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PubMed2006

Targeting proliferating tumor cells via the transcriptional control of therapeutic genes.

We have previously reported the construction of a cell cycle-regulated HSV-1 amplicon vector (denoted as pC8-36) that confers luciferase reporter gene activities dependent on cellular divisions. However, luciferase reporter gene is well known for its relatively high sensitivity, thus, it is crucial to evaluate the therapeutic efficacy of a transcriptional targeted vector. In this report, we have engineered the FasL and FADD genes into pC8-36 and demonstrated their efficacy for the treatment of human gliomas in vitro and in vivo. Using trypan blue dye exclusion and TUNEL assay, FasL expression mediated by pC8-36 was shown to induce a significantly higher percentage of cell death in proliferating cells than those observed in the G(1)-arrested cells. The observed cell killing effect correlated well with the level of FasL protein expression when analyzed by ELISA assay. Furthermore, the incorporation of both FasL and FADD into pC8-36 resulted in the enhancement of apoptosis in the target glioma cells both in vitro and in vivo. Targeting proliferating tumor cells via the transcriptional control of therapeutic genes could potentially improve the safety and efficacy of cancer gene therapy, and thus would allow the development of strategies for more effective anticancer therapies.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Use of adenoviral vectors as veterinary vaccines.

Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

Adenoviridae↗

PubMed2005

Conditional mutagenesis of the murine serum response factor gene blocks cardiogenesis and the transcription of downstream gene targets.

Serum response factor (SRF) homozygous-null embryos from our backcross of SRF(LacZ/)(+) "knock-in" mice failed to gastrulate and form mesoderm, similar to the findings of an earlier study (Arsenian, S., Weinhold, B., Oelgeschlager, M., Ruther, U., and Nordheim, A. (1998) EMBO J. 17, 6289-6299). Our use of embryonic stem cells provided a model system that could be used to investigate the specification of multiple embryonic lineages, including cardiac myocytes. We observed the absence of myogenic alpha-actins, SM22alpha, and myocardin expression and the failure to form beating cardiac myocytes in aggregated SRF null embryonic stem cells, whereas the appearance of transcription factors Nkx2-5 and GATA4 were unaffected. To study the role of SRF during heart organogenesis, we then performed cardiac-specific ablation of SRF by crossing the transgenic alpha-myosin heavy chain Cre recombinase line with SRF LoxP-engineered mice. Cardiac-specific ablation of SRF resulted in embryonic lethality due to cardiac insufficiency during chamber maturation. Conditional ablation of SRF also reduced cell survival concomitant with increased apoptosis and reduced cellularity. Significant reductions in SRF (> or =95%), atrial naturetic factor (> or =80%), and cardiac (> or =60%), skeletal (> or =90%), and smooth muscle (> or =75%) alpha-actin transcripts were also observed in the cardiac-conditional knock-out heart. This was consistent with the idea that SRF directs de novo cardiac and smooth muscle gene activities. Finally, quantitation of the knock-in LacZ reporter gene transcripts in the hearts of cardiac-conditional knock-out embryos revealed an approximately 30% reduction in gene activity, indicating SRF gene autoregulation during cardiogenesis.

Actins↗

PubMed2003

Functional interaction trap: a strategy for validating the functional consequences of tyrosine phosphorylation of specific substrates in vivo.

Protein tyrosine phosphorylation controls diverse signaling pathways, and disregulated tyrosine kinase activity plays a direct role in human diseases such as cancer. Because activated kinases exert their effects by phosphorylating multiple substrate proteins, it is difficult or impossible to assess experimentally the contribution of a particular substrate to a cellular response or activity. To overcome this problem, we have developed a novel approach termed the "functional interaction trap," in which two proteins are induced to interact in a pairwise fashion through an engineered, highly specific binding interface. We show that the functional interaction trap can be used to direct a modified tyrosine kinase to specifically phosphorylate a single substrate of choice in vivo, permitting analysis of the resulting biological output. This strategy provides a powerful tool for validating the functional significance of tyrosine phosphorylation and other post-translational modifications identified by proteomic discovery efforts.

Animals↗

PubMed2006

In vivo confocal microscopy of failing and functioning filtering blebs: Results and clinical correlations.

PURPOSE: To correlate clinical filtering bleb function with characteristics as detected by in vivo confocal microscopy. METHODS: In a case-matched cross-sectional study, 52 eyes of 48 patients were examined 1 day to 12.8 years after primary trabeculectomy (mean 375 d). The patients were examined clinically and by in vivo confocal microscopy (Rostock Cornea Module/Heidelberg Retina Tomograph II, Heidelberg Engineering, Inc, Heidelberg, Germany). Nine early and 17 late functioning blebs were pair-matched with malfunctioning blebs. Stromal fiber patterns, the number of intraepithelial and stromal cystic spaces, and the amount of cellular infiltrates were evaluated. RESULTS: Four stromal patterns (trabecular, reticular, corrugated, compacted) invisible to slit-lamp biomicroscopy could be distinguished by in vivo confocal microscopy. The trabecular pattern occurred only in functioning blebs, particularly early postoperatively. Intraepithelial cystic spaces were associated with functioning late blebs, whereas they were equally distributed in early blebs. In contrast, stromal cystic spaces indicate function in early blebs, whereas in late blebs the number of these cavities was similar in both groups. The density of intraepithelial and stromal round cells was higher in functioning late blebs compared with malfunctioning late blebs. CONCLUSIONS: In vivo confocal microscopy allows to assess filtering bleb structures that are invisible biomicroscopically. Some morphologic features detected by this technique seem to indicate filtering bleb function and time after surgery. The predictive value of these features deserves further clarification in a prospective longitudinal study.

Aged↗

PubMed2003

Costimulatory molecule immune enhancement in a plasmid vaccine model is regulated in part through the Ig constant-like domain of CD80/86.

There is great interest in understanding the role of costimulatory molecules in immune activation. In both the influenza and HIV DNA immunization models, several groups have reported that coimmunization of mice with plasmids encoding immunogen and CD86, but not CD80, effectively boosts Ag-specific T cell activation. This difference in immune priming provided an opportunity to examine the functional importance of different regions of the B.7 molecules in immune activation. To examine this issue, we developed a series of chimeric CD80 and CD86 constructs as well as deletion mutants, and examined their immune activating potential in the DNA vaccine model. We demonstrate that the lack of an Ig constant-like region in the CD80 molecule is critically important to the enhanced immune activation observed. CD80 C-domain deletion mutants induce a highly inflammatory Ag-specific cellular response when administered as part of a plasmid vaccine. The data suggest that the constant-like domains, likely through intermolecular interactions, are critically important for immune regulation during costimulation and that engineered CD80/86 molecules represent more potent costimulatory molecules and may improve vaccine adjuvant efficacy.

Adjuvants, Immunologic↗

PubMed2025

Understanding proneural-mesenchymal transition using patient-derived glioma stem-like cell (GSC) organoids and engineered extracellular matrix.

Glioblastoma multiforme (GBM) is a highly aggressive, angiogenic WHO grade IV glioma marked by rapid progression, therapeutic resistance, and poor prognosis. A defining feature of GBM is the presence of glioma stem-like cells (GSCs), which reside in specialized perivascular niches and drive tumor progression, recurrence, and therapeutic resistance. The blood-brain barrier, coupled with the complex and dynamic tumor microenvironment, poses significant challenges for both treatment and mechanistic investigation. Current in vitro GBM models inadequately recapitulate the structural and biochemical cues of the native perivascular niche due to the absence of functional vasculature and brain-mimetic extracellular matrix (ECM), limiting their physiological relevance and predictive power. To address the limitations of existing in vitro GBM models, we developed a patient-derived glioma stem cells (GSC) derived Matrigel spheroid system that transitions into organoids and enables integration into engineered microenvironments. Our model incorporates GSC organoids representing proneural and mesenchymal GBM subtypes, a synthetic engineered extracellular matrix (eECM), and endothelial cells (ECs) seeded on the matrix surface. We evaluated the expression of subtype-specific, pro-angiogenic, stemness, and differentiation markers under increasingly complex co-culture conditions. Our results show that Matrigel-derived GSC spheroids progressively differentiate into organoids over two weeks, with significantly enhanced expression of cell-specific markers in the presence of ECs. Encapsulation of these organoids within eECM, combined with EC co-culture, further promoted cellular invasion and induction of GBM associated genes. This in situ encapsulation strategy enables real-time observation of GSC behavior in a tunable microenvironment that mimics key features of the native tumor niche. Together, this platform provides a physiologically relevant and modular in vitro system for investigating GBM pathophysiology. It holds promise for uncovering tumor-specific cellular dependencies, studying GSC-vascular interactions, and conducting high-throughput drug screening under controlled, biomimetic conditions.

Engineered extracellular matrix↗

PubMed1999

Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP.

RNA transcripts containing the hammerhead ribozyme have been engineered to self-destruct in the presence of specific nucleoside 3',5'-cyclic monophosphate compounds. These RNA molecular switches were created by a new combinatorial strategy termed 'allosteric selection,' which favors the emergence of ribozymes that rapidly self-cleave only when incubated with their corresponding effector compounds. Representative RNAs exhibit 5,000-fold activation upon cGMP or cAMP addition, display precise molecular recognition characteristics, and operate with catalytic rates that match those exhibited by unaltered ribozymes. These findings demonstrate that a vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion. Moreover, optimized allosteric ribozymes could serve as highly selective sensors of chemical agents or as unique genetic control elements for the programmed destruction of cellular RNAs.

Acids↗

PubMed2000

Expressing active ribozymes in cells.

Artificially engineered ribozymes can be used to specifically regulate expression of target genes. Such ribozymes can be synthesized chemically and delivered into the cell exogeneously. Alternatively, ribozymes can be produced by the cell endogenously, after introduction of the artificial gene into the cellular genome. In the latter case, the design of the artificial gene defines the ribozyme properties, such as: expression level, intracellular localization, folding and association with proteins. Generally speaking, design of the expression vector is critical to obtain active ribozyme molecules. This paper first describes factors that are known or predicted to affect ribozyme activity in the cell, then reviews various expression systems that have been specifically developed for ribozymes. Lastly, a recently developed ribozyme system termed snorbozymes (small nucleolar RNA:ribozyme hybrids) will be discussed. This powerful test system has generated several important observations that are likely to affect the future development of ribozyme technology.

Animals↗

PubMed2004

Identification of cytoskeletal [14C]carboplatin-binding proteins reveals reduced expression and disorganization of actin and filamin in cisplatin-resistant cell lines.

Cisplatin resistant (CP-r) cells often show decreased uptake of cisplatin in association with reduced cell surface proteins and decreased endocytosis. In this report, two major [14C]carboplatin-binding proteins were identified as filamin and actin by photoaffinity labeling and mass spectrometry. Decreased expression of these two proteins was found in two different human CP-r cell lines (KB-CP20 and 7404-CP20), in comparison with their parental cell lines (KB-3-1 and BEL-7404), respectively. Disorganization of beta-actin and filamin 250 and 90 was also detected in these CP-r cells by confocal microscopy. Transfection of a wild-type actin-enhanced green fluorescent protein (EGFP) expression vector into 7404-CP20 cells resulted in a nonfilamentous actin-EGFP distribution compared with a normal distribution in the cisplatin-sensitive BEL-7404 cells, suggesting that cytoskeletal organization is disturbed in the CP-r cells. The identification of actin and filamin as [14C]carboplatin-binding proteins and decreased expression and disorganization of several cytoskeletal proteins in CP-r cells provide a molecular and cellular basis for the known defect in endocytosis in these cells.

Actins↗

PubMed2006

Chondrocyte phenotype in engineered fibrous matrix is regulated by fiber size.

A biomaterial scaffold acting as a functional substitute for the native extracellular matrix provides space for cell accommodation. In this study, we seeded chondrocytes, isolated from 4- to 6-month-old calves, in 2 types of poly(L-lactide) scaffolds, composed of micro- and nanofibers, and compared the effects on cellular activities. Scanning electron microscopy revealed a well-spread morphology for chondrocytes grown on microfibers. In contrast, chondrocytes on the nanofibers were found to have a rounded morphology and displayed a disorganized actin cytoskeletal structure compared to the organized cytoskeleton seen in well-spread chondrocytes culture on the microfibrous scaffold. Both scaffolds supported chondrocyte proliferation, with a higher rate seen in cultures in nanofibrous scaffold. Quantitative reverse transcription-polymerase chain reaction analysis showed that both cultures supported expression of collagen types I and II and aggrecan. Biochemical analysis showed a higher level of sulfated glycosaminoglycan in the nanofiber culture, confirmed by more intense alcian blue histologic staining. The nanofiber cultures also showed higher immunostaining for collagen types II and IX, aggrecan, and cartilage proteoglycan link protein. Based on these results, we conclude that chondrocytes respond differently to fibrous scaffolds of varying diameters, and that the scaffolds made of nanofibrous biomaterial promote efficient cell-based cartilage tissue engineering.

Aggrecans↗

PubMed1998

Genetic engineering in the pig. Gene knockout and alternative techniques.

Since endothelial cells (EC) are the major target cells during hyperacute rejection and are likely in delayed graft rejection, most of the genetic engineering of the xenotransplant donor is aimed at modifying their properties. Among the various strategies that are reviewed are the genotypic or phenotypic knockout of the alpha 1,3Gal antigen, which is a major target of xenoantibodies and is also probably involved in innate cellular response. In addition, the success of the transgeny of complement regulatory proteins is well established. In vitro data from analyses of the mechanisms of endothelial cell activation also suggest that other molecules could be used to regulate apoptosis or thrombotic microenvironment or to minimize recipient T-cell activation by inhibiting costimulatory proteins such as CD40 or B7. Alternative to usual knockout techniques (thus far not available in pigs, where no ES cells have been derived) will be presented.

Animals↗

PubMed2003

Engineering an artificial nerve graft for the repair of severe nerve injuries.

Nerve repair with tubes has a limit to regeneration depending upon the length of the gap. The characteristics of the guide, in terms of permeability, durability and adhesiveness, also influence regeneration. Considering the importance of the cellular component in regeneration, the development of artificial grafts, composed of a biocompatible nerve guide filled with a neurotropic matrix and seeded with Schwann cells (SCs), is an interesting option to enhance nerve regeneration and provide an alternative to the classical autologous nerve graft. We evaluated the ability of SCs transplanted into a nerve guide to improve regeneration after sciatic nerve resection, leaving a 6-mm gap, in the mouse. Syngeneic, isogeneic and autologous SCs were suspended in Matrigel and seeded in resorbable guides, and compared to acellular guides and to nerve autografts. The immunogenicity of the transplanted SCs clearly influenced the outcome. Transplants of autologous SCs resulted in only slightly lower levels of reinnervation than autografts, but higher recovery and number of regenerated axons than transplants of isologous and syngeneic SCs, and than acellular guides. Thus, by combined developments on nerve guides, extracellular matrix components and cell transplantation, an artificial graft has been designed that allows axonal regeneration across long gaps to levels comparable with an autograft.

Absorbable Implants↗

PubMed2005

Pharmacological analysis of CCK2 receptors up-regulated using engineered transcription factors.

Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.

Cell Line↗

PubMed2003

A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse.

Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse. The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites, has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted. Many Cre-expressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event. Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced, be affected by position-effect variegation, or reside in a chromatin configuration inaccessible for recombination. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites, and the availability of both wild-type (WT) and mutant loxP sites, we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.

Animals↗

PubMed2002

Vascular assembly in natural and engineered tissues.

With the advent of molecular embryology and exploitation of genetic models systems, many genes necessary for normal blood vessel formation during early development have been identified. These genes include soluble effectors and their receptors, as well as components of cell-cell junctions and mediators of cell-matrix interactions. In vitro model systems (2-D and 3-D) to study paracrine and autocrine interactions of vascular cells and their progenitors have also been created. These systems are being combined to study the behavior of genetically altered cells to dissect and define the cellular role(s) of specific genes and gene families in directing the migration, proliferation, and differentiation needed for blood vessel assembly. It is clear that a complex spatial and temporal interplay of signals, including both genetic and environmental, modulates the assembly process. The development of real-time imaging and image analysis will enable us to gain further insights into this process. Collaborative efforts among vascular biologists, biomedical engineers, mathematicians, and physicists will allow us to bridge the gap between understanding vessel assembly in vivo and assembling vessels ex vivo.

Adult↗

PubMed2003

Effects of innate immunity on herpes simplex virus and its ability to kill tumor cells.

Several clinical trials have or are being performed testing the safety and efficacy of different strains of oncolytic viruses (OV) for malignant cancers. OVs represent either naturally occurring or genetically engineered strains of viruses that exhibit relatively selective replication in tumor cells. Several types of OV have been derived from herpes simplex virus 1 (HSV1). Tumor oncolysis depends on the processes of initial OV infection of tumor, followed by subsequent propagation of OV within the tumor itself. The role of the immune responses in these processes has not been extensively studied. On the contrary, effects of the immune response on the processes of wild-type HSV1 infection and propagation in the central nervous system have been studied and described in detail. The first line of defense against a wild-type HSV1 infection in both naive and immunized individuals is provided by innate humoral (complement, cytokines, chemokines) and cellular (macrophages, neutrophils, NK cells, gammadelta T cells, and interferon-producing cells) responses. These orchestrate the lysis of virions and virus-infected cells as well as provide a link to effective adaptive immunity. The role of innate defenses in curtailing the oncolytic effect of genetically engineered HSV has only recently been studied, but several of the same host responses appear to be operative in limiting anticancer effects by the replicating virus. The importance of this knowledge lies in finding avenues to modulate such initial innate responses, in order to allow for increased oncolysis of tumors while minimizing host toxicity.

Animals↗

PubMed2006

Bio-electrosprays: the next generation of electrified jets.

Biological electrosprays are rapidly becoming a robust means by which to engineer living organisms for applications ranging from tissue repair to developmental biology. We previously reported the ability to electrospray living organisms without compromising their viability, but found it challenging to achieve stability in the jetting of these organisms as a result of the chemical properties of the living cellular suspensions. Jet stability is required for the generation of a near-mono distribution of droplets, which is necessary for the development of electrospray technology as a "drop and place" biotechnique. Recently, we determined the conditions needed to achieve jet stability and were able to generate droplets with a near-mono distribution (<50 microm). In this communication, we elucidate the relationship between jet behaviour and droplet size under stable jetting conditions, with a view to further reducing the droplet size to deposit a single living cell within a droplet. We believe that this level of resolution will make electrospray jetting superior amongst the jet-based biotechnologies presently being developed for the engineering of biological architectures comprised of living cells.

Animals↗