Richard P. Harbottle

Nuclear Localisation Sequence Templated Nonviral Gene Delivery Vectors: Investigation of Intracellular Trafficking Events of LMD and LD Vector Systems†

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3β‐[N‐(N′, N′‐dimethylaminoethane)carbamoyl]cholesterol/dioleoyl‐L‐α‐phosphatidyl ethanolamine (DC‐Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent‐labelled lipid and peptides plus dye‐labelled pDNA components were used to investigate gene delivery in dividing and S‐phase growth‐arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu‐β‐galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30–45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth‐arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC‐Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. “Stealth” systems obtained by post‐coating LMD particles with fluorescent‐labelled PEG molecules (0.5, 5 and 10 mol % fluorescein‐PEG5000‐N‐hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.

Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo

Gene transfer to the trachea and airways by adenoviral vectors is limited by the basolateral localization of viral receptors, resulting in relatively low levels of transduction. Modification of paracellular permeability by sodium caprate, which opens tight junctions, enhances gene transfer from the apical side of cultured human airway epithelial cells. Based on this observation we investigated whether Na-caprate could also increase gene transfer when applied to the luminal surface of the airway epithelia in vivo and compared these results with EGTA, which has previously been shown to enhance adenovirus transduction. Transgene expression in the trachea and upper airways was increased 25-fold by a 10-min pretreatment with 50 mM Na-caprate, corresponding to a 3-fold improvement over EGTA. In the more peripheral airways EGTA had no effect, whereas expression of beta-gal was increased 3-fold by Na-caprate. When the adenovirus was complexed with DEAE dextran, transduction of the airway epithelia after Na-caprate pretreatment was increased 45-fold over virus alone. In conclusion, Na-caprate facilitates gene transfer to airway epithelia, particularly when adenovirus is complexed with DEAE dextran, and may in future be used in a clinical setting to enhance the efficiency of vectors for gene therapy of cystic fibrosis via airway delivery.

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr‐His‐Ala‐Leu‐Trp‐His‐Thr (THALWHT). Subsequent binding studies confirmed that THALWHT‐displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non‐airway‐derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non‐viral and viral gene therapy vectors.

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo

To develop more efficient non‐viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR‐NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV‐1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle.

Delivery and long-term expression of a 135 kb LDLR genomic DNA locus in vivo by hydrodynamic tail vein injection.

The delivery of a complete genomic DNA locus in vivo may prove advantageous for complementation gene therapy, especially when physiological regulation of gene expression is desirable. Hydrodynamic tail vein injection has been shown to be a highly efficient means of non‐viral delivery of plasmid DNA to the liver. Here, we apply hydrodynamic tail vein injection to deliver and express large genomic DNA inserts > 100 kb in vivo.

LDLR-Gene therapy for familial hypercholesterolaemia: problems, progress, and perspectives.

Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first ex vivo clinical trial of gene therapy in FH was conducted nearly 18 years ago. Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patients own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency.

Development of a Self-assembling Nuclear Targeting Vector System Based on the Tetracycline Repressor Protein

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR·tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2–8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.

pH-Triggered Nanoparticle Mediated Delivery of siRNA to Liver Cells in Vitro and in Vivo

Recently, we reported for the first time the development of pH-triggered nanoparticles for the functional delivery of small interfering RNA (siRNA) to liver for treatment of hepatitis B virus infections in vivo. Here, we report on systematic formulation and biophysical studies of three different pH-triggered nanoparticle formulations looking for ways to improve on the capabilities of our previous nanoparticle system. We demonstrate how pH-triggered, PEGylated siRNA nanoparticles stable with respect to aggregation in 80% serum can still release siRNA payload at pH 5.5 within 30 min. This capability allows functional delivery to cultured murine hepatocyte cells in vitro, despite a high degree of PEGylation (5 mol %). We also demonstrate that pH-triggered, PEGylated siRNA nanoparticles typically enter cells by clathrin-coated pit endocytosis, but functional delivery requires membrane fusion events (fusogenicity). Biodistribution studies indicate that >70% of our administered nanoparticles are found in liver hepatocytes, post intravenous administration. Pharmacodynamic experiments show siRNA delivery to murine liver effecting maximum knockdown 48 h post administration from a single dose, while control (nontriggered) nanoparticles require 96 h and two doses to demonstrate the same effect. We also describe an anti-hepatitis C virus (HCV) proof-of-concept experiment indicating the possibility of RNAi therapy for HCV infections using pH-triggered, PEGylated siRNA nanoparticles.

A novel peptide, PLAEIDGIELTY, for the targeting of α9β1-integrins

Targeting gene therapy vectors to abundant receptors on airway epithelia may allow a significant enhancement of gene delivery and thereby be of particular importance for the gene therapy of cystic fibrosis. α9β1‐Integrins are highly expressed throughout the human airway epithelia in vivo, irrespective of any particular clinical status. Aiming to improve the targeting of our non‐viral integrin‐mediated gene transfer systems to airway epithelia, we searched for a short tenascin C‐derived peptide which would bind to these integrins. By utilizing recombinant bacteriophages that display overlapping regions of the third fibronectin type III repeat of tenascin C (TNfn3), we were able to localize its α9β1‐integrin binding site to the B‐C loop of TNfn3. A synthetic Pro‐Leu‐Ala‐Glu‐Ile‐Asp‐Gly‐Ile‐Glu‐Leu‐Thr‐Tyr peptide (PLAEIDGIELTY) was shown to displace α9β1‐integrin‐expressing cells completely from binding to TNfn3. This peptide, therefore, may prove useful both for the examination of the functional importance of α9β1‐integrins in vivo and the development of gene therapy vectors or drugs targeting these integrins.

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (alpha V beta 3/beta 5) and fibronectin (alpha 5 beta 1) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.