scholarly journals Construction and Molecular Analysis of Gene Transfer Systems Derived from Bovine Immunodeficiency Virus

2001 ◽  
Vol 75 (7) ◽  
pp. 3371-3382 ◽  
Author(s):  
Robert Berkowitz ◽  
Heini Ilves ◽  
Wei Yu Lin ◽  
Karl Eckert ◽  
Andrea Coward ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Target Cell ◽  
Expression System ◽  
Cell Types ◽  
Human Cells ◽  
Bovine Immunodeficiency Virus ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Immunodeficiency Virus ◽  
Immortalized Cell

ABSTRACT Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 × 105infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.

scholarly journals Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1016 ◽  
Author(s):  
Alejandra Gutierrez-Guerrero ◽  
François-Loïc Cosset ◽  
Els Verhoeyen
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Mammalian Cells ◽  
Measles Virus ◽  
Cell Types ◽  
Transduction Efficiency ◽  
Human Immune Deficiency Virus ◽  
Hematopoietic Stem ◽  
Monogenic Diseases ◽  
Viral Envelopes

Viruses have been repurposed into tools for gene delivery by transforming them into viral vectors. The most frequently used vectors are lentiviral vectors (LVs), derived from the human immune deficiency virus allowing efficient gene transfer in mammalian cells. They represent one of the safest and most efficient treatments for monogenic diseases affecting the hematopoietic system. LVs are modified with different viral envelopes (pseudotyping) to alter and improve their tropism for different primary cell types. The vesicular stomatitis virus glycoprotein (VSV-G) is commonly used for pseudotyping as it enhances gene transfer into multiple hematopoietic cell types. However, VSV-G pseudotyped LVs are not able to confer efficient transduction in quiescent blood cells, such as hematopoietic stem cells (HSC), B and T cells. To solve this problem, VSV-G can be exchanged for other heterologous viral envelopes glycoproteins, such as those from the Measles virus, Baboon endogenous retrovirus, Cocal virus, Nipah virus or Sendai virus. Here, we provide an overview of how these LV pseudotypes improved transduction efficiency of HSC, B, T and natural killer (NK) cells, underlined by multiple in vitro and in vivo studies demonstrating how pseudotyped LVs deliver therapeutic genes or gene editing tools to treat different genetic diseases and efficiently generate CAR T cells for cancer treatment.

Efficient Oncoretroviral Gene Transfer in Primary, Secondary and Tertiary NOD/SCID Mice under Serum-Free Conditions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5252-5252
Author(s):  
Loretta Gammaitoni ◽  
Simona Lucchi ◽  
Stefania Bruno ◽  
Melania Tesio ◽  
Monica Gunetti ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Clinical Application ◽  
Ex Vivo ◽  
Scid Mice ◽  
Transduction Efficiency ◽  
Proliferative Potential ◽  
Major Step ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Serum Free

Abstract Stable oncoretroviral gene transfer into hematopoietic stem cells (HSC) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSC to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells the role of ex-vivo expansion was investigated. For a possible clinical application all experiments were done according to good anufacturing practice (GMP) guidelines. The combination of Flt3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO), and Interleukin-6 (IL6) has been shown to stimulate proliferation and self-renewal of very primitive (SCID-repopulating cells, SRC) hematopoietic cells. We asked whether it is possible to efficiently transduce HSC with oncoretroviral vector, to expand them and whether transduced cells retain their self-renewal potential, as demonstrated by their capacity to efficiently and serially engraft NOD/SCID mice A Gibbon ape leukemia virus (GALV)-pseudotyped vectors already approved for clinical application has been used to efficiently and durably deliver a defective, non functional form of the cell surface marker truncated low affinity nerve growth factor receptor (LNGFR) into primitive cord blood (CB) HSC. The transduction was performed, following an up-to 24 hour exposure to FL, TPO, IL-6 and SCF, in the presence of the growth factors in serum-free (SF) medium on retronectin (RT) coated plates. At day 3 post-transduction, total cells and CD34+ cells were expanded 24-fold and 8.5-fold respectively. More than 40% of the cells were CD34+. Transduction efficiency was >55%. Serial transplantation is the most reliable method to assess the stable expression of a gene in cells with high proliferative potential. Mice transplanted with transduced or mock-transduced, expanded cells showed higher levels of human engraftment than those transplanted with unmanipulated cells (56.7%, 55% and 39.9% respectively). LNGFR expression of CD45+ cells was 14.35± 4.27%. All secondary mice transplanted with cells from primary recipient BM resulted engrafted (21.7%, 13.5 and 2.8% respectively). LNGFR expression was 47.54± 3.1% respectively of human CD45+ cells. BM cells from secondary recipients were used for tertiary transplants. Only mice transplanted with expanded cells were positively engrafted. Two mice out of five transplanted with secondary recipient BM cells derived from mice transplanted with transduced and expanded cells, showed good levels of human engraftment (6.15% CD45+). LNGFR expression was 49.1± 4.4% of human CD45+ cells. FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34+), B (CD19+), myeloid (CD14+), erythroid (GpA+) and megakaryocyte cells (CD41+) in equivalent proportion. BM of the engrafted mice was placed in a human colony assay. Human colonies also were generated from the murine BM. In conclusion, we have validated a SF-protocol for efficient gene transfer into human CB HSC using a retroviral vector. Under these conditions, transduced and expanded cells repopulated NOD/SCID mice for 3 generations with a human multilineage graft stably expressing the transgene. In a view of future clinical applications, this protocol represents a major step towards the achievement of this goal.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 3960-3967 ◽  
Author(s):  
Peter A. Horn ◽  
Max S. Topp ◽  
Julia C. Morris ◽  
Stanley R. Riddell ◽  
Hans-Peter Kiem
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Cell Line ◽  
Flow Cytometric Analysis ◽  
Lentiviral Vectors ◽  
Transduction Efficiency ◽  
Packaging Cell Line ◽  
Hematopoietic Stem ◽  
Packaging Cell ◽  
Cd34 Cells

Vector-containing medium harvested from murine packaging cell lines has been shown to contain factors that can negatively influence the transduction and maintenance of hematopoietic stem cells. Thus, we generated a human packaging cell line with a gibbon ape leukemia virus pseudotype (Phoenix-GALV), and we evaluated vectors produced by Phoenix-GALV for their ability to transduce hematopoietic progenitor/stem cells. In 3 baboons, we used a competitive repopulation assay to directly compare GALV-pseudotype retrovirus vectors produced by either Phoenix-GALV or by the NIH 3T3–derived packaging cell line, PG13. In 3 additional baboons we compared Phoenix-GALV–derived vectors to more recently developed lentiviral vectors. Gene transfer efficiency into hematopoietic repopulating cells was assessed by evaluating the number of genetically modified peripheral blood and marrow cells using flow cytometry and real-time polymerase chain reaction. Transduction efficiency of hematopoietic repopulating cells was significantly higher using the Phoenix-GALV–derived vector as compared with the PG13-derived vectors or lentiviral vectors, with stable transduction levels up to 25%. We followed 2 animals for more than one year. Flow cytometric analysis of hematopoietic subpopulations in these animals revealed transgene expression in CD13+ granulocytes, CD20+ B lymphocytes, CD3+ T lymphocytes, CD61+ platelets, as well as red blood cells, indicating multilineage engraftment of cells transduced by Phoenix-GALV–pseudotype vectors. In addition, transduction of human CD34+ cells was significantly more efficient than transduction of baboon CD34+ cells, suggesting that Phoenix-GALV–derived oncoretroviral vectors may be even more efficient in human stem cell gene therapy applications.

scholarly journals Transduction of Human Progenitor Hematopoietic Stem Cells by Human Immunodeficiency Virus Type 1-Based Vectors Is Cell Cycle Dependent

1999 ◽  
Vol 73 (5) ◽  
pp. 3649-3660 ◽  
Author(s):  
Richard E. Sutton ◽  
Michael J. Reitsma ◽  
Nobuko Uchida ◽  
Patrick O. Brown
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Human Immunodeficiency Virus ◽  
Hematopoietic Stem Cells ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Immunodeficiency Virus ◽  
Lentivirus Vectors ◽  
Cycle Dependent

ABSTRACT Human immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G2/M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G1 or S/G2/M fraction were more readily transduced than G0 cells. Maintaining the cells in G0 by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G0 cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G0 and enter G1. Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.

Long-Term Polyclonal and Stable Gene Transfer into Rhesus Repopulating Hematopoietic Stem Cells Using a Simian Immunodeficiency Virus-Based Lentiviral Vector System.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3259-3259
Author(s):  
Yoo-Jin Kim ◽  
Nadia L. Hussein ◽  
Peiman Hematti ◽  
Bum-Kee Hong ◽  
Boris Calmels ◽  
...  
Keyword(s):  
B Cells ◽  
Gene Transfer ◽  
Simian Immunodeficiency Virus ◽  
Vector System ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
High Level

Abstract Murine leukemia virus (MLV) vectors have been studied extensively in animal models and utilized for over a decade in clinical trials of gene therapy directed at hematopoietic stem and progenitor cells MLV have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression. More recently concerns have arisen regarding their safety regarding activation of adjacent proto-oncogenes and resultant leukemogenesis. We have previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus CD34+ cells, resulting in high-level in vivo marking with transduced progeny cells up to one year post-transplantation.(Hanawa et al, 2004) A comparison of vector integration sites in these animals compared to animals receiving MLV-transduced cells revealed different patterns, showing that SIV integrants strongly favored entire transcription units and gene-dense regions of the genome, compared to MLV that favored regions surrounding transcription start sites.(Hematti et al, 2004). Animals receiving MLV-transduced cells had highly non-random engraftment with integrants in or near the the MDS1/EVI1 gene complex. To evaluate long-term safety implications of the SIV vector-mediated CD34+ cell gene transfer, we analyzed the insertional sites in granulocytes, T cell, and B cells from 3 rhesus macaques which were transplanted three years ago with transduced, autologous cytokine-mobilized peripheral blood CD34+ cells. All three animals continued to show significant marking and expression levels in T cells, B cells and granulocytes, with mean GFP + levels of 6.7% (range, 3.3–13.0%), 7.4% (4.2–13.4%) and 5.6% (3.1–10.5%), respectively. Vector insertion site analysis by linear amplification-mediated PCR method at three years continued to show highly polyclonal reconstitution. Subsequent cloning and sequencing data confirmed long-term polyclonality with vector-containing cells and there was no evidence for any worrisome common integration sites, with no integrants detected in the MDS1/EVI1 region, in contrast to results with the MLV vector. These results indicate that the SIV vector system can result in stable and efficient long-term expression in progeny of transduced CD34+ cells, without the worrisome integration profile previously reported in our model with MLV vectors.

scholarly journals In Vivo Gene Transfer Using a Nonprimate Lentiviral Vector Pseudotyped with Ross River Virus Glycoproteins

2002 ◽  
Vol 76 (18) ◽  
pp. 9378-9388 ◽  
Author(s):  
Yubin Kang ◽  
Colleen S. Stein ◽  
Jason A. Heth ◽  
Patrick L. Sinn ◽  
Andrea K. Penisten ◽  
...  
Keyword(s):  
Gene Transfer ◽  
G Protein ◽  
Lentiviral Vector ◽  
Transduction Efficiency ◽  
Ross River Virus ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Pseudotyped Vector ◽  
In Vivo Gene Transfer

ABSTRACT Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 × 108 TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.

scholarly journals Efficient Gene Transfer into Human CD34+ Cells by a Retargeted Adenovirus Vector

2000 ◽  
Vol 74 (6) ◽  
pp. 2567-2583 ◽  
Author(s):  
Dmitry M. Shayakhmetov ◽  
Thalia Papayannopoulou ◽  
George Stamatoyannopoulos ◽  
André Lieber
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Transduction Efficiency ◽  
Limiting Factor ◽  
Stable Gene ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Specific Subset

ABSTRACT Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and αv integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34+ cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34+ cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an αv integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34+ cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34+cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34+ cells expressing αv integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34+ cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34+ cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34+ c-Kit+cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34+ c-Kit+ cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

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