Stoichiometric limitations in assembly of active recombinant retrovirus

Lei, Pedro; Andreadis, Stelios T.

doi:10.1002/bit.20468

Cited by 9 publications

(4 citation statements)

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“…The next day, the medium was replaced with 30mL of DMEM+10%FBS containing sodium butyrate (10mM) that was previously shown to improve virus production [43]. Two days later, the supernatant was harvested, filtered, ultra-centrifuged at 50,000g for 2 hr and the lentiviral pellet was dissolved by 300μL fresh medium (DMEM+10%FBS) to yield 100x concentrated lentivirus stock.…”

Section: Methodsmentioning

confidence: 99%

Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs

Liang¹,

Andreadis²

2011

Biomaterials

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We present a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-β1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-β1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-β1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.

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Section: Methodsmentioning

confidence: 99%

Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs

Liang¹,

Andreadis²

2011

Biomaterials

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“…The absence of one of these properties results in the production of deficient, noninfectious vectors. The packaging cell lines currently used to produce RVs are known to yield low infectious titers (10 6 -10 7 infectious particle (IP)/mL) and heterogeneous RVs populations lacking one or more of the elements mentioned above [5]. In addition, RVs biological activity is intrinsically very susceptible to environmental conditions, mainly temperature [6,7].…”

Section: Introductionmentioning

confidence: 99%

Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

Rodrigues

Alves

Lopes

et al. 2008

J of Separation Science

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Original PaperRemoval of envelope protein-free retroviral vectors by anion-exchange chromatography to improve product qualityWe have investigated the role of the retroviral lipid bilayer and envelope proteins in the adsorption of retroviral vectors (RVs) to a Fractogel TM DEAE matrix. Intact RVs and their degradation components (envelope protein-free vectors and solubilized vector components) were adsorbed to this matrix and eluted using a linear gradient. Envelope protein-free RVs (Env -) and soluble envelope proteins (gp70) eluted in a significantly lower range of conductivities than intact RVs (Env + ) (13.7 -30 mS/cm for Env -and gp70 proteins vs. 47 -80 mS/cm for Env + ). The zeta (f)-potential of Env + and Env -vectors was evaluated showing that envelope proteins define the pI of the viral particles (pI (Env + ) a 2 versus 3 a pI (Env -) a 4) and that Env + and Env -vectors have similar f-potentials within pH 5 and 8. The results presented herein indicate that the adsorption of retroviral particles occurs through multi-point interaction of the envelope proteins with the cationic groups on the chromatographic matrix. The strength of this adsorption is thus dependent on the amount of envelope protein present in the viral lipid bilayer. In conclusion, AEXc enables the separation of gp70 proteins as well as envelope protein-free vectors constituting a significant improvement to the quality of retroviral preparations for gene therapy applications.

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“…The cells were resuspended in PBS and analyzed for transduction efficiency and fluorescence intensity using flow cytometer (FACSCalibur; Becton Dickinson, San Jose, California) as described previously [6, 30–31]. …”

Section: Methodsmentioning

confidence: 99%

Fibrin-mediated lentivirus gene transfer: Implications for lentivirus microarrays

Raut

Lei

Padmashali

et al. 2010

Journal of Controlled Release

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We employed fibrin hydrogel as bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 – 7.5 mg/mL. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine.

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Stoichiometric limitations in assembly of active recombinant retrovirus

Cited by 9 publications

References 50 publications

Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs

Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs

Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

Fibrin-mediated lentivirus gene transfer: Implications for lentivirus microarrays

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