Search PubMed⌕ Search

Biomedical subjects

Andrew D Miller

Publications and source records attributed to Andrew D Miller.

At least 37 records · Page 2Linked to original sources
PubMed2005

What Role Can Chemistry Play in Cationic Liposome-Based Gene Therapy Research Today?

Gene therapy research is still in trouble owing to a paucity of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but may be too dangerous for routine clinical use. Synthetic non-viral vectors are inherently much safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors based upon well-found platform technologies and a thorough understanding of the barriers to efficient gene delivery and expression (transfection) relevant to clinical applications of interest. Here we introduce and interpret synthetic non-viral vector systems through the ABCD nanoparticle structural paradigm that represents, in our view, an appropriate lens through which to view all synthetic, non-viral vector systems applicable to in vitro use or in vivo applications and gene therapy. Our intention in introducing this paradigm is to shift the focus of organic and physical chemists away from the design of yet another cytofectin, and instead encourage them to appreciate the wider challenges presented by the need to produce tool kits of meaningful chemical components from which to assemble viable, tailor-made nanoparticles for in vivo applications and gene therapy, both now and in the future.

Journal Article↗
PubMed2005

What role can chemistry play in cationic liposome-based gene therapy research today?

Gene therapy research is still in trouble owing to a paucity of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but may be too dangerous for routine clinical use. Synthetic non-viral vectors are inherently much safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors based upon well-found platform technologies and a thorough understanding of the barriers to efficient gene delivery and expression (transfection) relevant to clinical applications of interest. Here we introduce and interpret synthetic non-viral vector systems through the ABCD nanoparticle structural paradigm that represents, in our view, an appropriate lens through which to view all synthetic, non-viral vector systems applicable to in vitro use or in vivo applications and gene therapy. Our intention in introducing this paradigm is to shift the focus of organic and physical chemists away from the design of yet another cytofectin, and instead encourage them to appreciate the wider challenges presented by the need to produce tool kits of meaningful chemical components from which to assemble viable, tailor-made nanoparticles for in vivo applications and gene therapy, both now and in the future.

Biogenic Polyamines↗
PubMed2004

Facile preparation of an orthogonally protected, pH-sensitive, bioconjugate linker for therapeutic applications.

We describe the facile, three-step synthesis of an orthogonally protected, pH-sensitive linker (8), based on maleic acid, and report its application to the preparation of a pH-sensitive phospholipid (20) for potential use in drug and gene delivery. In addition, we highlight the benefits of our linker over the use of the commercially available cis-aconitic anhydride (4).

Hydrogen-Ion Concentration↗
PubMed2004

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA.

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Apoptosis↗
PubMed2004

Synthesis of novel fluorescent-labelled dinucleoside polyphosphates.

A novel tandem synthetic-biosynthetic procedure is described for the synthesis of four new fluorescent dinucleoside polyphosphates: mant-Ap4A, mant-AppCH2ppA, TNP-Ap4A and TNP-AppCH2ppA. These compounds are expected to supplement the existing etheno (epsilon) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) labelled derivatives, being the fluorescent probes of choice to investigate polyphosphate/enzyme binding behaviour.

Boron Compounds↗
PubMed2004

De-novo design of complementary (antisense) peptide mini-receptor inhibitor of interleukin 18 (IL-18).

Complementary (antisense) peptide mini-receptor inhibitors are complementary peptides designed to be receptor-surrogates that act by binding to selected surface features of biologically important proteins thereby inhibiting protein-cognate receptor interactions and subsequent biological effects. Previously, we described a complementary peptide mini-receptor inhibitor of interleukin-1beta (IL-1beta) that was designed to bind to an external surface loop (beta-bulge) of IL-1beta (Boraschi loop) clearly identified in the X-ray crystal structure of this cytokine. Here, we report the de-novo design and rational development of a complementary peptide mini-receptor inhibitor of cytokine interleukin-18 (IL-18), a protein for which there is no known X-ray crystal structure. Using sequence homology comparisons with IL-1beta, putative IL-18 surface loops are identified and used as a starting point for design, including a loop region 1 thought to be equivalent with the Boraschi loop of IL-1beta. Only loop region 1 complementary peptides are found to be promising leads as mini-receptor inhibitors of IL-18 but these are prevented from being properly successful owing to solubility problems. The application of "M-I pair mutagenesis" and inclusion of a C-terminal arginine residue are then sufficient to solve this problem and convert one lead peptide into a functional complementary peptide mini-receptor inhibitor of IL-18. This suggests that the biophysical and biological properties of complementary peptides can be improved in a rational and logical manner where appropriate, further strengthening the potential importance of complementary peptides as inhibitors of protein-protein interactions, even when X-ray crystal structural information is not readily available.

Drug Design↗
PubMed2003

Kinetic study of DNA condensation by cationic peptides used in nonviral gene therapy: analogy of DNA condensation to protein folding.

Synthetic nonviral vector systems are attractive because of their apparent simplicity of preparation and use. However, there are many barriers to success at the moment, including the formulation of uniform and reproducible particles of transfection competent, condensed nucleic acids such as plasmid DNA (pDNA). For this reason, we have been studying the kinetics of cationic peptide-mediated pDNA condensation and the reverse process following peptide dissociation by stopped-flow techniques under conditions commonly used to prepare synthetic nonviral vector systems. We observe that the process of pDNA condensation and the reverse process of pDNA expansion appear to be equivalent to protein folding and unfolding, respectively. We also observe chaotic behavior at low peptide/pDNA ratios that becomes more uniform at higher ratios suggesting that with suboptimal ratios, pDNA is condensing in a multitude of conformations, each representing different stages of hydrophobic collapse in the search for the thermodynamically most stable (i.e., the fully condensed pDNA molecule). At higher ratios, peptide/pDNA complexes formed appear to be increasingly irreversible consistent with the formation of kinetically and/or thermodynamically stable, condensed pDNA molecules. Such stable states could create problems for the successful transcription of pDNA post delivery to cells.

Amino Acid Sequence↗
PubMed2003

Comparison between the interactions of adenovirus-derived peptides with plasmid DNA and their role in gene delivery mediated by liposome-peptide-DNA virus-like nanoparticles.

Previously we have described the development and applications of an important new platform system for gene delivery known as liposome-mu-DNA (LMD), prepared from cationic liposomes (L), plasmid DNA (D) and the mu(M) peptide derived from the adenovirus core. In an attempt to improve upon mu, an alternative peptide (pepV) derived from the adenovirus peptide/protein-DNA core complex was identified, synthesised and studied alongside mu using a number of biophysical techniques including gel retardation, ethidium bromide exclusion, CD binding titration, DNA melting, and plasmid protection assays. PepV binds to pDNA less efficiently than mu but is able to charge neutralise and condense pDNA into negatively charged pepVD particles comparable in dimension to MD particles. The results of CD studies and plasmid protection assays suggest that peptide-DNA interactions are likely to cause pDNA condensation by a combination of charge neutralisation, base pair tilting, double helix destabilisation and the induction of pDNA superfolding. Data suggest the pepVD particles may be formulated with cationic liposomes to give defined LpepVD particles that appear to transfect HeLa cells with marginally more efficiency than LMD particles suggesting that pepV may have some effect on the pDNA transcription process. Although pepV harbours a nuclear-nucleolar localisation sequence (NLS), transfection data show that this capacity is not being appropriately harnessed by the current LpepVD formulation. Further improvements may be required in terms of optimising LpepVD formulations--for instance, to ensure the integrity of the peptide-DNA complexes following cell entry--in order to fully exploit the full NLS capacity of the peptide, thereby facilitating the transfection of slowly dividing or quiescent cells.

Adenoviridae↗
PubMed2003

Chemical neuroimmunology: health in a nutshell bidirectional communication between immune and stress (limbic-hypothalamic-pituitary-adrenal) systems.

Stress is a ubiquitous and pervasive part of modern life that is frequently blamed for causing a plethora of diseases and other discomforting medical conditions. All higher organisms, including humans, experience stress in the form of a wide variety of stressors that range from environmental pollutants and drugs to traumatic events or self-induced trauma. Stressors registered by the central nervous system (CNS) generate physiological stress responses in the body (periphery) by means of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis. This LHPA axis operates through the use of chemical messengers such as the stress hormones corticotropin-releasing hormone (CRH) and glucocorticoids (GCs). Under conditions of frequent exposure to acute stress and/or chronic, long-term exposure to stress, the LHPA axis becomes dysfunctional and in the process frequently overproduces both CRH and GCs, which results in many mild to severely toxic side effects. Bidirectional communication between the LHPA axis and immune/inflammatory systems can dramatically potentiate these side effects and create environments in the CNS and periphery ripe for the triggering and/or promotion of tissue degeneration and disease. This review aims to present as far as possible a molecular view of the processes involved so as to provide a bridge from the diffuse range of studies on molecular structure and receptor interactions to the burgeoning biological and medical literature that describes the empirical interplay between stress and disease. We hope that our review of this fast-growing field, which we christen chemical neuroimmunology, will give a clear indication of the striking range and depth of current molecular, cellular and medical evidence linking stress hormones to degeneration and disease. In so doing, we hope to provide encouragement for others to become interested in this critical and far-reaching field of research, which is very much at the heart of many important disease processes and very much a critical part of the crucial interface between chemistry and biology.

Animals↗
PubMed2003

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment.

BACKGROUND: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. RESULTS: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. CONCLUSIONS: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism.

Adenosine Triphosphate↗
PubMed2003

Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes.

The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.

Biophysical Phenomena↗
PubMed2003

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 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-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-beta-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-PEG(5000)-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.

Adenoviridae↗
PubMed2003

The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy.

Gene therapy research is in crisis owing to the lack of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but currently appear to be too dangerous for routine clinical use. Synthetic non-viral vectors are inherently much safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors based upon well-found platform technologies and a thorough understanding of the barriers to efficient gene delivery and expression (transfection) relevant to clinical applications of interest. In this review, the current status and prospects for cationic liposome/micelle-based synthetic non-viral vector systems are discussed including a description of the barriers to efficient transfection, a summary of the main structure/activity studies and mention of ternary cationic liposome/micelle-nucleic acid (LD) systems. The review culminates with a description of two promising cationic liposome/micelle-based non-viral vector platform systems known as liposome:mu:DNA (LMD) and stabilised plasmid-lipid particles (SPLP) that should create a real opportunity for the development of clinically viable synthetic vector systems within the next few years.

Animals↗
PubMed2002

Specific interactions between sense and complementary peptides: the basis for the proteomic code.

The discovery of the genetic code was one of the milestone events in biology: a conserved, universal code defining the primary amino acid sequences of all proteins of all organisms. However, this code has been thought to be limited, unable to provide additional information appropriate to defining the three-dimensional structure and function of these proteins. This raises important questions. Can there be more to the genetic code? Is there a code embedded within the code? Does a two-dimensional genetic code exist? In our view, the answer to all three of these questions is a qualified "yes". This review describes how sense and complementary peptides coded for by mutually complementary nucleic acid sequences are capable of interacting specifically, thereby suggesting the existence of a second, two-dimensional genetic code (proteomic code). Theories attempting to explain such specific interactions between sense and complementary peptides are discussed including the Mekler-Idlis (M-I) pair theory that suggests that each codon-directed amino acid residue in a sense peptide may make a specific pair-wise interaction with the corresponding complementary codon-directed residue in the complementary peptide. In effect, through-space interactions between pairs of amino acid residues are suggested as being specified by the genetic code and its complement. The biological implications of sense/complementary peptide interactions are potentially vast but still to be fully understood and appreciated. That such peptide/peptide interactions could provide the basis for understanding and constructing the proteomic code remains to be properly established but research to date suggests that we should be able to make a start in that direction.

Amino Acids↗
PubMed2002

Biophysical characterization of the DNA binding and condensing properties of adenoviral core peptide mu.

Cationic peptides containing Lys and Arg residues interact with DNA via charge-charge interactions and are known to play an important role in DNA charge neutralization and condensation processes. In this paper, we describe investigations of the interaction of the cationic adenovirus core complex peptide mu with a dodecameric ODN (12 bp) and pDNA (7528 bp) using a combination of fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and photon correlation spectroscopy. Comparisons are made with protamine, a cationic peptide well-known for DNA charge neutralization and condensation. Equilibrium dissociation constants are derived independently by both CD and ITC methods for the interaction between protamine or mu with pDNA (K(d) = 0.6-1 microM). Thermodynamic data are also obtained by ITC, indicating strong charge-charge interactions. The interaction of protamine with pDNA takes place with decreasing entropy (-28.7 cal mol(-1) K(-1)); unusually, the interaction of mu with pDNA takes place with increasing entropy (Delta S degrees (bind) = 11.3 cal mol(-1) K(-1)). Although protamine and mu appear to destabilize pDNA double helix character to similar extents, according to CD thermal titration analyses, PCS studies show that interactions between mu and pDNA result in the formation of significantly more size-stable condensed particles than protamine. The enhanced flexibility and size stability of mu-DNA (MD) particles (80-110 nm) compared to protamine counterparts suggest that MD particles are ideal for use as a part of new nonviral gene delivery vectors.

Adenoviridae↗