EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins

Specific delivery to tumors and efficient cellular uptake of nucleic acids remain major challenges for gene-targeted cancer therapies. Here we report the use of a designed ankyrin repeat protein (DARPin) specific for the epithelial cell adhesion molecule (EpCAM) as a carrier for small interfering RNA (siRNA) complementary to the bcl-2 mRNA. For charge complexation of the siRNA, the DARPin was fused to a truncated human protamine-1 sequence. To increase the cell binding affinity and the amount of siRNA delivered into cells, DARPin dimers were generated and used as fusion proteins with protamine. All proteins expressed well in Escherichia coli in soluble form, yet, to remove tightly bound bacterial nucleic acids, they were purified under denaturing conditions by immobilized metal ion affinity chromatography, followed by refolding. The fusion proteins were capable of complexing four to five siRNA molecules per protamine, and fully retained the binding specificity for EpCAM as shown on MCF-7 breast carcinoma cells. In contrast to unspecific LipofectAMINE transfection, down-regulation of antiapoptotic bcl-2 using fusion protein complexed siRNA was strictly dependent on EpCAM binding and internalization. Inhibition of bcl-2 expression facilitated tumor cell apoptosis as shown by increased sensitivity to the anticancer agent doxorubicin.

Fetal gene therapy: opportunities and risks

Advances in human prenatal medicine and molecular genetics have allowed the diagnosis of many genetic diseases early in gestation. In-utero transplantation of allogeneic hematopoietic stem cells (HSC) has been successfully used as a therapy in different animal models and recently also in human fetuses. Unfortunately, clinical success of this novel treatment is limited by the lack of donor cell engraftment in non-immunocompromised hosts and is thus restricted to diseases where the fetus is affected by severe immunodeficiency. Gene therapy using genetically modified autologous HSC circumvents allogeneic HLA barriers and constitutes one of the most promising new approaches to correct genetic deficits in the fetus. Recent developments of strategies to overcome failure of efficient transduction of quiescent hematopoietic cells include the use of new vector constructs and transduction protocols. These improvements open new perspectives for gene therapy in general and for prenatal gene transfer in particular. The fetus may be especially susceptible for successful gene therapy due to the immunologic naiveté of the immature hematopoietic system during gestation, precluding an immune reaction towards the transgene. Ethical issues, in particular those regarding treatment safety, must be taken into account before clinical trials with fetal gene therapy in human pregnancies can be initiated.

Heart failure gene therapy: the path to clinical practice

Gene therapy, aimed at the correction of key pathologies being out of reach for conventional drugs, bears the potential to alter the treatment of cardiovascular diseases radically and thereby of heart failure. Heart failure gene therapy refers to a therapeutic system of targeted drug delivery to the heart that uses formulations of DNA and RNA, whose products determine the therapeutic classification through their biological actions. Among resident cardiac cells, cardiomyocytes have been the therapeutic target of numerous attempts to regenerate systolic and diastolic performance, to reverse remodeling and restore electric stability and metabolism. Although the concept to intervene directly within the genetic and molecular foundation of cardiac cells is simple and elegant, the path to clinical reality has been arduous because of the challenge on delivery technologies and vectors, expression regulation, and complex mechanisms of action of therapeutic gene products. Nonetheless, since the first demonstration of in vivo gene transfer into myocardium, there have been a series of advancements that have driven the evolution of heart failure gene therapy from an experimental tool to the threshold of becoming a viable clinical option. The objective of this review is to discuss the current state of the art in the field and point out inevitable innovations on which the future evolution of heart failure gene therapy into an effective and safe clinical treatment relies.

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

Gene expression profile in newborn rat lungs after two days of recovery of mechanical ventilation.

BACKGROUND Preterm infants having immature lungs often require respiratory support, potentially leading to bronchopulmonary dysplasia (BPD). Conventional BPD rodent models based on mechanical ventilation (MV) present outcome measured at the end of the ventilation period. A reversible intubation and ventilation model in newborn rats recently allowed discovering that different sets of genes modified their expression related to time after MV. In a newborn rat model, the expression profile 48 h after MV was analyzed with gene arrays to detect potentially interesting candidates with an impact on BPD development. METHODS Rat pups were injected P4-5 with 2 mg/kg lipopolysaccharide (LPS). One day later, MV with 21 or 60% oxygen was applied during 6 h. Animals were sacrified 48 h after end of ventilation. Affymetrix gene arrays assessed the total gene expression profile in lung tissue. RESULTS In fully treated animals (LPS + MV + 60% O(2)) vs. controls, 271 genes changed expression significantly. All modified genes could be classified in six pathways: tissue remodeling/wound repair, immune system and inflammatory response, hematopoiesis, vasodilatation, and oxidative stress. Major alterations were found in the MMP and complement system. CONCLUSION MMPs and complement factors play a central role in several of the pathways identified and may represent interesting targets for BPD treatment/prevention.Bronchopulmonary dysplasia (BPD) is a chronic lung disease occurring in ~30% of preterm infants born less than 30 wk of gestation (1). Its main risk factors include lung immaturity due to preterm delivery, mechanical ventilation (MV), oxygen toxicity, chorioamnionitis, and sepsis. The main feature is an arrest of alveolar and capillary formation (2). Models trying to decipher genes involved in the pathophysiology of BPD are mainly based on MV and oxygen application to young mammals with immature lungs of different species (3). In newborn rodent models, analyses of lung structure and gene and protein expression are performed for practical reasons directly at the end of MV (4,5,6). However, later appearing changes of gene expression might also have an impact on lung development and the evolution towards BPD and cannot be discovered by such models. Recently, we developed a newborn rat model of MV using an atraumatic (orotracheal) intubation technique that allows the weaning of the newborn animal off anesthesia and MV, the extubation to spontaneous breathing, and therefore allows the evaluation of effects of MV after a ventilation-free period of recovery (7). Indeed, applying this concept of atraumatic intubation by direct laryngoscopy, we recently were able to show significant differences between gene expression changes appearing directly after MV compared to those measured after a ventilation-free interval of 48 h. Immediately after MV, inflammation-related genes showed a transitory modified expression, while another set of more structurally related genes changed their expression only after a delay of 2 d (7). Lung structure, analyzed by conventional 2D histology and also by 3D reconstruction using synchrotron x-ray tomographic microscopy revealed, 48 h after end of MV, a reduced complexity of lung architecture compared to the nonventilated rat lungs, similar to the typical findings in BPD. To extend these observations about late gene expression modifications, we performed with a similar model a full gene expression profile of lung tissue 48 h after the end of MV with either room air or 60% oxygen. Essentially, we measured changes in the expression of genes related to the MMPs and complement system which played a role in many of the six identified mostly affected pathways.

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero

Development of in utero gene transfer approaches may provide therapies for genetic disorders with perinatal morbidity. In hemophilia A, prenatal and postnatal bleeding may be catastrophic, and modest increments in factor VIII (FVIII) activity are therapeutic. We performed transuterine i.p. gene transfer at day 15 of gestation in a murine model of hemophilia A. Normal, carrier (XHX), and FVIII-deficient (XHY and XHXH) fetuses injected with adenoviral vectors carrying luciferase or β-galactosidase reporter genes showed high-level gene expression with 91% fetal survival. The live-born rates of normal and FVIII-deficient animals injected in utero with adenovirus murine FVIII (3.3 × 105 plaque-forming units) was 87%. FVIII activity in plasma was 50.7 ± 10.5% of normal levels at day 2 of life, 7.2 ± 2.2% by day 15 of life, and no longer detectable at day 21 of life in hemophilic animals. Injection of higher doses of murine FVIII adenovirus at embryonic day 15 produced supranormal levels of FVIII activity in the neonatal period. PCR analysis identified viral genomes primarily in the liver, intestine, and spleen, although adenoviral DNA was detected in distal tissues when higher doses of adenovirus were administered. These studies show that transuterine i.p. injection of adenoviral vectors produces therapeutic levels of circulating FVIII throughout the neonatal period. The future development of efficient and persisting vectors that produce long-term gene expression may allow for in utero correction of genetic diseases originating in the fetal liver, hematopoietic stem cells, as well as other tissues.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Gene-experience interaction alters the cholinergic septohippocampal pathway of mice

Spatial learning requires the septohippocampal pathway. The interaction of learning experience with gene products to modulate the function of a pathway may underlie use-dependent plasticity. The regulated release of nerve growth factor (NGF) from hippocampal cultures and hippocampus, as well as its actions on cholinergic septal neurons, suggest it as a candidate protein to interact with a learning experience. A method was used to evaluate NGF gene-experience interaction on the septohippocampal neural circuitry in mice. The method permits brain region-specific expression of a new gene by using a two-component approach: a virus vector directing expression of cre recombinase; and transgenic mice carrying genomic recombination substrates rendered transcriptionally inactive by a “floxed” stop cassette. Cre recombinase vector delivery into transgenic mouse hippocampus resulted in recombination in 30% of infected cells and the expression of a new gene in those cells. To examine the interaction of the NGF gene and experience, adult mice carrying a NGF transgene with a floxed stop cassette (NGFXAT) received a cre recombinase vector to produce localized unilateral hippocampal NGF gene expression, so-called “activated” mice. Activated and control nonactivated NGFXAT mice were subjected to different experiences: repeated spatial learning, repeated rote performance, or standard vivarium housing. Latency, the time to complete the learning task, declined in the repeated spatial learning groups. The measurement of interaction between NGF gene expression and experience on the septohippocampal circuitry was assessed by counting retrogradely labeled basal forebrain cholinergic neurons projecting to the hippocampal site of NGF gene activation. Comparison of all NGF activated groups revealed a graded effect of experience on the septohippocampal pathway, with the largest change occurring in activated mice provided with repeated learning experience. These data demonstrate that plasticity of the adult spatial learning circuitry can be robustly modulated by experience-dependent interactions with a specific hippocampal gene product.

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced β-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21–26 nt) corresponding to the 3′ proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3′ end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma.

A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.