895. Age-Dependent Increases in Lentiviral Vector Transduction and Transgene Expression In Vivo

AbstractUbiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

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Prolonged transgene expression in murine salivary glands following non-primate lentiviral vector transduction

Molecular Therapy ◽

10.1016/j.ymthe.2005.02.022 ◽

2005 ◽

Vol 12 (1) ◽

pp. 137-143 ◽

Cited By ~ 14

Author(s):

Ela Shai ◽

Aaron Palmon ◽

Amos Panet ◽

Yitzhak Marmary ◽

Yoav Sherman ◽

...

Keyword(s):

Salivary Glands ◽

Transgene Expression ◽

Lentiviral Vector ◽

Vector Transduction

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Abstract 2672: Serial in vivo Non-invasive Imaging of Intramyocardially Injected Autologous Mesenchymal Stem Cells, Modified for Transgene Expression of Positron-Emission Tomography (PET) Reporter in Porcine Myocardial Infarction

Circulation ◽

10.1161/circ.116.suppl_16.ii_592-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Mariann Gyongyosi ◽

Jeronimo Blanco ◽

Terez Marian ◽

Ors Petnehazy ◽

Rayyan Hemetsberger ◽

...

Keyword(s):

Myocardial Infarction ◽

Stem Cells ◽

Pet Imaging ◽

Transgene Expression ◽

Lentiviral Vector ◽

Border Zone ◽

Percutaneous Balloon ◽

Diffuse Distribution

Objective. The aim of our study was to elaborate an in vivo tracking method of the intramyocardially injected msenchymal stem cells (MSCs), modified for transgene expression of trifusion protein (lentiviral vector, expressing renilla luciferase /RL/, red fluoroscein protein /RFP/ and herpes simplex truncated thymidine kinase /tTK, positron emmission tomography PET-reporter gene/) using serial PET imaging in pig myocardial infarction (MI). Methods. Bone marrow (100 ml) was harvested from pigs immediately before induction of MI by percutaneous balloon occlusion of the LAD followed by reperfusion. The MSCs were selected and cultivated. The lentiviral vector LV-RL-RFP-tTK was inoculated into the MSCs under control of CMV promoter. The cells with the highest fluorescence intensity (after achieving appr. 50% transfection efficacy) were sorted, and tracked in vitro by PET using 9-(4-[18F]fluoro-3-hydroxymethylbutyl)-guanine (18FHBG). The transfected MSCs (at least 3 mio MSCs/ pigs) were then injected direct intramyocardially using NOGA electromagnetic guidance in pigs in 10 locations of the infarct border zone (min. 0.3 million cells/injection), followed by PET imaging 30 hours and 7 days later, after intravenous injection of 5 mCi 18FHBG. Results. The in vitro 18FHBG uptakes of the transgene modified MSCs by PET were 10 times larger than the control JY human B-lymphoblasts and T lymphocytes. The minimum number of the cells detectable with PET was 0.2 million. MRI of the pigs revealed a mean global EF of 47+/-3.5%. PET imaging displayed diffuse distribution of the injected MSCs with high activity of the PET tracer in the anterior wall and septum at 30h, and less tracer activity in the injections sites with diffuse distribution in the pericardium and pleura indicating the wandering of the living cells at 7 days. PET imaging did not show 18FHBG accumulation in the infarcted heart of the control animals. Myocardial histology with RL and RFP staining confirmed the distribution of the injected MSCs through an elongated track around the injected area, 9 days after delivery. Conclusion. In vivo tracking of gene-modified porcine MSCs by PET imaging is feasible and allows serial noninvasive imaging of homing and propagation of MSCs in pigs after MI.

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A lentiviral vector with novel multiple cloning sites: Stable transgene expression in vitro and in vivo

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2008.04.106 ◽

2008 ◽

Vol 371 (3) ◽

pp. 546-550 ◽

Cited By ~ 23

Author(s):

C.V. Santhosh ◽

Mayur C. Tamhane ◽

Rohan H. Kamat ◽

Vainav V. Patel ◽

Robin Mukhopadhyaya

Keyword(s):

Transgene Expression ◽

Lentiviral Vector ◽

Multiple Cloning Sites

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563. In Vivo Derivation of HIV-1 Resistant Transgenic T Cells by Lentiviral Vector Transduction of a Triple Combination of Anti-HIV Genes Anti-CCR5 Ribozyme, Tat-Rev siRNA and a TAR Decoy

Molecular Therapy ◽

10.1016/s1525-0016(16)44769-6 ◽

2007 ◽

Vol 15 ◽

pp. S217

Keyword(s):

T Cells ◽

Lentiviral Vector ◽

Triple Combination ◽

Anti Hiv ◽

Hiv 1 ◽

Vector Transduction

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Codon Swapping of Zinc Finger Nucleases Confers Expression in Primary Cells and In Vivo from a Single Lentiviral Vector

Current Gene Therapy ◽

10.2174/156652321405140926161748 ◽

2014 ◽

Vol 14 (5) ◽

pp. 365-376 ◽

Cited By ~ 5

Author(s):

Abarrategui-Pontes Cecilia ◽

Creneguy Alison ◽

Thinard Reynald ◽

Fine J. ◽

Thepenier Virginie ◽

...

Keyword(s):

Zinc Finger ◽

Lentiviral Vector ◽

Zinc Finger Nucleases ◽

Primary Cells

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Abrogation of postentry restriction of HIV-1-based lentiviral vector transduction in simian cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0337541100 ◽

2003 ◽

Vol 100 (3) ◽

pp. 1298-1303 ◽

Cited By ~ 128

Author(s):

N. A. Kootstra ◽

C. Munk ◽

N. Tonnu ◽

N. R. Landau ◽

I. M. Verma

Keyword(s):

Lentiviral Vector ◽

Hiv 1 ◽

Vector Transduction

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Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽

10.3390/v13071288 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1288

Author(s):

Wendy Dong ◽

Boris Kantor

Keyword(s):

Large Scale ◽

Lentiviral Vector ◽

Clinical Applications ◽

Scale Production ◽

Base Editing ◽

Epigenome Editing ◽

Vector Systems ◽

Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

Viruses ◽

10.3390/v13071387 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1387

Author(s):

Yukiko Otsuka ◽

Hitomi Tsuge ◽

Shiori Uezono ◽

Soshi Tanabe ◽

Maki Fujiwara ◽

...

Keyword(s):

Gene Transfer ◽

Immune Responses ◽

Envelope Glycoprotein ◽

Transgene Expression ◽

Histological Analysis ◽

Lentiviral Vector ◽

Cortical Input ◽

Input System ◽

Vesicular Stomatitis ◽

Retrograde Gene Transfer

For achieving retrograde gene transfer, we have so far developed two types of lentiviral vectors pseudotyped with fusion envelope glycoprotein, termed HiRet vector and NeuRet vector, consisting of distinct combinations of rabies virus and vesicular stomatitis virus glycoproteins. In the present study, we compared the patterns of retrograde transgene expression for the HiRet vs. NeuRet vectors by testing the cortical input system. These vectors were injected into the motor cortex in rats, marmosets, and macaques, and the distributions of retrograde labels were investigated in the cortex and thalamus. Our histological analysis revealed that the NeuRet vector generally exhibits a higher efficiency of retrograde gene transfer than the HiRet vector, though its capacity of retrograde transgene expression in the macaque brain is unexpectedly low, especially in terms of the intracortical connections, as compared to the rat and marmoset brains. It was also demonstrated that the NeuRet but not the HiRet vector displays sufficiently high neuron specificity and causes no marked inflammatory/immune responses at the vector injection sites in the primate (marmoset and macaque) brains. The present results indicate that the retrograde transgene efficiency of the NeuRet vector varies depending not only on the species but also on the input projections.

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Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing

Nucleic Acids Research ◽

10.1093/nar/gkaa1152 ◽

2020 ◽

Author(s):

Joost van Haasteren ◽

Altar M Munis ◽

Deborah R Gill ◽

Stephen C Hyde

Keyword(s):

Long Range ◽

Insertional Mutagenesis ◽

Lentiviral Vector ◽

Integration Site ◽

Low Cost ◽

Safety Evaluation ◽

Read Length ◽

Chromosomal Dna ◽

Wide Range

Abstract The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

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