Progress toward a nonviral gene therapy protocol for the treatment of anemia.

Anemia frequently accompanies chronic diseases such as progressive renal failure, acquired immunodeficiency syndrome, and cancer. Patients are currently treated with erythropoietin (EPO) replacement therapy, using various recombinant human EPO protein formulations. Although this treatment is effective, gene therapy could be more economical and more convenient for the long-term management of the disease. The objective of this study was to develop a naked DNA-based gene therapy protocol that could fill this need. Hydrodynamic limb vein technology has been shown to be an effective and safe procedure for delivering naked plasmid DNA (pDNA) into the skeletal muscles of limbs. Using this method, we addressed the major challenge of an EPO-based gene therapy of anemia: maintaining stable, long-term expression at a level that sufficiently promotes erythropoiesis without leading to polycythemia. The results of our study, using a rat anemia model, provide proof of principle that repeated delivery of small pDNA doses has an additive effect and can gradually lead to the correction of anemia without triggering excessive hematopoiesis. This simple method provides an alternative approach for regulating EPO expression. EPO expression was also proportional to the injected pDNA dose in nonhuman primates. In addition, long-term (more than 450 days) expression was obtained after delivering rhesus EPO cDNA under the transcriptional control of the muscle-specific creatine kinase (MCK) promoter. In conclusion, these data suggest that the repeated delivery of small doses of EPO expressing pDNA into skeletal muscle is a promising, clinically viable approach to alleviate the symptoms of anemia.

Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA.

Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used to induce antigen-specific antibodies in mice, rats, and rabbits. Both methods allowed the efficient generation of high-titer, antigen-specific antibodies with an overall success rate of Western detectable antibodies of 78% and 92%, respectively. High-titer antibodies were typically present after 3 hydrodynamic tail vein plasmid DNA deliveries, 5 weeks after the initial injection (i.e., prime). For hydrodynamic limb vein plasmid DNA delivery, two deliveries were sufficient to induce high-titer antibody levels. Tail vein delivery was less successful at generating antibodies directed against secreted proteins as compared with limb vein delivery. Material for screening was generated by,transfection of the immunization vector into mammalian cell lines. The cell line (COS-7) that produced the highest level of antigen expression performed best in Western blot analysis screens. In summary, intravenous delivery of antigen-expressing plasmid DNA vectors is an effective genetic immunization method for the induction of antigen-specific antibodies in small and large research animals.

Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.

The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 µg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA.

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 µg/g and maximal expression at approximately 400 µg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Evaluation of hydrodynamic limb vein injections in nonhuman primates.

The administration route is emerging as a critical aspect of nonviral and viral vector delivery to muscle, so as to enable gene therapy for disorders such as muscular dystrophy. Although direct intramuscular routes were used initially, intravascular routes are garnering interest because of their ability to target multiple muscles at once and to increase the efficiency of delivery and expression. For the delivery of naked plasmid DNA, our group has developed a hydrodynamic, limb vein procedure that entails placing a tourniquet over the proximal part of the target limb to block all blood flow and injecting the gene vector rapidly in a large volume so as to enable the gene vector to be extravasated and to access the myofibers. The present study was conducted in part to optimize the procedure in preparation for a human clinical study. Various injection parameters such as the effect of papaverine preinjection, tourniquet inflation pressure and duration, and rate of injection were evaluated in rats and nonhuman primates. In addition, the safety of the procedure was further established by determining the effect of the procedure on the neuromuscular and vascular systems. The results from these studies provide additional evidence that the procedure is well tolerated and they provide a foundation on which to formulate the procedure for a human clinical study.

Effect of tissue-specific promoters and microRNA recognition elements on stability of transgene expression after hydrodynamic naked plasmid DNA delivery.

Intravenous hydrodynamic injections into the liver and skeletal muscle have increased the efficacy of naked DNA delivery to a level that makes therapeutically relevant gene transfer attainable. Although there are no concerns about the immunogenicity of the delivered DNA itself, transgene products that are foreign to the host can trigger an immune response and hamper the therapeutic effect. Our goal was to determine whether and to what extent some known preventive measures are applicable to these delivery methods in order to achieve longterm expression of foreign proteins in immunocompetent mice. We designed plasmid DNA vectors that expressed a marker gene under the control of either a ubiquitous or a tissue-specific promoter. We also included microRNA (miR) target sites in the transcripts in order to silence expression in antigen-presenting cells (APCs). The constructs were delivered either into muscle or liver, using outbred ICR and inbred C57BL=6 mice. The data suggest that firefly luciferase, a potent immunogen, triggered a uniform immune response only in outbred ICR mice, and only when expressed from a ubiquitous promoter. This response could not be prevented by including APC-specific miR target sites in the transcript. In contrast, the probability of immune rejection in ICR mice could be significantly diminished by using tissue-specific promoters, and under these circumstances, the silencing of transgene expression in APCs did confer some benefits. After a single hydrodynamic injection, inbred mice did not reject luciferase under any of the tested conditions for at least 8 weeks. To test whether they became tolerized, they were challenged with a second boost of a cytomegalovirus promoter-driven luciferase construct. This triggered a strong immune response, suggesting that luciferase-reactive cells from the animals' T and B cell repertoire had not been eliminated. This secondary reaction could not be prevented by silencing expression in APCs. In conclusion, for the clinical application of hydrodynamic naked DNA delivery the use of tissue-specific promoters in combination with silencing expression in APCs will increase the probability of long-term expression, but the most desirable outcome, the establishment of transgene tolerance, appears unlikely to be achieved by any of these measures.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies.

BACKGROUND: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA. METHODS: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance. RESULTS: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them. CONCLUSIONS: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules.

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.

The effect of cell division on the cellular dynamics of microinjected DNA and dextran.

Gene delivery is a multistep process that is being studied to increase its efficiency, a major hurdle for effective gene therapy. Our study focused on the nuclear entry step by microinjecting a mixture of fluorescent dextran and the pEYFP-Nuc plasmid (encoding a nuclear-targeted, enhanced GFP) into the cytoplasm of nondividing and dividing cells that were selected using non-chemical means. After 10 and 1000 ng/microl of plasmid DNA (pDNA) were cytoplasmically injected, 28% and 50% of the cells that had not divided expressed GFP, respectively, compared with 50% and 90% for the cells that had divided. This result suggested that pDNA can enter the nonmitotic nuclei of mononucleated cells, albeit at a lower efficiency than mitotic nuclei. The ability of pDNA to enter the intact nuclei of nondividing cells is consistent with our previous experience using multinucleated myotubes and digitonin-permeabilized cells in culture and using intravascular naked pDNA delivery in vivo. An explanation for the small effect of cell division was provided by studies using fluorescently labeled molecules and confocal fluorescent microscopy. They showed that the bulk of large dextran, and similarly pDNA, was excluded from re-formed nuclei after mitosis, thereby limiting the effect of cell division on the nuclear entry of pDNA.