880 lipid substituted poly(l lysine) polymers for effective transfection of human skin fibroblasts: structure function relationships

880 Lipid Substituted Poly(L Lysine) Polymers for Effective Transfection of Human Skin Fibroblasts Structure Function Relationships Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The[.]

Molecular Therapy Volume 17, Supplement 1, May 2009

Pseudodendritic Oligoamines

Verena Russ,1 Alexander Philipp,2 Ernst Wagner,2 John J Rossi.1

1 Department of Molecular Biology, City of Hope - Beckman

Research Institute, Duarte, CA; 2 Pharmaceutical

Biology-Biotechnology, Ludwig-Maximilians University Munich, Munich,

Germany.

RNA interference (RNAi) is an endogenous process, which

employs small interfering RNAs (siRNA) to suppress

target-specific gene expression by mRNA degradation Reduction of

gene expression through RNAi has a great potential to create a

novel class of therapeutics that address the treatment of several

previously untreatable diseases and viral infections like e.g HIV

Significant efforts in terms of non-viral siRNA delivery have

been made by developing cationic lipid based carriers, but the

establishment of safe and effective methods both in vitro and in

vivo is still challenging Currently, there are no effi cient synthetic

vectors available for non-viral gene therapy of HIV infections using

therapeutic siRNAs Pseudodendrimers, degradable hyperbranched

polymers, have been shown to deliver pDNA effi ciently [1] Here,

we explore the potential of pseudodendrimers for siRNA delivery

We formed the pseudodendritic cores by coupling an excess of

1,6-hexandioldiacrylate (HD) to non-toxic 800Da oligoethylenimine

(OEI) OEI is responsible for mediating endosomal release via the

“proton-sponge effect” The labile ester bonds in HD are hydrolysed

by esterases, thus decreasing cytotoxicity and increasing the

intracellular unpackaging of the carrier, which releases the siRNA

We established a structure activity relationship by modifying the

surface of the pseudodendritic cores with different LMW oligoamines

including ethanolamine, ethylenediamine, spermidine, spermine,

pentaethylenhexamine and OEI Screening studies revealed that

siRNA delivered by the spermine surface-modified conjugate

showed the best down regulation of gene expression (up to 80%) in

stably luciferase transfected cells In comparison to standard siRNA

transfection agents (e.g Lipofectamine 2000) this pseudodendrimer

exhibits a similar cytotoxicity profi le and its siRNA complex yields

better target gene silencing in human cell lines such as HEK293 and

CEM, as shown by qRT-PCR This polymer can be exploited to create

a biocompatible, safe and effi cient siRNA carrier for HIV treatment

Reference: [1] Russ V, Elfberg H, Thoma C, Kloeckner J, Ogris M,

Wagner E Gene Ther 2008 Jan;15(1):18-29

Gene Delivery Carriers

Malavosklish Bikram,1 Mohamed I Nounou,1 Vicky Mody,1

Kalyopy Emmanouil,1 Zhuoyuan Lu.1

1 Pharmacological & Pharmaceutical Sciences, University of

Houston, Houston, TX.

The development of biodegradable gene delivery systems, which

have the ability to effectively deliver therapeutic DNA to a target

tissue, is paramount to the success of nonviral gene delivery One

approach to developing biodegradable polymers is to introduce

disulfi de bonds along the backbone of the polymers to ensure release

of the DNA in the reductive environment of the cytoplasm, whilst

simultaneously reducing the molecular weight of the polymers.1

Tantamount to the degradability of the polymers is the need to develop

biocompatible polymers, which have low cytotoxicities so as to

maintain cell viability and hence increase transfection effi ciencies

Therefore, to produce a biocompatible gene delivery system, we

have designed and synthesized novel reducible copolymers of the

type (AB)n, which consist of repeating units of the natural amino

acid, L-lysine and cystamine bisacrylamide (CBA) These novel

lysine-based reducible copolymers (LRCs) were then modifi ed with

ethylenediamine so as to introduce primary amines for effi cient

DNA condensation The molecular weight (MW) of the copolymers

was found to be ∼3.2 kDa with a polydispersity index of ∼1.2 A gel retardation assay showed complete condensation of DNA at N/P ratios [nitrogens of polymer/phosphates of DNA] higher than 20/1

To investigate the mechanism of DNA release from the polymer/ DNA complexes, fl uorescence spectroscopy studies were performed with 1,4-dithio-DL-threitol (DTT) These data showed a signifi cant reduction in fl uorescence intensity following the addition of LRCs

to DNA After the addition of DTT, there was a 95 % increase in

fl uorescence intensity, which indicated the reduction of the disulfi de bonds and the release of the DNA from the complexes The particle sizes of the LRC/DNA complexes were found to be between 100

to 231 nm with surface charges of 0.8 to 20 mV The transfection effi ciencies of the complexes as determined with a luciferase assay showed that LRC complexes produced ∼7-8 times higher transfection effi ciencies in human dermal fi broblasts (HDFs), ∼3 times higher transfection effi ciencies in human breast adenocarcinoma cells (MCF-7s), and ∼4 times higher transfection effi ciencies in metastatic mouse breast cancer cells (4T1s) as compared to the control In addition, confocal studies showed the association of released DNA with the nuclei of transfected cells Finally, the cytotoxicities of the LRCs showed signifi cantly lower cytotoxicities as compared to the control Therefore, these results suggest that these novel LRCs are very effi cient and biocompatible polymers for nonviral gene delivery

References 1 L V Christensen, C W Chang, J W Yockman, R

Conners, H Jackson, Z Zhong, J Feijen, D A Bull, and S W Kim Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF

delivery J Control Release 118: 254-61 (2007).

for Effective Transfection of Human Skin Fibroblasts: Structure-Function Relationships

Meysam Abbasi,1 Hasan Uludag,1,2,3 Vanessa Incani,3 Charlie Yu Ming Hsu,1 Andrea Jeffery.2

1 Biomedical Engineering, University of Alberta, Edmonton, AB, Canada; 2 Chemical & Materials Engineering, University of

Alberta, Edmonton, AB, Canada; 3 Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada.

Purpose: Enabling gene expression in skin fi broblasts using

safe nonviral delivery systems has the potential to stimulate wound healing and aid in skin tissue engineering efforts In this study, several lipid-substituted poly(L-Lysines) (PLL) were investigated for their ability to deliver a plasmid DNA (pEGFP) to human skin

fi broblasts Methods: Human CRL fi broblasts were used in this

study and pEGFP-N2 plasmid DNA was used as a reporter gene Lipid-substituted polymers were synthesized from caprylic, myristic, palmitic, oleic and linoleic acids, and characterized by NMR Gel electrophoresis was performed for assessment of (i) pEGFP binding effi ciency of polymers, (ii) pEGFP/polymer complex dissociation upon incubation with heparin, and (iii) pEGFP/polymer degradation

by DNases Cellular uptake of pEGFP was assessed by fl uorescent microscopy and fl ow cytometry Analysis of EGFP Expression

was assessed by fl ow cytometry and PCR Results: While native

and lipid-substituted PLLs showed complete complexation with pEGFP, polymers with higher lipid substitution were more resilient

to dissociation after heparin treatment All polymers showed good protection of pEGFP against DNase I and DNase II digestion in vitro DNA delivery studies using fl uorescently labeled pEGFP showed that native PLL lacked the ability to deliver pEGFP into cells, whereas most of the lipid-substituted PLLs gave effi cient pEGFP delivery into the cells Extent of lipid substitution was an important factor in DNA delivery effi ciency The intracellular pEGFP was intact after delivery with lipid-substituted polymers up to 7 days An RT-PCR methodology indicated successful transcription of the EGFP gene, which was not the case when the cells were transfected with a blank plasmid lacking a EGFP gene Further studies with fl ow cytometry

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S336

showed that successful protein expression was obtained with PLLs

substituted with myristic and stearic acid, the latter displaying a

relatively lower toxicity Conclusion: We conclude that substituting

lipids on PLL results in effective gene carriers and the extent of

substitution, rather than the individual lipid, appeared to be critical

for effective plasmid delivery

Antisense Oligoribonucleotides Allows

Restoration of Dystrophin Expression in the mdx

Mouse

Paola Rimessi,1 Patrizia Sabatelli,1,2 Marina Fabris,1 Paola

Braghetta,3 Elena Bassi,1 Pietro Spitali,1 Gaetano A Vattemi,4

Giuliano Tomelleri,4 Lara Mari,5 Daniela Perrone,6 Alessandro

Medici,6 Marcella Neri,1 Matteo Bovolenta,1 Elena Martoni,1

Nadir M Maraldi,7 Francesca Gualandi,1 Luciano Merlini,1 Marco

Ballestri,8 Luisa Tondelli,8 Katia Sparnacci,9 Paolo Bonaldo,3

Antonella Caputo,3 Michele Laus,9 Alessandra Ferlini.1

1 Department of Experimental and Diagnostic Medicine, Section

of Medical Genetics, University of Ferrara, Ferrara, FE, Italy;

2 IGM-CNR, CNR Bologna, Bologna, BO, Italy; 3 Department of

Histology, Microbiology, and Medical Biotechnology, University

of Padova, Padova, PD, Italy; 4 Department of Neurological

Sciences and Vision, Section of Clinical Neurology, University of

Verona, Verona, VR, Italy; 5 Department of Chemistry, University

of Ferrara, Ferrara, FE, Italy; 6 Department of Biology and

Evolution, University of Ferrara, Ferrara, FE, Italy; 7 Department

of Human Anatomical Sciences, University of Bologna, Bologna,

BO, Italy; 8 ISOF, Consiglio Nazionale delle Ricerche, CNR

Bologna, Bologna, BO, Italy; 9 Department of Environmental

and Life Sciences INSTM, University of Piemonte Orientale,

Alessandria, AL, Italy.

For subsets of Duchenne muscular dystrophy mutations, antisense

oligoribonucleotide mediated exon skipping has proven to be

effi cacious in restoring the expression of dystrophin protein In the

mdx murine model systemic delivery of antisense oligoribonucleotide,

recognising the splice donor of dystrophin exon 23, has shown proof

of concept We have been able to restore dystrophin expression in

body-wide striated muscles of mdx animal model using different

formulations of cationic nanoparticles These were loaded with low

doses of 2’OMePS antisense oligoribonucleotide, ranging from

0.9 to 4.5 mg/kg/week, and delivered by weekly intraperitoneal injection Transcription, western and immunohistochemical analysis showed increased levels of dystrophin transcript, protein and correct localisation at the sarcolemma We characterised the physical properties, the interaction between nanoparticles and AON, and their diffusion pathways both by fl uorescence and electron microscopy analysis We therefore showed that cationic nanoparticles have the capacity to both deliver antisense oligoribonucleotides in body-wide muscles and reduce the dose required for dystrophin rescue This non-viral approach may improve the therapeutic usage of antisense oligoribonucleotides in Duchenne muscular dystrophy as well as the delivery of RNA molecules with many implications in both basic research and medicine

1a: T1 nanoparticle scanning electron microscope 1b: Interaction between AON and T1 nanoparticles 1c: Biodistribution in the mdx mouse of fl uorescent T1 A: diaphragm; B: gastrocnemius; C: heart 1d: Biodistribution of T1 by electron microscope analysis

Poly(tetrahydrofuran-b-Ethylene Oxide) Copolymers for DNA Delivery into Skeletal Muscle

Catherine Pomel,2 Christian Leborgne,1 Hervé Cheradame,2 Daniel Scherman,1 Antoine Kichler,1 Philippe Guegan.2

1 Généthon - CNRS FRE 3087 - Université d’Evry, Centre de Recherche et d’Applications sur les Thérapies Géniques, Evry Cedex, France; 2 CNRS UMR 7581, Université d’Evry, Laboratoire Matériaux Polymères aux Interfaces, Evry, France.

Purpose Amphiphilic triblock copolymers such as the pluronic poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) L64 (pEO13-pPO30-pEO13) have been shown to mediate more effi cient gene transfer in muscle as compared to naked DNA We were interested in studying the effect of a chemical change of the central block of pluronic polymers on the transfection activity Methods We synthesized new amphiphilic copolymers in which the hydrophobic pPO block was replaced by poly(tetrahydrofuran) (pTHF) chains The resulting triblock pEO–pTHF–pEO polymers have been characterized

by NMR and SEC and assayed for in vitro and in vivo gene transfer Results The animal experiments showed that the new copolymers are able to signifi cantly increase the transfection effi ciency of plasmid DNA after intramuscular injection Conclusions These results indicate that the capacity to enhance plasmid DNA transfection in skeletal muscle is not restricted to pEO–pPO–pEO arrangements