High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2

Uchida, Naoya; Nassehi, Tina; Drysdale, Claire; Gamer, Jackson; Yapundich, Morgan; Demirci, Selami; Haro‐Mora, Juan J.; Leonard, Alexis; Hsieh, Matthew M.; Tisdale, John F.

doi:10.1016/j.omtm.2019.01.005

Cited by 33 publications

(25 citation statements)

References 27 publications

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“…PGE2 acts by enhancing intracellular cAMP leading to an improvement in viral transduction. The combination of these two molecules (P407 and PGE2) has been previously shown improve lentiviral transduction in CD34+ cells with high-efficiency and without significant cytotoxicity ( 32 ).…”

Section: Methodsmentioning

confidence: 99%

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol

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Lambert

Dykxhoorn

et al. 2021

Front. Cardiovasc. Med.

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The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches.

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Section: Methodsmentioning

confidence: 99%

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol

Capcha

Lambert

Dykxhoorn

et al. 2021

Front. Cardiovasc. Med.

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“…9). In addition, we recently demonstrated more efficient transduction methods in human CD34 + cells in high-density culture with adjuvants 47 . Improvement of transduction efficiency and transgene expression might allow for higher amounts of human β-globin production at the protein level in transplanted rhesus macaques and, ultimately, in humans.…”

Section: Discussionmentioning

confidence: 99%

Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders

et al. 2019

Self Cite

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Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented β-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and β-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.

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“…Among the tested three combinations, the one with the lowest content of cytokines showed the highest VCN and maintained the highest CD34+ percentage following LV transduction ( Figure 3A). Second, we assessed distinct transduction enhancers, including protamine, dmPGE2, UM171, and Poloxamer407 [40][41][42] and tested their individual or combinational effects on LV transduction in mPB HSCs. We found that dmPGE2 and Poloxamer407 combination (PGE-Pol) was the most efficient, which increased the VCN by 6-7 times when compared with enhancer free control ( Figure 3B).…”

Section: Optimization Of Lentiviral Transduction In Mobilized Periphementioning

confidence: 99%

Restoration of β-globin expression with optimally designed lentiviral vector for β-thalassemia treatment in Chinese patients

Ouyang

Dong

Zhao

et al. 2020

Preprint

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Abstractβ-thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for β–thalassemia is allogeneic hematopoietic stem cell transplantation (HSCT), which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-ependent β-thalassemia (TDT). However, successful gene therapy for β-thalassemia patients in China has not been reported. Here we present the results of preclinical studies of an optimally designed LV named LentiHBBT87Q in hematopoietic stem cells (HSCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q β-globin gene (HBBT87Q) driven by a specific reconstituted locus control region (rLCR) and efficiently express HBB mRNA and HBB protein in erythroblasts derived from cord blood (CB) HSCs. To facilitate clinical transformation, we manufactured clinical grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Qrestored expression of HBB monomer and adult hemoglobin (HbA) tetramer to relatively normal level in erythroblasts from bone marrow (BM) HSCs of Chinese TDT patients, that carry the most common mutation types and cover various genotypes, including β0/β0. Furthermore, viral integration sites (VIS) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials and the associated risk of tumorigenesis was not observed in cLentiHBBT87Q transduced HSCs through comprehensive analysis. Taken together, we have engineered the cLentiHBBT87Q that can restore β-globin expression in the HSCs-derived erythroblasts of Chinese TDT patients with minimal risk on tumorigenesis, providing a favorable starting point for future clinical application.

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High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2

Cited by 33 publications

References 27 publications

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol

Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders

Restoration of β-globin expression with optimally designed lentiviral vector for β-thalassemia treatment in Chinese patients

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