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At least 1,693 records · Page 94Linked to original sources

PubMed2002

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12.

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12.

Animals↗

PubMed2006

Rational design of hydrogels for tissue engineering: impact of physical factors on cell behavior.

When designing suitable biomaterials for tissue-engineering applications, biological and chemical parameters are frequently taken into account, while the equally important physical design variables have often been neglected. For a rational design of biomaterials, however, all variables influencing cell function and tissue morphogenesis have to be considered. This review will stress the development of cross-linked hydrogels and outline the impact of their physical properties on cell function and tissue morphogenesis. In the first part, the principles of cellular mechanosensitivity, as well as the influence of substrate mechanics on cell behavior, will be discussed. Afterwards, methods to characterize the mechanical properties of biomaterials will be presented. The subsequent chapters will address hydrogels that allow for the control of their physical qualities followed by a discussion of their use in tissue-engineering applications.

Biocompatible Materials↗

PubMed2006

Living autologous heart valves engineered from human prenatally harvested progenitors.

BACKGROUND: Heart valve tissue engineering is a promising strategy to overcome the lack of autologous growing replacements, particularly for the repair of congenital malformations. Here, we present a novel concept using human prenatal progenitor cells as new and exclusive cell source to generate autologous implants ready for use at birth. METHODS AND RESULTS: Human fetal mesenchymal progenitors were isolated from routinely sampled prenatal chorionic villus specimens and expanded in vitro. A portion was cryopreserved. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable leaflet scaffolds (n=12) and conditioned in a bioreactor. After 21 days, leaflets were endothelialized with umbilical cord blood-derived endothelial progenitor cells and conditioned for additional 7 days. Resulting tissues were analyzed by histology, immunohistochemistry, biochemistry (amounts of extracellular matrix, DNA), mechanical testing, and scanning electron microscopy (SEM) and were compared with native neonatal heart valve leaflets. Fresh and cryopreserved cells showed comparable myofibroblast-like phenotypes. Genotyping confirmed their fetal origin. Neo-tissues exhibited organization, cell phenotypes, extracellular matrix production, and DNA content comparable to their native counterparts. Leaflet surfaces were covered with functional endothelia. SEM showed cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves. CONCLUSIONS: Prenatal fetal progenitors obtained from routine chorionic villus sampling were successfully used as an exclusive, new cell source for the engineering of living heart valve leaflets. This concept may enable autologous replacements with growth potential ready for use at birth. Combined with the use of cell banking technology, this approach may be applied also for postnatal applications.

Absorbable Implants↗

PubMed1994

Enhanced interleukin-2 production in human tumor-infiltrating lymphocytes engineered by 3'-truncated interleukin-2 gene.

Tumor-infiltrating lymphocytes (TILs), T lymphocytes associated with solid tumors that can be grown with interleukin (IL)-2 in vitro, preferentially accumulate at tumor sites after adoptive transfer. Therefore, TILs can be considered for use as cellular vehicles in gene therapy. We transduced melanoma TILs with the IL-2 gene and clarified functional characteristics of the TIL transductants. TILs transduced with 3'-end-truncated IL-2 gene (480 bp) produced high amounts of IL-2 detected in supernatants when compared to TILs transduced with the native IL-2 gene containing 3'-end adenine-thymidine (AT)-rich sequences (650 bp). The level of IL-2 in supernatants was higher with the addition of anti-Tac antibody (Ab) to block the consumption of IL-2 by the TILs. These TILs could proliferate autonomously in the absence of exogenous IL-2, and the proliferation of TILs could be completely blocked by anti-IL-2 Ab or anti-IL-2 receptor Ab. Thus TILs transduced with IL-2 gene can proliferate through the autocrine loop. However, the expression of IL-2 from TILs transduced with the IL-2 gene was downregulated after 2 to 3 weeks of G418 selection. Our study indicates the feasibility of transduction and expression of a truncated 480-bp IL-2 gene into TILs and the possibility of employing adoptive immunotherapy protocols using TILs modified with this IL-2 gene.

Base Sequence↗

PubMed1998

Artificial juxtacrine stimulation for tissue engineering.

To endow biomaterials with the ability to regulate cell functions such as proliferation, differentiation, and apoptosis, growth factor proteins were covalently immobilized. The proteins were immobilized on various matrices using different chemical methods. It was shown that insulin and epidermal growth factor stimulated cellular functions even after immobilization. Pattern-immobilization of growth factor proteins clearly demonstrated the stimulation by immobilized proteins. In other words, this type of stimulation by non-diffusional growth factors enabled us to regulate tissue formation with artificial biomaterials. The stimulation was enhanced by coimmobilization with adhesion factors. These stimulations due to the immobilized growth factors may mimic juxtacrine stimulation of membrane-anchored growth factors such as heparin-binding epidermal growth factor, transforming growth factor-alpha, and tumor necrosis factor-alpha.

Animals↗

PubMed1990

Evaluation of vaccines designed to induce protective cellular immunity against the Plasmodium yoelii circumsporozoite protein: vaccinia, pseudorabies, and Salmonella transformed with circumsporozoite gene.

In an attempt to induce a protective cytotoxic T-cell mediated immunity against sporozoites of Plasmodium yoelii, the gene encoding the P. yoelii circumsporozoite (CS) protein was engineered into three live vectors: vaccinia, attenuated pseudorabies, and attenuated Salmonella typhimurium. Balb/c mice were immunized with 1-4 doses of 10(8) pfu of the vaccinia construct (IP), 3 doses of 10(5), 10(6) or 10(7) pfu of pseudorabies construct (IV), and 3 doses of 10(9) salmonella transformants (orally). In the case of vaccinia and pseudorabies constructs, an excellent immune response was obtained as measured by antibodies to sporozoites. No protection or delay in prepatent period was seen in any of the experimental animals when challenged with 200 (vaccinia, pseudorabies) or 100 (salmonella) sporozoites, although mice immunized with irradiation-attenuated sporozoites were consistently protected against challenge with greater than 10(4) sporozoites. Since other vaccinia, pseudorabies, and salmonella CS constructs have been shown to induce cytotoxic T lymphocytes (CTL) against the CS protein, it is likely that CTL against the CS protein were induced during these studies. It is currently unclear if the vaccines did not induce the appropriate CTL or inadequate numbers of CTL, or if CTL against the P. yoelii CS protein are inadequate to protect against sporozoite challenge.

Animals↗

PubMed2006

Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance.

Abiotic stresses including various environmental factors adversely affect plant growth and limit agricultural production worldwide. Minimizing these losses is a major area of concern for all countries. Therefore, it is desirable to develop multi-stress tolerant varieties. Salinity, drought, and cold are among the major environmental stresses that greatly influence the growth, development, survival, and yield of plants. UV-B radiation of sunlight, which damages the cellular genomes, is another growth-retarding factor. Several genes are induced under the influence of various abiotic stresses. Among these are DNA repair genes, which are induced in response to the DNA damage. Since the stresses affect the cellular gene expression machinery, it is possible that molecules involved in nucleic acid metabolism including helicases are likely to be affected. The light-driven shifts in redox-potential can also initiate the helicase gene expression. Helicases are ubiquitous enzymes that catalyse the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases). Most helicases are members of DEAD-box protein superfamily and play essential roles in basic cellular processes such as DNA replication, repair, recombination, transcription, ribosome biogenesis and translation initiation. Therefore, helicases might be playing an important role in regulating plant growth and development under stress conditions by regulating some stress-induced pathways. There are now few reports on the up-regulation of DEAD-box helicases in response to abiotic stresses. Recently, salinity-stress tolerant tobacco plants have already been raised by overexpressing a helicase gene, which suggests a new pathway to engineer plant stress tolerance [N. Sanan-Mishra, X.H. Pham, S.K. Sopory, N. Tuteja, Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc. Natl. Acad. Sci. USA 102 (2005) 509-514]. Presently the exact mechanism of helicase-mediated stress tolerance is not understood. In this review we have described all the reported stress-induced helicases and also discussed the possible mechanisms by which they can provide stress tolerance.

DNA Helicases↗

PubMed1998

The three-dimensional structure of a helix-less variant of intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic 15.1-kDa protein that appears to function in the intracellular transport and metabolic trafficking of fatty acids. It binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two five-stranded antiparallel beta-sheets and a helix-turn-helix domain. To investigate the role of the helical domain, we engineered a variant of I-FABP by deleting 17 contiguous residues and inserting a Ser-Gly linker (Kim K et al., 1996, Biochemistry 35:7553-7558). This variant, termed delta17-SG, was remarkably stable, exhibited a high beta-sheet content and was able to bind fatty acids with some features characteristic of the wild-type protein. In the present study, we determined the structure of the delta17-SG/palmitate complex at atomic resolution using triple-resonance 3D NMR methods. Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 25 degrees C and used to define the consensus 1H/13C chemical shift-derived secondary structure. Subsequently, an iterative protocol was used to identify 2,544 NOE-derived interproton distance restraints and to calculate its tertiary structure using a unique distance geometry/simulated annealing algorithm. In spite of the sizable deletion, the delta17-SG structure exhibits a backbone conformation that is nearly superimposable with the beta-sheet domain of the wild-type protein. The selective deletion of the alpha-helical domain creates a very large opening that connects the interior ligand-binding cavity with exterior solvent. Unlike wild-type I-FABP, fatty acid dissociation from delta17-SG is structurally and kinetically unimpeded, and a protein conformational transition is not required. The delta17-SG variant of I-FABP is the only wild-type or engineered member of the intracellular lipid-binding protein family whose structure lacks alpha-helices. Thus, delta17-SG I-FABP constitutes a unique model system for investigating the role of the helical domain in ligand-protein recognition, protein stability and folding, lipid transfer mechanisms, and cellular function.

Animals↗

PubMed2002

Emerging design principles in biomaterials and scaffolds for tissue engineering.

Biomaterials and scaffolds play an essential role in tissue engineering by guiding new tissue growth in vivo and in vitro. While adaptation of existing surgical materials has fulfilled some needs in the field, new applications demand better control of bulk properties such as degradation and of surface properties that control cell interactions. Advances in molecular cell biology are driving the incorporation of new biological moieties into materials, and a set of design principles based on quantitative analysis of key cellular processes involved in regeneration is emerging. At the same time, new materials-processing methodologies are emerging to allow fabrication of these fragile materials into devices appropriate for delivery.

Animals↗

PubMed1993

Expression of human factor IX by microencapsulated recombinant fibroblasts.

Deficiency of clotting factor IX (FIX) causes hemophilia B in humans. We propose a novel approach to its treatment by engineering FIX-secreting cell lines suitable for implantation in different allogeneic hosts. To prevent graft rejection following implantation, the recombinant cells can be protected with biocompatible membranes that permit exit of FIX but not entry of cellular immune mediators. To explore the feasibility of this approach, we now report on the creation of mouse Ltk- fibroblast cell lines that can deliver FIX through such immune-protective membranes. Mouse fibroblasts (Ltk-) were transfected with the cDNA for human FIX and clones secreting high levels of FIX were isolated. About 70% of the secreted FIX was biologically active. Over 98% of the recovered biological activity was precipitable by barium citrate, indicating appropriate. gamma-carboxylation of the secreted FIX. The secreted FIX was similar in molecular weight and immunoreactivity to plasma-derived human FIX. Upon enclosure in microcapsules fabricated from the biocompatible polymers, alginate-polylysine-alginate, the cells survived the encapsulation procedure with about 70-90% viability, proliferated within the microcapsules to twice their original number within 2 weeks, and continued to secrete FIX into the culture medium at similar rates as the unencapsulated cells. The biological activity, degree of post-translational gamma-carboxylation, and immunoreactivity of the FIX recovered from the culture media of the encapsulated cells were identical to those of the FIX secreted by the unencapsulated cells. In conclusion, fibroblasts engineered to secrete recombinant human FIX can proliferate and continue to secrete biologically active FIX through the alginate microcapsules. This demonstrates the feasibility of using microencapsulated recombinant cells to deliver human FIX and the potential for allogeneic somatic gene therapy for hemophilia B.

Animals↗

PubMed2003

Accelerated clearance of PEGylated liposomes in rats after repeated injections.

Polyethylene glycol-modified liposomes (PEGylated liposomes) represent promising carrier systems for therapeutic agents. Herein, we report on a study of the effect of repeated injection of PEGylated liposomes on their pharmacokinetics in rats. The first dose resulted in a reduction in the circulation time and an increase in hepatic accumulation of the second dose in a time-interval of injection-dependent manner. No significant increases in the number of Kupffer cells were detectable, although the liver most likely played an important role in the accelerated clearance. Interestingly, the acceleration in clearance became less pronounced, when the third dose was injected at 4, 7 or 14 days after the second injection (the second dose was given 5 weeks after the first injection). An accelerated clearance was evoked in normal rats by the transfusion of serum from rats that had received PEGylated liposomes 5 days earlier, indicating that humoral serum factor(s) are also involved in causing the accelerated clearance. A complement consumption assay indicated that the complement system is not the factor. In summary, multiple injections of PEGylated liposomes clearly altered their pharmacokinetic behavior in rats. It is likely that cellular immunity (Kupffer cells) and humoral immunity are required to cause the phenomenon. The results reported here have a considerable impact in and important implications on the clinical application, design and engineering of PEGylated liposomes for use in repeated intravenous administration.

Animals↗

PubMed1997

Interleukin-7 enhances cell-mediated immune responses in vivo in an interleukin-2-dependent manner.

We have engineered recombinant vaccinia virus vectors expressing murine interleukin-7 (IL-7) in order to study the activity of this factor during virus infection. Virus-encoded IL-7 dramatically increased splenic cellularity in infected mice and enhanced the proliferative activity of T cells and their capacity to secrete IL-2 and IL-6, but not IFN-gamma, TNF-alpha or IL-4. Numbers of splenic CD4+ and CD8+ T lymphocytes were elevated two- to threefold. IL-7 also mediated a marked enhancement of both antigen-specific and nonspecific cellular immune activity. Total splenic antiviral cytotoxic T cells (CTL), natural killer (NK), and lymphokine-activated killer cells (LAK) responses were augmented significantly in mice given VV-HA-IL-7 compared with those given control virus, with accelerated clearance of the former. The enhanced antiviral cellular immune activity mediated by IL-7 was critically dependent on IL-2 produced by the host, but occurred independently of IFN-gamma. The ability of IL-7 to induce cellular immune responses in vivo may have applications in antiviral immunotherapy, particularly in cases of immunodeficiency.

Animals↗

PubMed2004

Use of porcine tumor necrosis factor receptor 1-Ig fusion protein to prolong xenograft survival.

BACKGROUND: Delayed rejection of xenografts is a major hurdle that needs to be addressed to achieve long-term engraftment in the pig-to-primate transplant setting. Both vascular and avascular xenografts are susceptible to a delayed rejection process that comprises humoral and cellular responses. Tumor necrosis factor (TNF) is believed to play a role in this process by promoting cell activation, apoptosis and the recruitment of inflammatory cells. To address this problem, we engineered the donor cell in such a way that it could block both human and porcine TNF. METHODS: We produced a recombinant fusion protein containing the extracellular domain of the porcine TNF-Receptor 1 and an IgG Fc moiety (pTNFR1Ig). We first evaluated by flow cytometry the pTNFR1Ig capacity to prevent TNF alpha-induced expression of SLAI, SLAII, VCAM-1, ICAM-1 and E-selectin on the cell surface of porcine aortic endothelial cells (PAEC). The effect on TNF alpha-mediated cell death was also assessed by propidium iodide staining after incubating PAEC with TNF alpha plus cycloheximide for 24 h. PAEC and porcine fibroblasts were subsequently engineered by retroviral infection to express and secrete pTNFR1Ig and their resistance to the TNF alpha effects was tested in vitro. Finally, we transplanted mock-control and pTNFR1Ig-expressing PAEC under the kidney capsule of BALB/c mice in the absence of immunosuppression and examined the degree of rejection at 2 and 3 weeks post-transplantation. RESULTS: Treatment with pTNFR1Ig resulted in a very potent blockade of human, porcine and murine TNF alpha activity on porcine cells. It inhibited the upregulation of all cell surface markers of activation tested as well as the TNF alpha-mediated cell death. Moreover, pTNFR1Ig-expressing PAEC showed prolonged engraftment in a pig-to-mouse xenotransplant model. CONCLUSIONS: Incorporation of strategies that block TNF may prove useful in the development of xenografts resistant to delayed rejection.

Animals↗

PubMed2003

Integrin-mediated preadipocyte adhesion and migration on laminin-1.

Cell adhesion and migration are key events in many biological processes and depend on extracellular matrix proteins. Understanding these cellular events in adipogenesis is paramount to elucidating the biological mechanisms underlying preadipocyte-specific aspects of obesity, diabetes, and adipose tissue development for the design of pharmaceutical screening and tissue engineering strategies. We quantitatively investigated preadipocyte adhesion and migration on laminin-1 surfaces and identified candidate cognate preadipocyte receptors for laminin-1. In adhesion studies, we found that preadipocytes readily adhered to laminin-1 as compared with other extracellular matrix proteins. In addition, immunocytochemical analysis demonstrated that an array of integrin molecules was present on the surface of preadipocytes. Preadipocyte adhesion on laminin-1 was quantitatively assessed using a sedimentation adhesion assay, and results suggested that preadipocyte adhesion to laminin-1 was mediated by the alpha1beta1 integrin. In addition, digital time-lapse microscopy and quantitative cell tracking revealed that inhibition of the alpha1beta1 integrin resulted in abrogation of preadipocyte migration on laminin-1 surfaces. These results strongly support the hypothesis that preadipocyte adhesion to and migration on laminin-1 substrata are regulated, in part, by integrins.

Adipocytes↗

PubMed2003

Motivation, characterization, and strategy for tissue engineering the temporomandibular joint disc.

The purpose of this review is to serve as the standard point of reference in guiding researchers investigating the tissue engineering of the temporomandibular joint (TMJ) disc. Tissue engineering of the TMJ disc is in its infancy, and currently there exists a gap between the tissue engineering community and the TMJ characterization community. The primary goal is to help bridge that gap by consolidating the characterization studies here as a reference to researchers attempting to tissue engineer the TMJ disc. A brief review of TMJ anatomy is provided, along with a description of relevant pathology, current treatment, and a rationale for engineering the TMJ disc. The biochemical composition and organization of the disc are reviewed, including glycosaminoglycan (GAG) and collagen content. The collagen of the disc is almost exclusively type I and primarily runs anteroposteriorly through the center and in a ringlike fashion around the periphery. The GAG content is approximately an order of magnitude less than that of hyaline cartilage, and although the distribution is not entirely clear, it seems as though chondroitin and dermatan sulfate are by far the primary GAGs. Cellular characterization and mechanical properties under compression, tension, and shear are reviewed as well. The cells of the disc are not chondrocytes, but rather resemble fibrocytes and fibrochondrocytes and may be of the same lineage. Mechanically, the disc is certainly anisotropic and nonhomogeneous. Finally, a review of efforts in tissue engineering and cell culture studies of the disc is provided and we close with a description of the direction we envision/propose for successful tissue engineering of the TMJ disc.

Animals↗

PubMed2005

Suppression of Huntington's disease pathology in Drosophila by human single-chain Fv antibodies.

Misfolded neuronal proteins have been identified in a number of neurodegenerative disorders and have been implicated in the pathogenesis of diseases that include Alzheimer's disease, Parkinson's disease, prion-based dementia, Huntington's disease (HD), and other polyglutamine diseases. Although underlying mechanisms remain the subject of ongoing research, it is clear that aberrant processing, protein degradation, and aggregate formation or spurious protein association of the abnormal neuronal proteins may be critical factors in disease progression. Recent work in these diseases has demonstrated in vitro that specific engineered antibody species, peptides, or other general agents may suppress the formation of aggregates. We have modified an approach with intracellularly expressed single-chain Fv (sFv) antibodies (intrabodies) that bind with unique HD protein epitopes. In cell and tissue culture models of HD, anti-N-terminal huntingtin intrabodies (C4 sFv) reduce aggregation and cellular toxicity. Here, we present the crucial experiment of intrabody-mediated in vivo suppression of neuropathology, using a Drosophila model of HD. In the presence of the C4 sFv intrabody, the proportion of HD flies surviving to adulthood increases from 23% to 100%, and the mean and maximum lifespan of adult HD flies is significantly prolonged. Neurodegeneration and formation of visible huntingtin aggregates are slowed. We conclude from this investigation that engineered intrabodies are a potential new class of therapeutic agents for the treatment of neurodegenerative diseases. They may also serve as tools for drug discovery and validation of sites on mutant neuronal proteins that could be exploited for rational drug design.

Animals↗

PubMed1996

Epitope-specific tolerance induction with an engineered immunoglobulin.

Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years. The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors. The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides. To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein. This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system. We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26. Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes. This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity.

Amino Acid Sequence↗

PubMed2006

Modulating target site selection during human immunodeficiency virus DNA integration in vitro with an engineered tethering factor.

The mechanisms controlling retroviral integration have been the topic of intense interest, in part because of adverse clinical events that occurred during retrovirus-mediated human gene therapy. Here we investigate the use of artificial tethering interactions to constrain retroviral integration site selection in an in vitro model. During normal infection, HIV DNA integration is favored in active cellular transcription units. One component of the targeting mechanism is the cellular LEDGF/p75 protein. LEDGF/p75 binds tightly to HIV integrase (IN) protein, and depletion of LEDGF/p75 from target cells results in reduced integration in transcription units, suggesting integration targeting by a tethering mechanism. We constructed and analyzed fusions of LEDGF/p75 or its IN-binding domain (IBD) to the DNA-binding domain of phage lambda repressor protein (lambdaR). In the presence of the lambdaR-LEDGF/p75 fusions, increased strand transfer by IN was seen in target DNA near lambdaR-binding sites in vitro . These data support the idea that a direct interaction between LEDGF/p75 and IN can mediate targeting via a tethering mechanism, and provide proof of concept for the idea that protein-protein interactions might be engineered to constrain integration site selection during human gene therapy.

Base Sequence↗