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.

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes.

Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.

Transcriptional and phenotypic comparisons of Ppara knockout and siRNA knockdown mice.

RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara-/- mice. Combining the profiles from mice treated with the PPARalpha agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARalpha regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara-/- mice. In contrast to Ppara-/- mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies.

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.

Rapid intravascular injection into limb skeletal muscle: a damage assessment study.

We have recently developed a simple and highly efficient methodology for delivering plasmid DNA (pDNA) to skeletal muscle cells of mammalian limbs. The procedure involves the rapid intravascular injection of a large volume of saline (containing pDNA) into the vasculature of the distal limb. As a result of the robust delivery methodology involved, it is important to understand the effects of the injection procedure on the skeletal muscle tissue in the targeted limb. In previous studies, only modest and transient muscle damage was noted. In this study we quantitatively assessed the degree of muscle damage in rat limbs following intravascular injections using muscle histology (H&E staining), membrane integrity (Evans blue staining), and leukocyte infiltration (immunohistochemistry) assays. The rapid extravasation of fluid during the injection process resulted in edema of the muscle tissue of the targeted limb; however, the edema was transient and resolved within 24 h. Consistent with observations from previous studies, minimal levels of myofiber damage were detected. Immunohistochemical labeling indicated that increased numbers of neutrophils (CD43+) and macrophages (ED1+ and ED2+) were present in the muscle tissue interstitium shortly after injection but that elevations were relatively modest and resolved by 2 weeks postinjection.

Delivery of small interfering RNA to mammalian cells in culture by using cationic lipid/polymer-based transfection reagents.

RNA interference (RNAi) has become a powerful tool for the knockdown of target gene expression and subsequent phenotypic analysis of gene function in mammalian cells in culture. Critical to the success of any small inhibitory RNA (siRNA)-mediated RNAi knockdown in mammalian cells is the efficient delivery of the siRNA to those cells. This chapter describes the use of popular cationic lipid?polymer-based transfection reagents for in vitro siRNA delivery and includes a general protocol with special emphasis on key transfection parameters important to the success of siRNA delivery.

A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs.

Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications.

Plasmid-based gene delivery to target tissues in vivo: the intravascular approach.

Over the past several years, significant progress has been made in the development of non-viral methodologies that can effectively deliver genes to target tissues in vivo. One of the most surprising successes has been the discovery that naked plasmid DNA (pDNA) can be delivered into tissues such as liver and muscle with high efficiency using the vascular system. The key breakthrough involved the realization that pDNA could be injected rapidly into blood vessels (using increased volumes) in a manner that facilitates extravasation of the DNA solution outside the blood vessel wall. The extravasation process places the DNA in contact with the plasma membranes of the underlying parenchymal cells of the target organ. This intravascular delivery technique, termed 'hydrodynamic delivery', has become established as the primary non-viral methodology for delivering pDNA expression constructs to target tissues in vivo. This review highlights many of the most recent studies in which increased volume/rapid injection procedures have been used. These include studies in which the technology was used as a new and powerful tool to address in vivo gene expression questions, as well as numerous studies that were designed to better understand or improve the methodology. It is these scientific efforts that have served to fuel the development of this delivery technology from simply an interesting phenomenon to a highly useful and broadly used gene delivery methodology.

Efficient in vitro and in vivo expression of covalently modified plasmid DNA.

The tracking of plasmid DNA (pDNA) movement within cells requires the attachment of labels to the DNA in a manner such that: (a) the pDNA remains intact during the labeling process and (b) the labels remain stably attached to the DNA. Keeping these two criteria in mind, we have recently developed a series of alkylating reagents that facilitate the one-step, covalent attachment of compounds directly onto nucleic acids in a nondestructive manner. Using these DNA-alkylating reagents, we have attached a wide range of both fluorescent and nonfluorescent reporter molecules onto pDNAs. We now show that even with the covalent attachment of various marker compounds, the pDNA remains expression competent. The ability to create labeled, expression-competent DNA allows for the simultaneous tracking of both pDNA location and reporter gene expression within living or fixed cells.

Efficient delivery of siRNA for inhibition of gene expression in postnatal mice.

It has recently been shown that RNA interference can be induced in cultured mammalian cells by delivery of short interfering RNAs (siRNAs). Here we describe a method for efficient in vivo delivery of siRNAs to organs of postnatal mice and demonstrate effective and specific inhibition of transgene expression in a variety of organs.

Self-quenched covalent fluorescent dye-nucleic acid conjugates as polymeric substrates for enzymatic nuclease assays.

A fluorescent method is described for assessing nuclease activity. The technique is based on the preparation of quenched fluorophore-nucleic acid covalent conjugates and their subsequent dequenching due to degradation by nucleases. The resulting fluorescence increase can be measured by a spectrofluorometer and exhibits subpicogram per milliliter sensitivity level for RNase A and low picogram per milliliter level for DNase I. The method is adaptable for quantitative nuclease inhibitor testing.