Wawa Vavava

Figure C.3. Controlling gene expression using regulated transcription.

The "transcriptional-switch" technology (described above) features an induction-decay response for the therapeutic protein that occurs on a time-scale of days: transgene-encoded protein in blood typically peaks at about 24 hours and then decreases to background over 4 to 14 days. This kinetic profile probably reflects the "early-point" of transgene regulation as well as the many potentially rate-

limiting steps after therapeutic gene delivery. These steps involve the pharmacokinetics and pharmacodynamics of rapamycin (Mahalati and Kahan 2001) as well as the dynamic processes of transgene transcription, therapeutic protein translation and secretion, and therapeutic protein bioavailability. Such prolonged kinetics may be appropriate for certain proteins (e.g., erythropoietin) that govern relatively slow physiological processes. Prolonged kinetics may not be as appropriate, however, for proteins that regulate processes such as glucose homeostasis, which tend to be much faster.

To address this potential limitation of the transcriptional-switch system, Rivera et al. (2000), recently developed a second technology that allows protein secretion from the endoplasmic reticulum (ER) to be rapidly regulated (Figure C.4). Therapeutic proteins are expressed as fusions with a conditional aggregation domain (CAD). CADs self-interact, and fusion proteins therefore form an aggregate in the ER that is far too large to be transported. Rivera and colleagues showed that the addition of cell-permeant ligand ("disaggregator") to transfected cells dissolves the aggregates and permits the rapid transport of therapeutic proteins from the ER via the constitutive secretory pathway.

To produce bioactive proteins, CAD moieties must be removed. Rivera et al. solved this problem by interposing a furin cleavage sequence between therapeutic protein and the CAD. In one example, Rivera et al. (2000) demonstrated that a natural version of hGH could be secreted in a controllable fashion using disaggregator technology. Thus, a single amino acid change (Phe36 to Met) converted monomeric FKBP12 into a CAD. Recombinant hGH was generated via a cDNA construct (Fig. C.3) consisting of a CMV promoter, signal sequence, four CAD motifs, a furin cleavage signal, and growth hormone (proinsulin was also used). Vectors were stably transfected into HT1080 cells and fluorescence microscopy was used to demonstrate ER retention of both insulin and growth hormone in the absence of disaggregator. Cells expressing fusion proteins were then treated with increasing concentrations of disaggregator for 2 hours. The authors showed that accumulated protein was released by disaggregator administration, and the rate of release was controllable over an ~20-fold dose range. In the absence of ligand, fusion proteins were found only in cell lysate samples, whereas 2 hours after addition of ligand, fusion proteins were cleaved appropriately and secreted, as determined by Western analysis. Finally, myoblast transfer was used to demonstrate feasibility of the system in animal models. To this end, engineered cells were implanted into mice made diabetic by treatment with streptozotocin. Administration of vehicle failed to normalize serum glucose concentrations. However, after intravenous administration of ligand insulin was detected in serum within 15 minutes and peaked by 2 hours. Indeed, 2 hours after administration of a 10.0-mg/kg dose of ligand, the circulating insulin concentration increased to greater than 200.0-pM and serum glucose decreased concomitantly to normal. Lower doses of ligand were less effective.

Figure C.4. Scheme for the pharmacologic control of protein secretion. (A) (left) Natural control of protein secretion (protein is stored in the secretory granules) is contrasted with the scheme for pharmacological control (protein is stored in the ER). (right) The therapeutic protein of interest (TP) is expressed as part of a fusion protein that contains, at its NH2-terminus, a signal sequence, a conditional aggregation domain (CAD), and a furin cleavage sequence (FCS). Processing and secretion of the TP is induced by ligand (Rivera et al. 2000).

Figure C.4. Scheme for the pharmacologic control of protein secretion. (A) (left) Natural control of protein secretion (protein is stored in the secretory granules) is contrasted with the scheme for pharmacological control (protein is stored in the ER). (right) The therapeutic protein of interest (TP) is expressed as part of a fusion protein that contains, at its NH2-terminus, a signal sequence, a conditional aggregation domain (CAD), and a furin cleavage sequence (FCS). Processing and secretion of the TP is induced by ligand (Rivera et al. 2000).

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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