Michael R. Jorgensen

Controlling HBV Replication in Vivo by Intravenous Administration of Triggered PEGylated siRNA-Nanoparticles

Harnessing RNA interference (RNAi) to inhibit hepatitis B virus (HBV) gene expression has promising application to therapy. Here we describe a new hepatotropic nontoxic lipid-based vector system that is used to deliver chemically unmodified small interfering RNA (siRNA) sequences to the liver. Anti HBV formulations were generated by condensation of siRNA (A component) with cationic liposomes (B component) to form AB core particles. These core particles incorporate an aminoxy cholesteryl lipid for convenient surface postcoupling of polyethylene glycol (PEG; C component, stealth/biocompatibility polymer) to give triggered PEGylated siRNA-nanoparticles (also known as siRNA-ABC nanoparticles) with uniform small sizes of 80-100 nm in diameter. The oxime linkage that results from PEG coupling is pH sensitive and was included to facilitate acidic pH-triggered release of nucleic acids from endosomes. Nanoparticle-mediated siRNA delivery results in HBV replication knockdown in cell culture and in murine hydrodynamic injection models in vivo. Furthermore repeated systemic administration of triggered PEGylated siRNA-nanoparticles to HBV transgenic mice results in the suppression of markers of HBV replication by up to 3-fold relative to controls over a 28 day period. This compares favorably to silencing effects seen during lamivudine treatment. Collectively these observations indicate that our PEGylated siRNA-nanoparticles may have valuable applications in RNAi-based HBV therapy.

A dialkynoyl analogue of DOPE improves gene transfer of lower-charged, cationic lipoplexes

Positively-charged gene delivery agents, such as cationic liposomes, typically prepared by mixing a cationic lipid and a neutral lipid in a 1 : 1 molar ratio, exhibit a fundamental flaw: on the one hand, the charge encourages cell uptake; on the other hand, the charge leads to aggregation in vivo with anionic serum components. We herein report a more phase-stable analogue of the zwitterionic and fusogenic lipid DOPE that allows for the reduction of the cationic lipid component of the liposome from 50 to 9 mol% with almost no apparent loss in transfection activity. This reduction in charge may induce important in vivo stability whilst still imparting high cell uptake and transgene expression.

MAGfect: a novel liposome formulation for MRI labelling and visualization of cells

Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojene.

Synthesis and Application of Integrin Targeting Lipopeptides in Targeted Gene Delivery

One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool‐kits of chemical components. First‐generation LMD systems were prepared from DC‐Chol/DOPE cationic liposomes (DC‐Chol=3β‐[N‐(N′,N′‐dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl‐L‐α‐phosphatidylethanolamine), μ peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide‐targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell‐surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target α9β1‐integrin proteins predominant on upper‐airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. “Targeted” LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide‐targeted, synthetic, nonviral gene delivery to be realised.

Biophysical Properties of CDAN/DOPE-Analogue Lipoplexes Account for Enhanced Gene Delivery

Typically, cationic liposomes are formulated from the combination of a synthetic cationic lipid (cytofectin) and a neutral, biologically available co‐lipid. However, the use of cationic liposome formulations to mediate gene delivery to cells is hampered by a paradox. Cationic lipids, such as N1‐cholesteryloxycarbonyl‐3‐7‐diazanonane‐1,9‐diamine (CDAN), are needed to ensure the formation of cationic liposome‐DNA (lipoplex, LD) particles by plasmid DNA (pDNA) condensation, as well as for efficient cell binding of LD particles and intracellular trafficking of pDNA post‐intracellular delivery by endocytosis. However, the same cationic lipids can exhibit toxicity, and also promote LD particle colloidal instability, leading to aggregation. This results from electrostatic interactions with anionic agents in biological fluids, particularly in vivo. One of the most commonly used neutral, bioavailable co‐lipids, dioleoyl L‐α‐phosphatidylethanolamine (DOPE), has been incorporated into many cationic liposome formulations owing to its fusogenic characteristics that are associated with a preference for the inverted hexagonal (HII) phase—a phase typical of membrane–membrane fusion events. However, these same fusogenic characteristics also destabilize LD particles substantially with respect to aggregation, in vitro and especially in vivo. Therefore, there is a real need to engineer more stable cationic liposome systems with lower cellular toxicity. We hypothesize that one way to achieve this goal should be to find the means to reduce the mol fraction of cationic lipid in cationic liposomes without impairing the overall transfection efficiency, by replacing DOPE with an alternative co‐lipid with fusogenic properties “tuned” with a greater preference for the more stable lamellar phases than DOPE is able to achieve. Herein, we document the syntheses of triple bond variants of DOPE, and their formulation into a range of low charge, low cationic lipid containing LD systems. The first indications are that our hypothesis is correct in vitro.

The facile preparation of primary and secondary amines via an improved Fukuyama–Mitsunobu procedure. Application to the synthesis of a lung-targeted gene delivery agent

An efficient modification of the Fukuyama-Mitsunobu procedure has been developed whereby primary or secondary amines can be synthesized from alkyl alcohols and the corresponding nosyl-protected/activated amine. Most importantly, the use of the DTBAD and diphenylpyridinylphosphine, as Mitsunobu reagents, generates reaction by-products that can be easily removed, providing a remarkably clean product mixture. This improved technique was implemented in the synthesis of a complex lipopeptide designed to target alpha9beta1-integrin proteins predominant on upper airway epithelial cells.

Novel phospholipid analogues of pan-PPAR activator tetradecylthioacetic acid are more PPARα selective

Tetradecylthioacetic acid (TTA) is a modified fatty acid that appears to improve insulin sensitivity, lower blood lipid levels, enhance fatty acid oxidation and promote anti-inflammatory action in vivo, through mechanisms partly dependent upon peroxisome proliferator-activated receptors (PPARs). In order to improve the biological efficacy of TTA as a PPAR agonist, two novel phospholipid analogue lyso tetradecylthioacetyl-L-alpha-phosphatidylcholine and di-tetradecylthioacetyl-L-alpha-phosphatidylglycerol have been developed. Here we report on the syntheses of these novel phospholipids and their relative potential to act as PPAR agonists in vitro, in comparison to TTA and other positive controls.

Synthesis and Analysis of Novel Glycerolipids for the Treatment of Metabolic Syndrome

Tetradecylthioacetic acid (TTA) 1 is a peroxisome proliferator-activated receptor (PPAR) agonist found to improve insulin sensitivity, lower blood lipid levels, enhance fatty acid oxidation, and promote anti-inflammation in vivo. In an attempt to enhance these properties, two key thioether fatty acid (Thefa) lipids, ditetradecylthioacetyl phosphatidylcholine 2 and tritetradecylthioacetyl glycerol 3, are synthesized and administered po to male Wistar rats at two different doses to study and compare metabolic outcomes relative to the administration of 1 alone after 6 days. Liposomal formulations of 1 and 2 are also prepared to evaluate acute metabolic responses (at 3 h) post i.v. injection. Across all metrics measured, 1-induced responses post po administration are in line with previous data. Responses induced from 3 are mostly equivalent to 1-induced responses. By contrast, 2-induced responses almost always outperform those of 1 and 3. Therefore, 2 may represent a new lead for the treatment of metabolic syndrome.

321. siFECT|[trade]|: Optimised Formulation for the Delivery of siRNA at Minimum Doses and Maximum Specificity

RNA interference (RNAi) has become a popular tool to down-regulate specific gene expression in many species including mammalian cells. Synthetic double stranded RNA sequences (siRNA) of 21–23 nucleotides were shown to have the potential to specifically silence gene function in cultured mammalian cells. This finding surged a great interest to apply siRNA in functional genomics programs to decipher specific gene function. However, literally no studies have been published in the literature on the impact of the lipid formulation on siRNA mediated gene silencing. For functional genomics studies to be valuable, specific gene down-regulation of one single target gene devoid of non-specific down-regulation of many cellular proteins has to be achieved.