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Methodologies for monitoring, nanoparticle formation by self-assembly of DNA with poly(L-lysine) Parker AL, Oupicky D, Dash PR, Seymour LW ANALYTICAL BIOCHEMISTRY 302 (1): 75-80 MAR 1 2002

The complexes formed between plasmid DNA and polycationic peptides such as those based on poly(L-lysine) form the basis of numerous non-viral gene delivery vectors.  In this paper, we describe several distinct methodologies for monitoring the formation of such nanoparticles formed by electrostatic interation between plasmid DNA and polycations such as those based on poly(L-lysine).  Effects of N:P (charge) ratio and polycation molecular weight are also examined with respect to complex stability to dilution and physiological levels of salt (150mM), ultimately drawing the conclusion that polyelectrolyte complexes for targeted application in vivo should be based on high molecular weight polycations, or should be stabilised to prevent their dissociation under physiological salt concentrations.  Click the link above to view this paper.

Laterally stabilized complexes of DNA with linear reducible polycations: Strategy for triggered intracellular activation of DNA delivery vectors Oupicky D, Parker AL, Seymour LW JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (1): 8-9 JAN 9 2002

This paper addresses one of the most significant quandries of systemic application of gene therapy vectors, namely how to achieve systemic inertness of the applied vectors (ie procluding interactions with the bodies immune system and blood serum factors) whilst providing a high degree of intracellular activity following uptake into the target cells.  By using a combination of pHPMA, a amino-reactive hydrophilic polymer that can (at least theorectically) be used to surface modify any gene therapy vectors with available surface amino groups, increasing their solubility whilst decreasing their interaction with blood and immune components, with a high molecular weight poly(L-lysine) designed to incorporate regular disulphide bonds (that undergo reduction at intracellular gluatathione levels), we have created nanoparticles that show excellent stability in the extracellular compartment coupled with high levels of intracellular activity.  Furthermore, cell specific targeting ligands (such as bFGF) can be incorporated into the vector (via spare amino-reactive ester groups on the polymer) to increase cell association and specificity.  Click the link above to view this paper.

Identification of FGF receptor-binding peptides for cancer gene therapy Maruta F, Parker AL, Fisher KD, Hallissey MT, Ismail T, Rowlands DC, Chandler LA, Kerr DJ, Seymour LW CANCER GENE THERAPY 9 (6): 543-552 JUN 2002

In this paper we describe how small (7-mer) oligopeptides were iterated using phage display technology that bind to bFGF receptors.  The receptors for bFGF have been shown to be upregulated in numerous tumours in vivo, presumably to help stimulate angiogensis.  Therefore, the possibility of using bFGF to target gene delivery vectors (or chemotherapeutic drugs) to the the tumour vasculature is an appealing one.  However, the likelihood of being able to use bFGF systemically is limited by the large, hydrophobic nature of the molecule, its difficulty (and cost) to mass produce, its potential angiogenic effects, and potential interaction with the bodies natural vasculature, presumably lowering the dose limiting toxicity.  Therefore, we reasoned that small, oligopeptides that bind to the same receptor (both on cells in vitro, and resected tumours ex vivo), could easily be incorporated into either viral, or non-viral gene delivery vectors mediating selective delvery of their DNA payload to the bFGF recptor expressing cells of the tumour vasculature. Importantly, whilst the peptides were shown to bind to bFGF receptors, they did not stimulate the strong mitogenic effect observed using bFGF.  Click the link above to veiw this paper.

Targeting of polyelectrolyte RNA complexes to cell surface integrins as an efficient cytoplasmic transfection mechanism Parker AL, Seymour LW JOURNAL OF BIOACTIVE AND COMPATIBLE POLYMERS 17 (4): 229-238 JUL 2002

Translocation of exogenous DNA from the cytosolic compartment to the nucleus of non-dividing cells is considered to be the greatest rate limiting step in successful gene delivery.  In the absence of nuclear translocation of the exogenous DNA, transcription and translation cannot occur, and phenotypic correction of the diseased cell will not be achieved.  An alluring alternative to the use of DNA to mediate cellular correction is to bypass the requirement for cytoplasmic to nuclear translocation by delivering mRNA, that can readily undergo translation in the cytosol.  In this paper we demonstrate the feasibility of this approach using reporter mRNA.  This "cytoplasmic expression system" provides a short term burst of expression but could feasibly be used to set up an autonomous cytoplasmic expression system such as those based upon T7 polymerase.  Click on the link above to view this paper.

Use of a phage display library to identify oligopeptides binding to the lumenal surface of polarized endothelium by ex vivo perfusion of human umbilical veins Maruta F, Parker AL, Fisher KD, Murray PG, Kerr DJ, Seymour LW JOURNAL OF DRUG TARGETING 11 (1): 53-59 2003

In this paper we address one of the fundamental difficulties in developing targeted gene delivery vectors for in vivo application, namely that cells cultured in vitro show loss of polarisation (compared to in vivo) and deregualtion of cellular receptors (compared to in vivo).  Frequently, vector targeting strategies that are shown to be efficient in vitro are twarted by lack of in vivo efficacy, attributable to the significant changes in cellular phenotype exhibited in the two very different environments.  In order to achieve targeted gene delivery in vivo, we have utilised phage display technology to identify receptor binding oligopeptides that are expressed on polarised cells in vivo.  To achieve this, receptor binding oligopeptides were recovered by biopanning against the endothelial cells of human umbilical veins.  The resulting peptides bound not only to the polarised cells ex vivo, but also to cultured HUVECs in culture, but showed little or no affinity for other cultured cell lines indicating selectivity for the vascular endothelial cells.  To view this paper, click on the link above. 

Nonviral gene delivery: techniques and implications for molecular medicine. Parker AL, Newman C, Briggs S, Seymour L, Sheridan PJ. Expert Rev Mol Med. 2003 Sep 3;2003:1-15.

In this article, the current status in the field of non-viral gene therapy is reviewed.  Initially focussing upon the multitude of different approaches applied in the field (lipoplexes, polyplexes, ultrasound etc), the review then goes on to discuss the numerous pitfalls en-route to achieving targeted gene delivery in vivo, both at the extracellular and intracellular levels.  To view this article, click the link above. 

Enhanced gene transfer activity of peptide-targeted gene-delivery vectors Parker AL, Fisher KD, Oupicky D, Read ML, Nicklin SA, Baker AH, Seymour LW JOURNAL OF DRUG TARGETING 13 (1): 39-51 JAN 2005

This paper addresses the intriguing possibility that small, synthetic peptides iterated using phage display technology could be incorporated into gene therapy vectors (both viral and non-viral) to retarget the vector tropism to alternative cellular receptors. This paper demonstrates that vectors can be modified chemically (using pHPMA) rendering them inert. The incorporation of the targeting peptide, however, resulted in marked restoration of gene expression in various cell types. Retargeting of viral vectors using this construct resulted in high levels of gene expression in HUVECs (Human Umbilical Vein Endothelial Cells) - the cell line against which the targeting peptide was selected.

12th Annual Congress of the European Society of Gene Therapy Read ML, Spice R, Parker AL, Mir S, Logan A EXPERT OPINION ON BIOLOGICAL THERAPY 5 (1): 137-141 JAN 2005

This paper reviews the main highlights of the European Society for Gene Therapy (ESGT) conference 2004 (Tampere, Finland).  Click the link above to view this paper.

Exploration of peptide motifs for potent non-viral gene delivery highly selective for dividing cells. Parker AL, Collins L, Zhang X, Fabre JW. JOURNAL OF GENE MEDICINE (2005);7:1545-1554

This paper addresses the transfection promoting capacity of an influenza derived fusogenic  peptide (FUSO) on non-viral gene delivery, coupled with the ability of (Lys)16 peptides to mediate nuclear transloaction of plasmid DNA.  A composite peptide of the two peptides (polyfuso) mediated high levels of gene expression that was selective for dividing cells, and therefore is likely to have implications for the selective delivery of therapeutic DNA to dividing cells (eg in cancer).  Click the link above to view this paper.

Identification of oligopeptides binding to peritoneal tumors of gastric cancer Noriyuki Akita, Fukuto Maruta, Leonard W. Seymour, David J. Kerr, Alan L. Parker, Tomohiro Asai, Naoto Oku, Jun Nakayama, Shinichi Miyagawa. Cancer Science (2006) 97(10):1075-81.

Multiple Vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes in vitro and in vivo. Parker AL, Waddington SN, Nicol C, Shayakhmetov DM, Buckley SM, Denby L, Kemball-Cook G, Ni S, Lieber A, McVey JH, Nicklin SA, Baker AH. Blood 108 (8); 2554-2561 (2006).

Identifiation of an oligopeptide binding to hepatocellular carcimona. Shimizu A, Maruta F, Akita N, Miwa S, Seymour LW, Kerr DJ, Parker AL, Miyagawa S. Oncology 6(71): 136-145 (2007)

Influence of coagulation factor zymogens on the infectivity of adenoviruses pseudotyped with fibers from subgroup D. Parker AL, McVey JH, Doctor JH, Lopez-Franco O, Waddington SN, Havenga MJE, Nicklin SA, Baker AH. Journal of Virology. 81 (7): 3627-3631 (2007)

Bacteriophage Biopanning in Human Tumour Biopsies to Identify Cancer-Specific Targeting Ligands. Fukuto Maruta, Noriyuki Akita, Jun Nakayama, Shinichi Miyagawa, Tariq Ismail, David C. Rowlands, David J. Kerr, Kerry D. Fisher, Leonard W. Seymour and Alan L. Parker. Journal of Drug Targeting 15(4): 311-319 (2007).

Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: A fundamental involvement of coagulation factors and redundancy of CAR binding by Ad5. Waddington SN, Parker AL, Havenga M, Nicklin SA, Buckley SM, McVey JH, Baker AH. Journal of Virology 81 (17) pp 9568-9571 (2007).

(LYS)(16)-based reducible polycations provide stable polyplexes with anionic fusogenic peptides and efficient gene delivery to post mitotic cells. Parker AL, Eckley L, Singh S, Preece JA, Collins L, Fabre JW. Biochim Biophys Acta. 1770 (9), pp 1331-1337 (2007)

Adenovirus Serotype 5 Hexon Mediates Liver Gene Transfer Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, Atoda H, Pink R, Buckley SMK, Greig JA, Denby L, Custers J, Morita T, Francischetti IMB, Monteiro RQ, Barouch DH, van Rooijen N, Napoli C, Havenga MJE, Nicklin SA, Baker AH. CELL Vol 132, 397-409 (2008).

Identification of oligopeptide binding to colon cancer cells separated from patients using laser capture microdissection. KUBO N, AKITA N, SHIMIZU A, KITAHARA H, PARKER AL , MIYAGAWA S. Journal of Drug Targeting 16(5): 396-404 (2008)

Interactions of adenovirus vectors with blood: Implications for intravascular gene therapy applications Parker AL, Nicklin SA & Baker AH. Current Opinions in Molecular Therapeutics 10 (5) (2008)

The effect of neutralising sera on factor X-mediated adenoviral serotype 5 gene transfer. Parker AL, Waddington SN, Buckley SM, Custers J, Havenga MJ, van Rooijen N, Goudsmit J, McVey JH, Nicklin SA, Baker AH. J Virol, 83(1): pp479-483 (2009)

Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer. Alba R, Bradshaw AC, Parker AL, Bhella D, Waddington SN, Nicklin SA, van Rooijen N, Custers J, Goudsmit J, Barouch DH, McVey JH, Baker AH. Blood, 114 (5): 965-971 (2009)

Influence of Coagulation Factor X on In Vitro and In Vivo Gene Delivery by Adenovirus (Ad) 5, Ad35, and Chimeric Ad5/Ad35 Vectors. Greig JA, Buckley SM, Waddington SN, Parker AL, Bhella D, Pink R, Rahim AA, Morita T, Nicklin SA, McVey JH, Baker AH. Molecular Therapy, 17 10, 1683-1691 (2009)

Self-assembly of peptides into spherical nanoparticles for delivery of hydrophilic moieties to the cytosol. Collins L*, Parker AL*, Gehman JD*, Eckley L, Perugini MA, Separovic F, Fabre JW. ACS Nano. 4(5):2856-64 (2010). *=authors contributed equally

Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X. Bradshaw AC, Parker AL, Duffy MR, Coughlan L, van Rooijen N, Kähäri VM, Nicklin SA, Baker AH. PLoS Pathog. Oct 7;6(10). pii: e1001142. (2010)

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors. Coughlan L, Alba R, Parker AL, Bradshaw AC, McNeish IA, Nicklin SA, Baker AH. Viruses 2(10): 2290-2355 (2010)

Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors. Alba R, Bradshaw AC, Coughlan L, Denby L, McDonald RA, Waddington SN, Buckley SM, Greig JA, Parker AL, Miller AM, Wang H, Lieber A, van Rooijen N, McVey JH, Nicklin SA, Baker AH. Blood. Oct 14;116(15):2656-64. (2010)

Functional characterization of a 13 bp deletion (c.-1522_-1510del13) in the promoter of the von Willebrand factor gene in type 1 von Willebrand disease. Othman M, Chirinian Y, Brown C, Notley C, Hickson N, Hampshire D, Buckley S, Waddington S, Parker AL, Baker A, James P, Lillicrap D. Blood. 116(18):3645-52 (2010)

COP35, a Cholangiocarcinoma-Binding Oligopeptide, Interacts with the Clathrin Heavy Chain Accompanied by GRP78. Kitahara H, Matsumoto J, Parker AL, Maruta F, Kubo N, Shimizu A, Akita N, Miwa S, Kobayashi N, Nakayama J, Miyagawa S. Molecular Cancer Research, 9 (6): 688-701 (2011)

A Cluster of Basic Amino Acids in the Factor X Serine Protease Mediates Surface Attachment of Adenovirus/FX Complexes. Duffy MR, Bradshaw AC, Parker AL, McVey JH, Baker AH. Journal of Virology, 85 (20):10914-9 (2011)