The Engraftment of Lentiviral Vector-Transduced Human CD34+ Cells into Humanized Mice

2019 ◽  
pp. 91-100
Author(s):  
Yoon-Sang Kim ◽  
Matthew Wielgosz ◽  
Byoung Ryu
Keyword(s):  
Lentiviral Vector ◽  
Humanized Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesis

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3326-3335 ◽  
Author(s):  
Emmanuel Ravet ◽  
Damien Reynaud ◽  
Monique Titeux ◽  
Brigitte Izac ◽  
Serge Fichelson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Lentiviral Vector ◽  
Erythroid Differentiation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Gene Expressions ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Colony Forming Units ◽  
Independent Functions

Abstract The transcription factor TAL1 has major functions during embryonic hematopoiesis and in adult erythropoiesis and megakaryocytopoiesis. These functions rely on different TAL1 structural domains that are responsible for dimerization, transactivation, and DNA binding. Previous work, most often done in mice, has shown that some TAL1 functions do not require DNA binding. To study the role of TAL1 and the relevance of the TAL1 DNA-binding domain in human erythropoiesis, we developed an approach that allows an efficient enforced wild-type or mutant TAL1 protein expression in human hematopoietic CD34+ cells using a lentiviral vector. Differentiation capacities of the transduced cells were studied in a culture system that distinguishes early and late erythroid development. Results indicate that enforced TAL1 expression enhances long-term culture initiating cell (LTC-IC) potential and erythroid differentiation of human CD34+ cells as shown by increased βglobin and porphobilinogen deaminase (PBGD) gene expressions and erythroid colony-forming units (CFU-Es), erythroid burst-forming units (BFU-Es), and glycophorin A-positive (GPA+) cell productions. Enforced expression of a TAL1 protein deleted of its DNA-binding domain (named ΔbTAL1) mimicked most TAL1 effects except for the LTC-IC enhancement, the down-regulation of the CD34 surface marker, and the GPA+ cell production. These results provide the first functional indications of DNA-binding-dependent and -independent roles of TAL1 in human erythropoiesis. (Blood. 2004;103:3326-3335)

scholarly journals Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4378-4378
Author(s):  
Yasuyuki Saito ◽  
Jana M. Ellegast ◽  
Rouven Müller ◽  
Richard A. Flavell ◽  
Markus G. Manz
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Organs ◽  
Humanized Mice ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Lymphoid System ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014). To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice. Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals. Disclosures No relevant conflicts of interest to declare.

scholarly journals Preclinical Safety and Efficacy of Human CD34+ Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome

2013 ◽  
Vol 21 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Samantha Scaramuzza ◽  
Luca Biasco ◽  
Anna Ripamonti ◽  
Maria C Castiello ◽  
Mariana Loperfido ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Safety And Efficacy ◽  
Wiskott Aldrich Syndrome ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Preclinical Safety

scholarly journals Human CD34+ cell isolation from fetal liver, and fetal thymus preparation v1

2021 ◽  
Author(s):  
Austin Chen ◽  
Mohsen Khosravi-Maharlooei ◽  
Markus Holzl ◽  
Nichole Danzl ◽  
Chris Parks ◽  
...  
Keyword(s):  
Immune Cells ◽  
Mixed Lymphocyte Reaction ◽  
Fetal Liver ◽  
Humanized Mice ◽  
Hematopoietic Progenitor ◽  
Fetal Thymus ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Bone Marrow Ablation ◽  
Marrow Ablation

This protocol details the steps for isolating human CD34+ cells from human fetal liver. It also explains how to prepare human fetal thymus for immediate use or for freezing, as well as the process for thawing. The CD34+ cells are hematopoietic progenitor cells and can be used to generate humanized mice through reconstitution of immune cells via IV injection after bone marrow ablation. These cells can also be used for mixed lymphocyte reaction experiments.

scholarly journals Kinetics of lentiviral vector transduction in human CD34+ cells

2016 ◽  
Vol 44 (2) ◽  
pp. 106-115 ◽  
Author(s):  
Naoya Uchida ◽  
Rashidah Green ◽  
Josiah Ballantine ◽  
Luke P. Skala ◽  
Matthew M. Hsieh ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Kinetics Of ◽  
Vector Transduction

Regulatory-Approval Driven Safety and Efficacy of MGMTP140K Lentivirus Transduction of Human CD34+ Cells for a Phase I Clinical Trial for Upfront Treatment of Glioma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4177-4177
Author(s):  
Yulan Qing ◽  
Jane Reese ◽  
Min Liu ◽  
Amar Desai ◽  
Heather Embree ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Clinical Grade ◽  
Clinical Trial Application ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Selection Of

Abstract Abstract 4177 Temozolomide (TMZ) is a standard of care treatment for patients with glioma post surgical resection, but induces dose limiting myelosuppresion. Gene transfer of O6-benzylguanine (BG) resistant mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs in mice, dogs and primates. We designed a clinical trial to improve HSC tolerance of TMZ in patients with glioma [clinicaltrials.gov]. This trial uses a lentiviral backbone for gene delivery, and infuses cells during the active phase of treatment to maximize the potential for stem cell protection and selection. The viral construct was synthesized by Lentigen, with an EF1-alpha promoter driving MGMTP140K (LV-P140K MGMT). Clinical grade vector was subject to safety and toxicity analysis at the request of both NCI and FDA. Transduction conditions were optimized towards a high rate of gene transfer and expression with ≤2 copies per cell to minimize adverse insertions. This study presents the validation of the vector by assessing the engraftment, drug resistance and copy number after gene transfer into human CD34+ cells followed by in vivo selection using a nonobese diabetic (NOD)/severe combined immunodeficient (SCID) gammaC (NSG) mice. We are currently analyzing the insertion sites of the clinical grade LV-P140K MGMT in human cord blood progenitors as a safety monitoring tool for LV-HSC gene therapy. Engraftment of LV-P140K MGMT (LG631) transduced CD34+ cells in irradiated NSG mice, and the analysis of engrafted human CD45+ cells in peripheral blood, spleen and bone marrow showed consistent but slightly decreased (10%) engraftment of transduced cells compared to the freshly isolated non transduced cells and was multilineage. There was no toxicity to the mice or human HSC as a result of transduction with lentiviral vector. The NSG recipients of the LG631 transduced CD34+ cells received two rounds of 3-consecutive day treatments of 30 mg/kg of BG and 60 mg/kg of TMZ, and three weeks after the second round of treatment, expression of MGMT was examined by PCR in the CFUs from the BM of the recipient mice. Combined treatment with BG and TMZ showed efficient selection of human cells and human CFU expressing MGMT (100% after treatment vs 44+/−4.1% without treatment). Copy number and characterization of insertion sites of the clinical grade LV-P140K MGMT in human CD34 cells serve as a critical safety monitoring tool for LV-HSC gene therapy. Insertion site analysis showed a polyclonal pattern and selection did not result in oligoclonality. The insertion database will continue to be made publically available as the data is generated prior to and during clinical trial application. We have fully characterized the preclinical safety and efficacy of a clinical grade lentiviral vector for clinical trial application in patients with glioma. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document