Predictive modeling of non‐viral gene transfer

Schwake, Gerlinde; Youssef, Simon; Kuhr, Jan-Timm; Gude, Sebastian; David, Maria Pamela C.; Mendoza, Eduardo; Frey, Erwin; Rädler, Joachim O.

doi:10.1002/bit.22604

Cited by 48 publications

(59 citation statements)

References 45 publications

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“…Co-transfection efficacy and balanced expression of 2 transgenes in vitro has been demonstrated to be highly variable (Schwake et al, 2010), with only a low number of cells positive for both transgenes when using individual plasmids for co-transfection, thus indicating a potential advantage of delivering 2 transgenes via single vector strategies (Kerrigan et al, 2011).…”

Section: Ga Feichtinger Et Al Non-viral Osteoinductive Gene Therapymentioning

confidence: 99%

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Feichtinger

Hacobian²,

Hofmann³

et al. 2014

eCM

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Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously.Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue.Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46 % bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo µCT and histological evaluation.In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicate that co-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future.

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Section: Ga Feichtinger Et Al Non-viral Osteoinductive Gene Therapymentioning

confidence: 99%

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Feichtinger

Hacobian²,

Hofmann³

et al. 2014

eCM

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“…Some specialized models also address the spatial distribution and active transport along microtubules [21]. The stochastic nature of in the delivery process has been investigated for nanoparticles [22] and for plasmid DNA [23]. The use of movies for the analysis of single-cell tracking experiments has been reviewed [24].…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we showed that quantitative analysis of transfection at the single-cell level makes it possible to analyze the stochastic aspects of transfection quantitatively [23], [44]. The single cell exhibits time courses that are characterized by a distinct delay time before the onset of expression, a phase of GFP increase and finally a steady state level.…”

Section: Introductionmentioning

confidence: 99%

Multi-Level Kinetic Model of mRNA Delivery via Transfection of Lipoplexes

2014

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Recent work on the use of mRNA lipoplexes for gene delivery demonstrates the need for a mathematical model that simulates and predicts kinetics and transfection efficiency. The small copy numbers involved make it necessary to use stochastic models and include statistical analysis of the variation observed in the experimental data. The modeling requirements are further complicated by the multi-level nature of the problem, where mRNA molecules are contained in lipoplexes, which are in turn contained in endosomes, where each of these entities displays a behavior of its own. We have created a mathematical model that reproduces both the time courses and the statistical variance observed in recent experiments using single-cell tracking of GFP expression after transfection. By applying a few key simplifications and assumptions, we have limited the number of free parameters to five, which we optimize to match five experimental determinants by means of a simulated annealing algorithm. The models demonstrate the need for modeling of nested species in order to reproduce the shape of the dose-response and expression-level curves.

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“…For nonviral gene delivery, the kinetic constants (Table I) are reported as averages in trafficking studies in literature [15], [18], [19], [22]- [24], [44], [50]- [57]. To make the simulation model more realistic, random noise is added to the constants by using a 10% standard deviation applying a Gaussian distribution to account for the variability reported in the literature, which is presumably due to a variety of factors including differences in cell culture conditions and packet formulations.…”

Section: Theoretical Aspects Of Modelmentioning

confidence: 99%

Identifying Intracellular pDNA Losses From a Model of Nonviral Gene Delivery

Martin

Wysocki

et al. 2015

IEEE Trans.on Nanobioscience

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Abstract-Nonviral gene delivery systems are a type of nanocommunication system that transmit plasmid packets (i.e., pDNA packets) that are programmed at the nanoscale to biological systems at the microscopic cellular level. This engineered nanocommunication system suffers large pDNA losses during transmission of the genetically encoded information, preventing its use in biotechnological and medical applications. The pDNA losses largely remain uncharacterized, and the ramifications of reducing pDNA loss from newly designed gene delivery systems remain difficult to predict. Here, the pDNA losses during primary and secondary transmission chains were identified utilizing a MATLAB model employing queuing theory simulating delivery of pEGFPLuc transgene to HeLa cells carried by Lipofectamine 2000 nonviral DNA carrier. Minimizing pDNA loss during endosomal escape of the primary transmission process results in increased number of pDNA in the nucleus with increased transfection, but with increased probability of cell death. The number of pDNA copies in the nucleus and the amount of time the pDNAs are in the nucleus directly correlates to improved transfection efficiency. During secondary transmission, pDNAs are degraded during distribution to daughter cells. Reducing pDNA losses improves transfection, but a balance in quantity of nuclear pDNA, mitosis, and toxicity must be considered in order to achieve therapeutically relevant transfection levels.

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Predictive modeling of non‐viral gene transfer

Cited by 48 publications

References 45 publications

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Multi-Level Kinetic Model of mRNA Delivery via Transfection of Lipoplexes

Identifying Intracellular pDNA Losses From a Model of Nonviral Gene Delivery

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