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PubMed2026

Engineering extracellular vesicles for targeted siRNA delivery: Advances, therapeutic applications, and clinical translation.

Small interfering RNA (siRNA) therapeutics have emerged as a transformative approach for sequence-specific gene silencing, offering the potential to treat a broad spectrum of diseases by selectively suppressing disease-associated genes. However, the clinical translation of siRNA remains limited by rapid enzymatic degradation, poor cellular uptake, inadequate endosomal escape, and off-target effects, necessitating the development of efficient delivery systems. Extracellular vesicles (EVs) have gained considerable attention as natural nanocarriers owing to their excellent biocompatibility, low immunogenicity, intrinsic targeting capability, and ability to protect therapeutic cargo while traversing complex biological barriers. This review comprehensively discusses the biological characteristics of EVs, the molecular basis of RNA interference, and the major challenges associated with siRNA delivery [Fig. 1]. Recent advances in EV engineering, including cargo-loading strategies such as electroporation, sonication, extrusion, parent-cell engineering, and microfluidic approaches, together with surface functionalization using peptides, antibodies, aptamers, and hybrid nanoplatforms, are critically evaluated for improving targeting specificity and intracellular delivery. Furthermore, the therapeutic applications of engineered EV-mediated siRNA delivery in cancer, neurological disorders, liver diseases, cardiovascular diseases, inflammatory disorders, and infectious diseases are systematically summarized, highlighting their potential to enhance gene silencing while minimizing systemic toxicity. Current challenges related to large-scale manufacturing, cargo-loading efficiency, standardization, quality control, regulatory approval, and clinical translation are also discussed, together with emerging technologies involving synthetic biology, genome engineering, artificial intelligence, and multifunctional hybrid vesicles. Overall, engineered extracellular vesicles represent a highly versatile and biologically inspired platform for targeted siRNA delivery, providing a promising foundation for the development of next-generation precision RNA therapeutics and accelerating the clinical translation of gene-silencing strategies.

Extracellular vesicle engineering↗
PubMed2002

A glovebox with three levels of containment and clean room facilities for growing and handling biological material at physiologically correct gas compositions and with optimal quality assessment for tissue-engineering, ex vivo expansion, manipulation and gene therapy.

Traditional two levels of containment provide enclosure and underpressure in order to avoid hazardous material to flow towards e.g. a crewmember and thereby cause severe harm. The present-day demands for laboratory safety have revealed a paradox: In the laboratory overpressure is needed to prevent contamination of biological material and under pressure is needed to prevent the pollution of the environment. A new type of combined workbench/incubator has been constructed to meet future regulatory demands for handling and growing human biological cellular material at safe constant physiological conditions: A so-called three levels of containment glovebox/workbench. This new invention avoids the hazards of prior technology. It sets new standards for proper handling of biological materials and will meet the coming safety demands from the growing field of tissue engineering and ex vivo biotechnology. The invention is computer controlled, has a build in cleaning facility for assuring a particle free and aseptic working facility. We now have invented a solution to the above paradox concerning laboratory safety that seems to fulfil the need for safe biological experiments in microgravity. This concept has already been applied into ground-based research and is expected in a few years also to be applied similarly in the ISS environment. Furthermore, handling biological material mimicking in vivo conditions ex vivo requires precise and stabile monitoring and regulation of the isotherm and isobar conditions. Handling stem cells requires in addition low to very low oxygen tension to mimic the stem cells natural habitats. Besides that, the ex vivo gaseous atmosphere and temperature surrounding the cells has to be of same correct composition and temperature as found in the body in order to mimic in vivo situations in such way, that scientifically correct, reproducible and comparable results can be achieved. This fact is strengthened by forthcoming regulations as being prepared by several international regulatory bodies. The new concept will find its use in microgravity biotechnology and will set new standards on ground and in microgravity in the field of basic research, tissue-engineering, production of patient specific cells and tissue, embryo-genesis and in vitro fertilisation, ex vivo expansion of blood progenitor cells, gene therapy etc.

Journal Article↗
PubMed1991

Engineering considerations for the application of extremophiles in biotechnology.

Biotechnology may soon take greater advantage of extremophiles--microorganisms that grow in high salt or heavy metal concentrations, or at extremes of temperature, pressure, or pH. These organisms and their cellular components are attractive because they permit process operation over a wider range of conditions than their traditional counterparts. However, extremophiles also present a number of challenges for the development of bioprocesses, such as slow growth, low cell yield, and high shear sensitivity. Difficulties inherent in designing equipment suitable for extreme conditions are also encountered. This review describes both the advantages and disadvantages of extremophiles, as well as the specialized equipment required for their study and application in biotechnology.

Amino Acid Sequence↗
PubMed1993

The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments.

Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.

Alternative Splicing↗
PubMed2006

Polymers as biomaterials for tissue engineering and controlled drug delivery.

The advent of biodegradable polymers has significantly influenced the development and rapid growth of various technologies in modern medicine. Biodegradable polymers are mainly used where the transient existence of materials is required and they find applications as sutures, scaffolds for tissue regeneration, tissue adhesives, hemostats, and transient barriers for tissue adhesion, as well as drug delivery systems. Each of these applications demands materials with unique physical, chemical, biological, and biomechanical properties to provide efficient therapy. Consequently, a wide range of degradable polymers, both natural and synthetic, have been investigated for these applications. Furthermore, recent advances in molecular and cellular biology, coupled with the development of novel biotechnological drugs, necessitate the modification of existing polymers or synthesis of novel polymers for specific applications. This review highlights various biodegradable polymeric materials currently investigated for use in two key medical applications: drug delivery and tissue engineering.

Biocompatible Materials↗
PubMed2001

Cellular overexpression of heme oxygenase-1 up-regulates p21 and confers resistance to apoptosis.

BACKGROUND: Induction of heme oxygenase-1 (HO-1) protects against diverse insults in the kidney and other tissues. We examined the effect of overexpression of HO-1 on cell growth, expression of p21, and susceptibility to apoptosis. METHODS: LLC-PK1 cells were genetically engineered to exhibit stable overexpression of HO-1. The effects of such overexpression on cell growth, the cell cycle, and the cell cycle-inhibitory protein, p21, were assessed; additionally, the susceptibility of these HO-1 overexpressing cells to apoptosis induced by three different stimuli (TNF-alpha/cycloheximide, staurosporine, or serum deprivation) was evaluated by such methods as the quantitation of caspase-3 activity, phase contrast microscopy, and the TUNEL method. RESULTS: HO-1 overexpressing LLC-PK1 cells demonstrated cellular hypertrophy, decreased hyperplastic growth, and growth arrest in the G0/G1 phase of the cell cycle. HO-1 overexpressing cells were markedly resistant to apoptosis induced by TNFalpha/cycloheximide or staurosporine as assessed by the caspase-3 activity assay. Such overexpression also conferred resistance to apoptosis induced by serum deprivation as evaluated by the TUNEL method; in these studies, inhibition of HO attenuated the resistance to apoptosis. Expression of the cyclin dependent kinase inhibitor, p21CIP1, WAF1, SDI1, as judged by Northern and Western analyses, was significantly increased in HO-1 overexpressing cells, and decreased as HO activity was inhibited. Moreover, this reduction in expression of p21 attendant upon the inhibition of HO activity in HO-1 overexpressing cells paralleled the loss of resistance of these cells to apoptosis when HO activity is inhibited. The pharmacologic inducer of HO-1, hemin, increased expression of p21 in wild-type cells and decreased apoptosis provoked by TNF-alpha/cycloheximide. CONCLUSION: Cellular overexpression of HO-1 up-regulates p21, diminishes proliferative cell growth, and confers marked resistance to apoptosis. We speculate that such up-regulation of p21 contributes to the altered pattern of cell growth and resistance to apoptosis. Our studies uncover the capacity of HO-1 to markedly influence the cell cycle in renal epithelial cells. In light of the profound importance of the cell cycle as a determinant of cell fate, we speculate that the inductive effect of HO-1 on p21 and the attendant inhibitory effect on the cell cycle provide a hitherto unsuspected mechanism underlying the cytoprotective actions of HO-1.

Animals↗
PubMed1997

Regulatory interactions of transcription factor YY1 with control sequences of the E6 promoter of human papillomavirus type 8.

Human papillomavirus type 8 (HPV-8) is a strictly cutaneous oncogenic virus known to induce malignant skin lesions in epidermodysplasia verruciformis patients. Our study shows that sequences surrounding transcription start sites of the HPV-8 oncogene E6 (nt 175-179) and comprising the presumable CCAAC and TATA boxes of the E6 promoter P175 contain a cluster of four motifs similar to the DNA recognition site of the multifunctional cellular transcription factor yin-yang 1 (YY1). Using DNase I footprinting and gel retardation tests it could be demonstrated that three of these motifs indeed act as YY1 binding sites. To test their functional relevance for P175 activity, engineered YY1 binding site mutants were analysed in the context of a P175 test vector using transient expression assays with human keratinocytes. YY1 turned out to exert both positive and negative effects upon the activity of the HPV-8 E6 promoter; binding of YY1 to a site upstream of the promoter's cap-position (BS1) activated transcription, whereas the downstream site (BS2) mediated repression. The second downstream YY1 binding site (BS3) seemed to play an auxiliary role, enhancing the negative effect of YY1 BS2. These observations define YY1 as an important cellular protein involved in the control of E6 oncogene expression of the skin-specific 'high risk' HPV-8 and emphasize the potential regulatory role of sequences located downstream of the transcription start site.

DNA-Binding Proteins↗
PubMed2002

Engineering liver therapies for the future.

Treatment of liver disease has been greatly improved by the advent and evolution of liver transplantation. However, as demand for donor organs continues to increase beyond their availability, the need for alternative liver therapies is clear. Several approaches including extracorporeal devices, cell transplantation, and tissue-engineered constructs have been proposed as potential adjuncts or even replacements for transplantation. Simultaneously, experience from the liver biology community have provided valuable insight into tissue morphogenesis and in vitro stabilization of the hepatocyte phenotype. The next generation of cellular therapies must therefore consider incorporating cell sources and cellular microenvironments that provide both a large population of cells and strategies to maintain liver-specific functions over extended time frames. As cell-based therapies evolve, their success will require contribution from many diverse disciplines including regenerative medicine, developmental biology, and transplant medicine.

Animals↗
PubMed1999

Chondrocyte aggregation and reorganization into three-dimensional scaffolds.

Articular cartilage has a very limited self-repairing capacity; thus, chondral lesions normally result in chronic degeneration and, eventually, osteoarthritis development. Currently, tissue engineering offers a new tool for the clinical treatment of osteochondral defects. The present investigation aimed to develop an in vitro engineered cartilage using a new class of semisynthetic scaffolds. Two nonwoven meshes of hyaluronan esters (Hyaff(R) derivatives) were seeded with sternal chick embryo chondrocytes cultured for up to 21 days, after which time they were assessed for both the cellular growth profile and histological features. Avian chondrocytes easily adhered and proliferated onto hyaluronan-based scaffolds, demonstrating a significant preference for the fully esterified benzylic form. Histochemical staining revealed the presence of a neosynthesized glycosaminoglycan-rich extracellular matrix, and immunohistochemistry confirmed the deposition of collagen type II. Moreover, ultrastructural observations supported evidence that chondrocytes grown onto a hyaluronan-derived three-dimensional scaffold maintained their unique phenotype and organization in a cartilage-like extracellular matrix. These findings support the further pursuit of a transplantable engineered cartilage using human chondrocytes for the regeneration of chondral lesions.

Animals↗
PubMed1994

Bioreactor engineering as an enabling technology to tap biodiversity. The case of taxol.

One barrier to exploiting the chemical and genetic diversity in nature is the difficulty of cultivating many organisms in a controlled manner. In some cases it is difficult to achieve growth. In many others, good growth is achieved, but the expression of the organism's genetic potential to make a desired product is not realized. The thesis of this paper is that a coupling of an understanding of reactor engineering principles with the basic knowledge of the biology is often necessary to circumvent these barriers. In many cases the construction of appropriate cultivation systems is a necessary step to better understanding of cellular physiology. In some cases the chemical of interest is of high social utility and comes from a natural source that is uncommon and difficult to secure. In these cases a method of controlled cultivation becomes a prerequisite for commercial exploitation. These points were illustrated using a taxol. Taxol is an important new anticancer drug whose development has been greatly impeded by supply problems. Taxol has been derived from the park of the pacific yew tree, a process that kills the tree. The pacific yew is a relatively uncommon tree and very slow growing. One alternative to the natural source is plant cell culture. Such cultures can produce significant levels of taxol with substantial release into the medium. Taxane products not observed in typical extracts from field-grown plants can be found in cell cultures, indicating the potential unmasking of pathways. These cultures are quite responsive to changes in their environments as illustrated by the summary of initial observations. With regard to natural compounds, biochemical engineers can play a major role in the capture and preservation of producing systems, in the discovery of useful compounds, and in providing the basis for commercial production of natural compounds.

Biological Products↗
PubMed2006

Measles virus V protein inhibits p53 family member p73.

Paramyxovirus V proteins function as host interference factors that inactivate antiviral responses, including interferon. Characterization of cellular proteins that copurify with ectopically expressed measles virus V protein has revealed interactions with DNA binding domains of p53 family proteins, p53 and p73. Specific transcriptional assays reveal that expression of measles virus V cDNA inhibits p73, but not p53. Expression of measles virus V cDNA can delay cell death induced by genotoxic stress and also can decrease the abundance of the proapoptotic factor PUMA, a p73 target. Recombinant measles virus with an engineered deficiency in V protein is capable of inducing more severe cytopathic effects than the wild type, implicating measles virus V protein as an inhibitor of cell death. These findings also suggest that p73-PUMA signaling may be a previously unrecognized arm of cellular innate antiviral immunity.

DNA-Binding Proteins↗
PubMed2004

Umbilical cord blood derived endothelial progenitor cells for tissue engineering of vascular grafts.

BACKGROUND: A substantial limitation regarding present pediatric cardiac surgery is the lack of appropriate materials for the repair of congenital defects. To address this shortcoming, tissue engineering is a scientific field that aims at in vitro fabrication of living autologous grafts with the capacity of growth, repair, and regeneration. Here we focused on tissue engineered vascular grafts using human umbilical cord blood derived endothelial progenitor cells (EPCs), as a noninvasive cell source for pediatric applications. METHODS: EPCs were isolated from 20 ml fresh human umbilical cord blood by Ficoll gradient centrifugation and cultured in endothelial basal medium containing growth factors. After proliferation and differentiation cells were analyzed by immunohistochemistry and seeded onto three-dimensional (3D) biodegradable vascular scaffolds (porosity > 95%, n = 22). Twenty-four hours after seeding the vascular grafts were positioned into a pulse-duplicator-in vitro system and grown for 48 hours under biomimetic conditions. A second group was grown 6 days statically and an additional 6 days biomimetically. Controls were cultured statically. Analysis of the grafts included immunohistochemistry, histology, and scanning electron microscopy. RESULTS: Preseeding differentiated EPCs indicated constant endothelial phenotypes including acetylated low-density lipoprotein, cluster of differentiation 31, von Willebrand factor, and endothelial nitric oxide synthetase. Seeded EPCs established favorable cell-to-polymer attachment and proliferation into the 3D tubular scaffolds. Both conditioned and static cellular constructs demonstrated positive staining for cluster of differentiation 31, von Willebrand factor, and expression of endothelial nitric oxide synthase. CONCLUSIONS: Human umbilical cord derived EPCs indicated exceptional growth characteristics used for tissue engineering of vascular grafts. These cells demonstrated a constant endothelial phenotype and related functional features. Based on these results EPCs seem to be a promising autologous cell source with regard to cardiovascular tissue engineering, particularly for the repair of congenital defects.

Absorbable Implants↗
PubMedDate not supplied

Estimation of the shear stress on the surface of an aortic valve leaflet.

The limited durability of xenograft heart valves and the limited supply of allografts have sparked interest in tissue engineered replacement valves. A bioreactor for tissue engineered valves must operate at conditions that optimize the biosynthetic abilities of seeded cells while promoting their adherence to the leaflet matrix. An important parameter is shear stress, which is known to influence cellular behavior and may thus be crucial in bioreactor optimization. Therefore, an accurate estimate of the shear stress on the leaflet surface would not only improve our understanding of the mechanical environment of aortic valve leaflets, but it would also aid in bioreactor design. To estimate the shear stress on the leaflet surface, two-component laser-Doppler velocimetry measurements have been conducted inside a transparent polyurethane valve with a trileaflet structure similar to the native aortic valve. Steady flow rates of 7.5, 15.0, and 22.5 L/min were examined to cover the complete range possible during the cardiac cycle. The laminar shear stresses were calculated by linear regression of four axial velocity measurements near the surface of the leaflet. The maximum shear stress recorded was 79 dyne/cm2, in agreement with boundary layer theory and previous experimental and computational studies. This study has provided a range of shear stresses to be explored in bioreactor design and has defined a maximum shear stress at which cells must remain adherent upon a tissue engineered construct.

Aortic Valve↗
PubMed2001

Telomeres and telomerase: implications for cancer and aging.

Maintenance of telomere stability is required for cells to escape from replicative senescence and proliferate indefinitely. Telomere length is maintained by a balance between processes that lengthen telomeres (telomerase) and processes that shorten telomeres (the end-replication problem). Telomerase is a cellular ribonucleoprotein reverse transcriptase which stabilizes telomere length by adding hexameric (TTAGGG) repeats to the telomeric ends of the chromosomes, thus compensating for the continued erosion of telomeres. Introduction of the telomerase catalytic protein component into normal telomerase-negative human cells results in restoration of telomerase activity and extension of cellular life span. Human cells with introduced telomerase maintain a normal chromosome complement and continue to grow in a normal manner. Telomerase-induced manipulations of telomere length may thus be important not only for cell and tissue engineering but also for dissecting the molecular mechanisms underlying inherited genetic diseases, as well as defining the genetic pathways leading to cancer. Because almost all human tumors express telomerase activity, inhibition of telomerase may result in gradual erosion of telomeres and eventual cessation of cell proliferation or induction of apoptosis. Thus telomerase may also be a promising target for cancer therapy.

Animals↗
PubMed2005

Engineering plants with increased disease resistance: how are we going to express it?

Precise control of transgene expression is pivotal to the engineering of plants with increased disease resistance. Many early attempts to boost disease resistance used constitutive overexpression of defence components but frequently this resulted in poor quality plants. It is now clear that the extensive cellular reprogramming associated with defence will reduce yields if uncontrolled defence reactions are activated in uninfected cells. Therefore, for many strategies pathogen-inducible promoters might be the most useful as they limit the cost of resistance by restricting expression to infection sites. Although progress to date has been hindered by a lack of suitable promoters, new research should reveal more potentially useful native promoters. Additionally, the first steps towards 'designer' synthetic promoters have proved encouraging.

Crops, Agricultural↗
PubMed2005

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation.

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli.

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.

Aerobiosis↗