Search

Therapeutic genome modification requires precise control over the introduced therapeutic functions. Current approaches of gene and cell therapy fail to deliver such command and rely on semi-quantitative methods with limited influence on timing, contextuality and levels of transgene expression, and hence on therapeutic function. Synthetic biology offers new opportunities for quantitative functionality in designing therapeutic systems and their components. Here, we discuss synthetic biology tools in their therapeutic context, with examples of proof-of-principle and clinical applications of engineered synthetic biomolecules and higher-order functional systems, i.e. gene circuits. We also present the prospects of future development towards advanced gene-circuit therapy.

Active pharmaceutical ingredients (API) with suboptimal pharmacokinetic properties may require formulation into nanoparticles. In addition to the quality of the excipients, production parameters are crucial for producing nanoparticles which reliably deliver APIs to their target. Microfluidic platforms promise increased control over the formulation process due to the decreased degrees of freedom at the micro- and nanoscale. Publications about these platforms usually provide only limited information about the soft- and hardware required to integrate the microfluidic chip seamlessly into an experimental set-up. We describe a modular, low-budget prototype for microfluidic mixing in detail. The prototype consists of four modules. The control module is a raspberry pi executing customizable python scripts to control the syringe pumps and the fraction collector. The feeding module consists of up to three commercially available, programable syringe pumps. The formulation module can be any macro- or microfluidic chip connectable to syringe pumps. The collection module is a custom-built fraction collector. We describe each feature of the working prototype and demonstrate its power with polyplexes formulated from siRNA and two different oligomers that are fed to the chip at two different stages during the assembly of the nanoparticles.

Non-viral delivery of nucleic acids for therapies based on RNA interference requires a rational design and optimal self-assembly strategies. Nucleic acid particles need to be small, stable and functional in terms of selective cell uptake and controlled release of encapsulated nucleic acids. Here we report on small (∼38 nm) monomolecular nucleic acid/lipid particles (mNALPs) that contain single molecules of short double-stranded oligonucleotides covered by a tight, highly curved lipid bilayer. The particles consist of DOPE, DOTAP, DOPC and DSPE-PEG(2000) and are assembled with 21 bp double-stranded DNA or small interfering RNA by solvent exchange on a hydrodynamic-focusing microfluidic chip. In comparison to vortex mixing by hand this method increases the encapsulation efficiency by 20%, and yields particles with a narrower size distribution, negligible aggregate formation and high stability in blood plasma and serum. Modification of mNALPs with folate-conjugated PEG-lipids results in specific binding and uptake by epithelial carcinoma KB cells overexpressing folate receptors. Binding is significantly reduced by competitive inhibition using free folate and is not observed with non-targeted mNALPs, revealing high specificity. The functionalized mNALPs show gene silencing in the presence of chloroquine, an endosome-destabilizing agent. Together, the robust self-assembly of small-sized mNALPs with their high stability and receptor-specific cell uptake demonstrate that the tight, PEG-grafted lipid-bilayer encapsulation may offer a promising approach towards the delivery of short double-stranded oligonucleotides.

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed ‘oligomers’) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure - function relationships. We chose nanoparticle shielding PEG and cell targeting folic acid (termed ‘PEG-ligands’) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide – alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core - lipid anchor - PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined polyethylene glycol (PEG) domains of in sum 24 and 36 ethylene oxide (EO) repetitions. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core - PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with DBCO-PEG-ligand. Hence successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed “oligomers”) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding polyethylene glycol (PEG) and cell targeting folic acid (termed “PEG-ligands”) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide—alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core—lipid anchor—PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined PEG domains of in sum 24 and 36 ethylene oxide repetitions, 12 EOs each from the lipid anchor and 12 or 24 EOs from the PEG-ligand, respectively. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core—PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with aforementioned PEG-ligands. Hence, successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

Amphiphilic dicationic surfactants, known as gemini surfactants, are currently studied for gene delivery purposes. The gemini surfactant molecule is composed of two hydrophilic “head” groups attached to hydrophobic chains and connected via molecular linker between them. The influence of different concentrations of 1,5-bis (1-imidazolilo-3-decyloxymethyl) pentane chloride (gemini surfactant) on the thermotropic phase behaviour of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers with and without the presence of DNA was investigated using Fourier transformed infrared (FTIR) and circular dichroism (CD) spectroscopies, small angle scattering of synchrotron radiation and differential scanning calorimetry. With increasing concentration of surfactant in DMPC/DNA systems, a disappearance of pretransition and a decrease in the main phase transition enthalpy and temperature were observed. The increasing intensity of diffraction peaks as a function of surfactant concentration also clearly shows the ability of the surfactant to promote the organisation of lipid bilayers in the multilayer lamellar phase.