNs), the CRISPR/Cas9 components, or the AAV2-derived Rep 78 protein.
Targeted gene delivery involves broadening the in vivo tropism of vectors to transduce previously non-permissive cells or replacing the viral tropism to transduce specific target cells exclusively. These approaches offer potential advantages of enhanced therapeutic effects and reduced side effects. We are in the process of testing targetable lentiviral vectors bearing a cell-binding domain linked to the vector's membrane and a separate fusion domain. Our focus has been on using interleukin 13 (IL-13)-displaying vectors to target IL-13-receptor-alpha 2 -overexpressing cancer cells in vitro and in vivo. We use a broad range of approaches, including cell culture, and molecular biological and virological techniques to construct, manufacture and test new HIV-1-based lentiviral vectors. Our work emphasizes targetable vectors for cell-specific transduction and vectors capable of site-specific integration.
Our research allows us to evaluate the technologies used to construct, manufacture, and test novel lentiviral 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 lentiviral vectors for clinical use.
Publications
- Mol Ther Methods Clin Dev 2020 Jul 9;18:631-8
Design and testing of vector-producing HEK293T cells bearing a genomic deletion of the SV40 T antigen coding region.
Bae DH, Marino M, Iaffaldano B, Fenstermaker S, Afione S, Argaw T, McCright J, Kwilas A, Chiorini JA, Timmons AE, Reiser J - Oncotarget 2018 Aug 24;9(66):32718-29
A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis.
Gill J, Moret R, Zhang X, Nelson J, Maresh G, Hellmers L, Canter D, Hudson M, Halat S, Matrana M, Marino MP, Reiser J, Shuh M, Laborde E, Latsis M, Talwar S, Bardot Li L - Hum Gene Ther Methods 2018 Jun;29(3):135-45
Efficiency and specificity of targeted integration mediated by the AAV2 Rep 78 protein.
Li P, Marino MP, Zou J, Argaw T, Morreale MT, Iaffaldano BJ, Reiser J - 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