Integrated non‐invasive system for quantifying secreted human therapeutic hIL2

Abstract

Cell encapsulation has been used to treat diabetes, amyotrophic lateral sclerosis, and other chronic ailments by the secretion of therapeutic proteins in vivo. Detection of these proteins typically requires invasive procedures such as blood sampling or device extraction, however. In this article, a non-invasive means of measuring secreted protein concentration using a co-expressed red fluorescent protein marker is developed. A bicistronic expression vector was constructed for the intracellular production of a red fluorescent protein marker and the secreted production of human interleukin-2 (hIL2). The destabilized red fluorescent protein, DsExDR, was selected for its rapid turnover, as well as its ability to emit red light, which is readily transmitted through mammalian tissue. Transfections of this bicistronic vector into three cell lines C2C12, HEK293, and Jurkat showed linear correlations between the expressed proteins, DsExDR (intracellular) and hIL2 (secreted), with transfection DNA concentration. Correspondingly, there was a linear correlation between secreted product (hIL2) and intracellular marker (DsExDR). As transfection DNA was increased, Jurkat cells were found to increase secreted hIL2 in direct proportion to the accumulated DsExDR. HEK293 and C2C12 cells expressed and secreted significantly more hIL2 than the Jurkat cells, while still maintaining a linear relationship. Thus, all three cell lines were suitable hosts for the bicistronic expression of DsExDR and expression and secretion of therapeutic hIL2. This reporting strategy may find the greatest use in cell encapsulation therapy.

Diffusion of interleukin‐2 from cells overlaid with cytocompatible enzyme‐crosslinked gelatin hydrogels

“…Cultures transfected with pF3 rather than pF2 had slightly higher levels of red fluorescence at each overlay thickness, demonstrating the protective effects of Bcl‐2Δ 30–32. This finding is consistent with previous results, which showed that Bcl‐2Δ not only lessened necrosis and apoptosis but also augmented protein production in various mammalian cultures 25…”

“…The pF2 vector allowed constitutive secretion of hIL2 using a CMV promoter and tandem intracellular expression of DsExDR using an internal ribosome entry site (IRES). 25 The pF3 vector is the same as pF2 with the addition of an antiapoptotic gene, bcl-2D, placed downstream of a hypoxia inducible promoter, 5HRE-hCMVmp (5HRE). pF3 was constructed by modifying a previously constructed plasmid, pO2Ybp, which contains a 5HRE-EYFP-bcl-2D sequence.…”

“…To model the diffusional environment, human embryonic kidney cells (HEK293) were genetically engineered to secrete hIL2 via two genetic vectors and overlaid with 4% mTG‐gels. The first vector (pF2) encodes a secreteable hIL2 gene along with a bicistronically expressed destabilized DsRed‐Express (DsExDR) fluorescent protein, which we have previously demonstrated to be a highly quantitative marker for this system 25. The second vector (pF3) adds the ability to selectively induce the expression of an antiapoptotic protein, Bcl‐2Δ, through a hypoxia sensitive promoter.…”

“…The first vector (pF2) encodes a secreteable hIL2 gene along with a bicistronically expressed destabilized DsRed-Express (DsExDR) fluorescent protein, which we have previously demonstrated to be a highly quantitative marker for this system. 25 The second vector (pF3) adds the ability to selectively induce the expression of an antiapoptotic protein, Bcl-2D, through a hypoxia sensitive promoter. The diffusion coefficient of hIL2 was determined from steady-state data derived from HEK cells overlaid by various thicknesses of hydrogel (0, 0.10, 0.13, 0.16, 0.19, 0.22, 0.25, and 0.28 cm).…”

Directed assembly of a bacterial quorum

“…Signal transduction-expression of DsRed on per cell basis Plasmids pCT6 and pET200/DsRed (Yung et al, 2006;Tsao et al, 2010) were sequentially transformed into the E. coli W3110 luxS deletion mutant, W3110ΔluxS, and grown in LB media with appropriate antibiotics. Plasmid pCT6 senses and responds to the presence of phosphorylated AI-2 by the expression of T7 RNA polymerase, which, in turn, activates T7 polymerase-mediated overexpression of DsRed from plasmid pET200/DsRed.…”