Biomedical subjects

Martin Fussenegger

Publications and source records attributed to Martin Fussenegger.

At least 19 recordsLinked to original sources

PubMed2007

A gas-inducible expression system in HEK.EBNA cells applied to controlled proliferation studies by expression of p27(Kip1).

We describe an efficient inducible gene expression system in HEK.EBNA cells, a well-established cell system for the rapid transient expression of research-tool proteins. The transgene control system of choice is the novel acetaldehyde-inducible regulation (AIR) technology, which has been shown to modulate transgene levels following exposure of cells to acetaldehyde. For application in HEK.EBNA cells, AlcR transactivator plasmids were constructed and co-expressed with the secreted alkaline phosphatase (SEAP) gene under the control of a chimeric mammalian promoter (P(AIR)) for acetaldehyde-regulated expression. Several highly inducible transactivator cell lines were established. Adjustable transgene induction by gaseous acetaldehyde led to high induction levels and tight repression in transient expression trials and in stably transfected HEK.EBNA cell lines. Thus, the AIR technology can be used for inducible expression of any desired recombinant protein in HEK.EBNA cells. A possible application for inducible gene expression is a controlled proliferation strategy. Clonal HEK.EBNA cell lines, expressing the fungal transactivator protein AlcR, were engineered for gas-adjustable expression of the cell-cycle regulator p27(Kip1). We show that expression of p27(Kip1) via transient or stable transfection led to a G1-phase specific growth arrest of HEK.EBNA cells. Furthermore, production pools engineered for gas-adjustable expression of p27(Kip1) and constitutive expression of SEAP showed enhanced productive capacity.

Acetaldehyde↗

PubMed2007

Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform.

Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.

Gene Expression↗

PubMed2006

A novel generic dipstick-based technology for rapid and precise detection of tetracycline, streptogramin and macrolide antibiotics in food samples.

Excessive use of antibiotics in veterinary medicine and as growth promoters in stock farming has been associated with the dramatically increasing prevalence of multidrug-resistant human pathogenic bacteria. European community legislators have therefore restricted the veterinary use of antibiotics and banned them as growth-promoting food additives in stock breeding (1831/2003/EC). The monitoring of such legislation requires technology for precise and straightforward on-site quantification of antibiotics in farm samples and food products without the need for extensive laboratory equipment and trained personnel. Capitalizing on bacterial transcriptional regulators (TetR, PIP, E), which are dose-dependently released from their cognate operators (tetO, PIR, ETR) upon binding of specific classes of antibiotics (tetracycline, streptogramins, macrolides) we have designed an easy-to-handle dipstick-based assay for detection of antibiotic levels in serum, meat and milk whose detection limits are up to 40-fold below licensed threshold values. The generic dipstick consists of either nitrocellulose, nylon or polyvinylidenfluorid (PVDF) membrane strips coated with streptavidin and immobilized biotinylated operator DNA, which acts as capture DNA to bind hexa-histidine (His(6))-tagged bacterial biosensors. Antibiotics present in specific samples triggered the dose-dependent release of the capture DNA-biosensor interaction, which, after dipping into two different solutions, results in a correlated conversion of a chromogenic substrate by a standard His(6)-targeted enzyme complex. This can be quantified by comparison of the dipstick to a standardized color scale or by assessing the terminal solution at 450nm. As demonstrated using serum, meat and milk samples spiked with 14 different antibiotics, the dipstick technology provided sensitive detection in a rapid assay format, and could be employed to monitor non-authorized use of antibiotics and to discover novel antibiotics.

Animals↗

PubMed2006

A novel synthetic mammalian promoter derived from an internal ribosome entry site.

Introduction of specific mutations into a synthetic internal ribosome entry site (IRES(GTX)) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P(GTX) promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF(121)), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in P(GTX2)) doubled the SEAP expression levels in CHO-K1. P(GTX2) reached near 70% of P(SV40)-driven expression levels and outperformed constitutive phosphoglycerate kinase (P(PGK)) and human ubiquitin C (P(hUBC)) promoters in CHO-K1. Also, P(GTX2) was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, P(GTX2) is one of the smallest eukaryotic promoters. Although P(GTX2) was found to be a potent promoter, it retained its IRES(GTX)-specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells.

Animals↗

PubMed2006

Impact of RNA interference on gene networks.

Small endogenous RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been found to post-transcriptionally control cellular gene networks by targeting complementary mRNAs for translation impairment (miRNA) or destruction (siRNA). We have developed a computational model, coordinated to molecular and biochemical parameters of RNA interference pathways, to provide (semi-) quantitative insight into the molecular events managing siRNA-mediated gene expression silencing in native and synthetic gene networks. Based on mass-conservation principles and kinetic rate laws, we converted biochemical RNA interference pathways into a set of ordinary differential equations that describe the dynamics of siRNA-mediated translation-regulation in mammalian cells. Capitalizing on mechanistic details of synthetic transactivator operation, we wired this model into a transcription control circuitry in which the siRNA and its target mRNA are independently regulated at the transcriptional level. In this context, we studied the impact of siRNA transcription timing on the onset of target gene transcription and production kinetics of target mRNA-encoded proteins. We also simulated the rate of siRNA-induced mRNA depletion and demonstrated that the relative concentrations of interacting siRNAs/mRNAs and the number of siRNA-specific target sites on a transcript modulate (i) the rate of target mRNA disappearance, (ii) the steady-state mRNA levels and (iii) induction dynamics of mRNA-encoded protein production. As our model predictions are consistent with available biochemical parameters, extrapolations may improve our understanding of how complex regulatory gene networks are impacted by small endogenous RNAs.

Gene Expression Regulation↗

PubMed2006

6-hydroxy-nicotine-inducible multilevel transgene control in mammalian cells.

The precise control of transgene expression is essential for biopharmaceutical manufacturing, gene therapy and tissue engineering. We have designed a novel conditional transcription technology, which enables reversible induction, repression and adjustment of desired transgene expression using the clinically inert 6-hydroxy-nicotine (6HNic). The 6-hydroxy-nicotine oxidase (6HNO) repressor (HdnoR), which manages nicotine metabolism in Arthrobacter nicotinovorans pAO1 by binding to a specific operator of the 6-hydroxy-nicotine oxidase (O(NIC)), was fused to the Krueppel-associated box protein of the human kox-1 gene (KRAB) to create a synthetic 6HNic-dependent transsilencer (NS) that controls chimeric mammalian promoters, which are assembled by cloning tandem O(NIC) operators 3' of a constitutive promoter. In the absence of 6HNic, NS binds to O(NIC) and silences the constitutive promoter, which otherwise drives high-level transgene expression when the NS-O(NIC) interaction stops in the presence of 6HNic. Generic NICE(ON) technology was compatible with a variety of constitutive viral and mammalian housekeeping promoters, each of which enabled specific induced, repressed, adjusted and reversible transgene expression profiles in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) as well as in human fibrosarcoma (HT-1080) cells. NICE(ON) also proved successful in controlling multicistronic expression units for coordinated transcription of up to three transgenes and in the fine-tuning of transcription-translation networks, in which RNA polymerase II- and III-dependent promoters, engineered for 6HNic responsiveness, drove expression of siRNAs that triggered specific transgene knockdown. NICE(ON) represents a robust and versatile technology for the precise tuning of transgene expression in mammalian cells.

Animals↗

PubMed2006

Improved transgene expression fine-tuning in mammalian cells using a novel transcription-translation network.

Following the discovery of RNA interference (RNAi) and related phenomena, novel regulatory processes, attributable to small non-protein-coding RNAs, continue to emerge. Capitalizing on the ability of artificial short interfering RNAs (siRNAs) to trigger degradation of specific target transcripts, and thereby silence desired gene expression, we designed and characterized a generic transcription-translation network in which it is possible to fine-tune heterologous protein production by coordinated transcription and translation interventions using macrolide and tetracycline antibiotics. Integration of siRNA-specific target sequences (TAGs) into the 5' or 3' untranslated regions (5'UTR, 3'UTR) of a desired constitutive transcription unit rendered transgene-encoded protein (erythropoietin, EPO; human placental alkaline phosphatase, SEAP; human vascular endothelial growth factor 121, VEGF(121)) production in mammalian cells responsive to siRNA levels that can be fine-tuned by macrolide-adjustable RNA polymerase II- or III-dependent promoters. Coupling of such macrolide-responsive siRNA-triggered translation control with tetracycline-responsive transcription of tagged transgene mRNAs created an antibiotic-adjustable two-input transcription-translation network characterized by elimination of detectable leaky expression with no reduction in maximum protein production levels. This transcription-translation network revealed transgene mRNA depletion to be dependent on siRNA and mRNA levels and that translation control was able to eliminate basal expression inherent to current transcription control modalities. Coupled transcription-translation circuitries have the potential to lead the way towards composite artificial regulatory networks, to enable complex therapeutic interventions in future biopharmaceutical manufacturing, gene therapy and tissue engineering initiatives.

Alkaline Phosphatase↗

PubMed2006

A genetic redox sensor for mammalian cells.

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5' of a minimal eukaryotic promoter (P(ROP)). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent P(ROP)-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX-P(ROP) affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX-P(ROP) interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD+ poise could also be detected using the REDOX-P(ROP) sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing.

Animals↗

PubMed2006

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles.

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Animals↗

PubMed2006

Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues.

A precise understanding of processes managing heterologous protein production in vitro and in vivo is essential for the manufacture of sophisticated biopharmaceuticals as well as for future gene therapy and tissue engineering initiatives. Capitalizing on the gravity-enforced self-assembly of monodispersed cells into coherent (multicellular) microtissues we studied heterologous protein production of microtissues and monolayers derived from cell lines and primary cells engineered/transduced for (i) constitutive, (ii) proliferation-controlled, (iii) macrolide-, or (iv) gas-inducible expression of the human placental secreted alkaline phosphatase (SEAP) and of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Specific productivity of cells assembled in microtissues was up to 20-fold higher than isogenic monolayer cultures. Diffusion across microtissues could be further increased by HUVEC-mediated vascularization. As well as higher specific protein productivities, microtissues were also more efficient than monolayer cultures in assembling transgenic lentiviral particles. Our results showed that mammalian cells embedded in a tissue-like three-dimensional (3D) microenvironment exhibit increased production capacity. This observation should be considered for gene therapy and tissue engineering scenarios as well as for biopharmaceutical manufacturing.

Alkaline Phosphatase↗

PubMed2006

Pharmacologic transgene control systems for gene therapy.

Pharmacologic transgene-expression dosing is considered essential for future gene therapy scenarios. Genetic interventions require precise transcription or translation fine-tuning of therapeutic transgenes to enable their titration into the therapeutic window, to adapt them to daily changing dosing regimes of the patient, to integrate them seamlessly into the patient's transcriptome orchestra, and to terminate their expression after successful therapy. In recent years, decisive progress has been achieved in designing high-precision trigger-inducible mammalian transgene control modalities responsive to clinically licensed and inert heterologous molecules or to endogenous physiologic signals. Availability of a portfolio of compatible transcription control systems has enabled assembly of higher-order control circuitries providing simultaneous or independent control of several transgenes and the design of (semi-)synthetic gene networks, which emulate digital expression switches, regulatory transcription cascades, epigenetic expression imprinting, and cellular transcription memories. This review provides an overview of cutting-edge developments in transgene control systems, of the design of synthetic gene networks, and of the delivery of such systems for the prototype treatment of prominent human disease phenotypes.

Animals↗

PubMed2006

Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple-transcript transgene expression in mammalian cells and microtissues.

BACKGROUND: Adenoviral particles can efficiently transduce a broad spectrum of cell types, so they are widely used in basic research and clinical trials. METHODS: We have developed a novel adenoviral vector platform for delivery of constitutive or streptogramin-inducible expression of up to three therapeutic transgenes into a variety of murine and human cell lines, primary cells and microtissues. RESULTS: Coordinated expression of three independent transgenes in a compact genetic format was achieved by two different expression configurations: (i) The multicistronic expression format consisting of a single constitutive (simian virus 40 promoter, P(SV40); murine or human cytomegalovirus immediate-early promoter, P(mCMV), P(hCMV)) or regulated (streptogramin-inducible) promoters (P(PIR)ON2) driving the expression of a single multicistronic transcript of which the first cistron is translated in a cap-dependent manner and the two subsequent ones by internal ribosome entry site (IRES)-mediated translation initiation. (ii) The triple-transcript expression configuration, in which a combination of well-established (P(SV40), P(hCMV), P(mCMV)) and novel synthetic constitutive promoters (P(GTX)) control transcription of three expression units. The constitutive multigene expression design enabled coordinated high-level expression of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY), the human vascular endothelial growth factor 121 (VEGF(121)) and the human placental secreted alkaline phosphatase (SEAP) in monolayer populations and microtissues of Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary neonatal rat cardiomyocytes (NRCs) and primary human aortic fibroblasts (HAFs). Streptogramin-inducible tricistronic SAMY-VEGF(121)-SEAP expression provided excellent regulation performance-high-level induction in the presence of the streptogramin antibiotic pristinamycin I (PI), near-undetectable basal expression in the absence of PI, optimal adjustability and perfect reversibility-in all cell types, in particular in NRCs and NRC-derived myocardial microtissues. CONCLUSIONS: Triple-transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size-constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios.

Adenoviridae↗

PubMed2006

Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells.

A variety of successful transcription and translation engineering strategies implemented during the past decade have driven the specific productivity of mammalian cells to an apparent limit. Restricted post-translation competence has since been considered the major bottleneck preventing mammalian cells from fully exploiting their physiologic production capacity in a biopharmaceutical manufacturing scenario. Through ectopic expression of the human transcription factor Xbp1 (X-box-binding-protein 1), evolved to manage plasma cell differentiation and coordinate the unfolded protein response, we have specifically expanded the endoplasmic reticulum and the Golgi of transgenic Chinese hamster ovary (CHO-K1)-derived cell lines with a resulting increase in overall production capacity. Xbp-1-based engineering of secretory bottlenecks was compatible with a variety of different promoter–product gene configurations suggesting that Xbp-1 induces generic production increases in CHO-K1 cell derivatives. Secretion engineering, illustrated here by Xbp1-based reprogramming of the post-translational processing machinery, provides a first insight into mastering a major system bottleneck which impacts biopharmaceutical manufacturing of secreted protein therapeutics.

Animals↗

PubMed2006

Design of custom-shaped vascularized tissues using microtissue spheroids as minimal building units.

Tissue engineering strategies are gathering clinical momentum in regenerative medicine and are expected to provide excellent opportunities for therapy for difficult-to-treat human pathologies. Being aware of the requirement to produce larger artificial tissue implants for clinical applications, we used microtissues, produced using gravity-enforced self-assembly of monodispersed primary cells, as minimal tissue units to generate scaffold-free vascularized artificial macrotissues in custom-shaped agarose molds. Mouse myoblast, pig and human articular-derived chondrocytes, and human myofibroblast (HMF)-composed microtissues (microm3 scale) were amalgamated to form coherent macrotissue patches (mm3 scale) of a desired shape. Macrotissues, assembled from the human umbilical vein endothelial cell (HUVEC)-coated HMF microtissues, developed a vascular system, which functionally connected to the chicken embryo's vasculature after implantation. The design of scaffold-free vascularized macrotissues is a first step toward the scale-up and production of artificial tissue implants for future tissue engineering initiatives.

Animals↗

PubMed2006

Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats.

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.

Animals↗

PubMed2005

Design of high-throughput-compatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa.

With a rate exceeding 90% in cattle, artificial insemination (AI) is the prime reproduction technology in stock farming. AI success is expected to increase with extended persistence of sperms in utero. In order to enable controlled sperm release during artificial insemination we have designed two strategies for the automated microencapsulation of bovine spermatozoa in either alginate-Ca2+ or cellulose sulfate (CS)-poly-diallyldimethyl ammonium chloride (pDADMAC) capsules using standard encapsulation hardware. Animal protein- and citric acid-free sperm extenders and encapsulation protocols have been developed to ensure encapsulation compatible with sperm physiology. Bovine spermatozoa have showed high motility rates inside CS-pDADMAC-based capsules, were preserved by standard cryoconservation and rescued with high viability/motility following disintegration of the thawed capsules. CS-pDADMAC-based capsules break up within 72 h after addition of either purified cellulase or cellulase-filled alignate-Ca2+ capsules. The controlled release, associated with the microencapsulation of bovine spermatozoa, may be a promising approach to increase the success rate of artificial insemination.

Alginates↗

PubMed2005

Self-assembly of sensory neurons into ganglia-like microtissues.

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

Animals↗

PubMed2005

Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles.

Transgenic HIV-1-derived lentiviral particles are at the forefront of current gene therapy and tissue engineering initiatives, which will require optimal protocols for large-scale production of clinical-grade therapeutic lentiviruses. Production of latest-generation self-inactivating lentiviral particles requires cotransfection of mammalian production cell lines with two helper plasmids along with the lentivector, whose transgene-encoding expression cassette is the only genetic information stably transduced into target chromosomes. Capitalizing on a recently designed lentiviral expression vector family, we conducted rigorous analysis of production-relevant parameters including transfection, cell density, media composition, temperature, relative (helper) vector concentrations and genetic configuration. Comparative analysis of lentiviral particle performance (VP) was based on the viral titer (reflecting the number of transduction-competent lentiviral particles) relative to the number of lentiviral particles produced (correlating with p24 production levels) (VP=titer/viral particle number). Optimal lentiviral production parameters, resulting in up to 132-fold greater VP compared to standard protocols, required (i) CaPO4-based transfection (ii) of helper plasmids and lentivector at a fixed concentration ratio (helper plasmid I:helper plasmid II:lentivector=1:1:2) (iii) into 1x10(5) human embryonic kidney cells/cm2 (HEK293-T) (iv) cultivated at 37 degrees C (v) in Advanced D-MEM medium supplemented with (vi) 2% fetal calf serum, (vii) and a culture additive containing 0.01 mM cholesterol, 0.01 mM egg's lecithin and 1x chemically defined lipid concentrate. (viii) Furthermore, constitutive transgene expression units placed in a forward polyadenylation site (pA)-free orientation relative to the lentivector backbone resulted in optimal transgene transduction/expression. Our studies suggest that detailed knowledge of lentivector design and the production of lentiviral particles will advance large-scale manufacturing of clinically relevant lentiviruses for future gene therapy applications.

Cell Culture Techniques↗