Spatial control of gene expression within a scaffold by localized inducer release.

Gene expression can be controlled in genetically modified cells by employing an inducer/promoter system where presence of the inducer molecule regulates the timing and level of gene expression. By applying the principles of controlled release, it should be possible to control gene expression on a biomaterial surface by the presence or absence of inducer release from the underlying material matrix, thus avoiding alternative techniques that rely upon uptake of relatively labile DNA from material surfaces. To evaluate this concept, a modified ecdysone-responsive gene expression system was transfected into B16 murine cells and the ability of an inducer ligand, which was released from elastomeric poly(ester urethane) urea (PEUU), to initiate gene expression was studied. The synthetic inducer ligand was first loaded into PEUU to demonstrate extended release of the bioactive molecule at various loading densities over a one year period in vitro. Patterning films of PEUU variably-loaded with inducer resulted in spatially controlled cell expression of the gene product (green fluorescent protein, GFP). In porous scaffolds made from PEUU by salt leaching, where the central region was exclusively loaded with inducer, cells expressed GFP predominately in the loaded central regions whereas expression was minimal in outer regions where ligand was omitted. This scaffold system may ultimately provide a means to precisely control progenitor cell commitment in a spatially-defined manner in vivo for soft tissue repair and regeneration.

Semi-synthetic ecdysteroids as gene-switch actuators: synthesis, structure-activity relationships, and prospective ADME properties.

The ligand-inducible, ecdysteroid receptor (EcR) gene-expression system can add critical control features to protein expression in cell and gene therapy. However, potent natural ecdysteroids possess absorption, distribution, metabolism and excretion (ADME) properties that have not been optimised for use as gene-switch actuators in vivo. Herein we report the first systematic synthetic exploration of ecdysteroids toward modulation of gene-switch potency. Twenty-three semi-synthetic O-alkyl ecdysteroids were assayed in both a natural insect system (Drosophila B(II) cells) and engineered gene-switch systems in mammalian cells using Drosophila melanogaster, Choristoneura fumiferana, and Aedes aegypti EcRs. Gene-switch potency is maintained, or even enhanced, for ecdysteroids methylated at the 22-position in favourable cases. Furthermore, trends toward lower solubility, higher permeability, and higher blood-brain barrier penetration are supported by predicted ADME properties, calculated using the membrane-interaction (MI)-QSAR methodology. The structure-activity relationship (SAR) of alkylated ecdysteroids indicates that 22-OH is an H-bond acceptor, 25-OH is most likely an H-bond donor, and 2-OH and 3-OH are donors and/or acceptors in network with each other, and with the EcR. The strategy of alkylation points the way to improved ecdysteroidal actuators for switch-activated gene therapy.

Ecdysteroid ligand-receptor selectivity--exploring trends to design orthogonal gene switches.

A set of thirty-two natural and ten semisynthetic ecdysteroids was assayed in murine 3T3 cells across ten different ecdysteroid receptor (EcR) ligand-binding domains derived from nine arthropod species in an engineered gene switch format. Among the ecdysteroids tested, the most biologically widespread ecdysteroid, 20-hydroxyecdysone (20E), was moderately and consistently potent across the tested EcRs. The most potent ligand-receptor combination (EC(50) = 0.3 nm) was ponasterone A (PoA) actuating the Nephotettix cincticeps EcR switch. The most robust ligand-receptor combination, as measured by potency and efficacy, was PoA actuating either the Bombyx mori EcR or a 'VY' (E274V/V390I/Y410E) mutant of Choristoneura fumiferana EcR. Parallel ecdysteroid structure-activity relationships were observed across species; addition of hydroxyl groups at positions 2, 3, 14, 20 and 22 incrementally enhanced potency, whereas hydroxylation at position 25 retarded potency. Nevertheless, several outlier ligand-EcR combinations, such as cyasterone actuating the VY C. fumiferana EcR mutant and canescensterone activating Bemisia argentifolii EcR, exhibited an inversion of relative potency, and therefore lend themselves to construction of orthogonal duplex gene switches. The potency inversion between these two ligand-receptor pairs can be accounted for by steroid-tail contact residues Tyr411 and Met502 in VY C. fumiferana EcR corresponding to two threonines in B. argentifolii EcR. Another potency inversion was also observed with cyasterone operating on the VY mutant of C. fumiferana EcR and polypodine B activating Aedes aegypti EcR. The ecdysteroid-EcR dataset, generated in a non-natural system, nevertheless invites conjecture regarding relative ecdysteroid potencies, plant species distribution of certain phytoecdysteroids, and the role of phytoecdysteroids as chemodefense against relevant insect herbivores.

Optimization of alpha-acylaminoketone ecdysone agonists for control of gene expression.

Fifteen new alpha-acylaminoketones were prepared by four different routes in an initial effort to optimize the potency of these compounds as ecdysone agonists. The compounds were assayed in mammalian cells expressing the ecdysone receptors from Bombyx mori (BmEcR) and Choristoneura fumiferana (CfEcR) for their ability to cause expression of a reporter gene downstream of an ecdysone response element. A new alpha-acylaminoketone was identified which had activity equal to that of the standard dibenzoylhydrazine ecdysone agonist GS()-E in the assay based on CfEcR.

Synthesis and SAR of cis-1-benzoyl-1,2,3,4-tetrahydroquinoline ligands for control of gene expression in ecdysone responsive systems.

A library of 35 cis-1-benzoyl-2-methyl-4-(phenylamino)-1,2,3,4-tetrahydroquinolines was prepared. The compounds bore various substitutuents on the benzoyl ring, at the 4-position of the phenylamino ring and at the 6-position of the tetrahydroquinoline ring. The compounds were assayed for their ability to cause expression of a reporter gene downstream of an ecdysone response element in a mammalian cell line engineered to express the ecdysone receptor from Aedes aegypti. In general, compounds with small lipophilic substituents at the meta and para-positions of the benzoyl ring and hydrogen or fluorine at the 4-position of the phenylamino ring and the 6-position of the tetrahydroquinoline ring were the most potent.

Synthesis and SAR of alpha-acylaminoketone ligands for control of gene expression.

A lead discovery library and a follow-up focused library of alpha-acylaminoketones were designed based on known dibenzoylhydrazine ecdysone agonists, including GS(TM)-E. The compounds were assayed in mammalian cells expressing the ecdysone receptor from Bombyx mori for their ability to cause expression of a reporter gene downstream of an ecdysone response element. The most potent alpha-acylaminoketones were comparable to GS(TM)-E in this assay.