489 a novel mixing device for the reproducible manufacture of non viral gene therapy formulations

489 A Novel Mixing Device for the Reproducible Manufacture of Non Viral Gene Therapy Formulations Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S188

486 Structure-Activity Relationship of a New

Class of Polyamines

Xiang Gao,1 Ramalinga Kuruba,1 Damodaran K Achary,2 Billy W

Day,1 Dexi Liu,1 Song Li.1

1 Center for Pharmacogenetics, Department of Pharmaceutical

Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh,

PA; 2 Department of Chemistry, University of Pittsburgh School of

Arts and Sciences, Pittsburgh, PA.

Cationic polymers constitute an important class of gene transfer

agent However, a detailed structure-activity relationship for these

agents is still murky We have synthesized a panel of structurally

related amine-containing polycations whose overall backbone

structures resemble polyethylenimines These are co-polymers

synthesized from a di -or oligoamine and a dichlorine cross-linker

by alkylation The choice of amines with different numbers of

amine groups, the length between these amine groups and different

substitutions, and the chain length and terminal group of the side

chain in the cross-linkers provide great diversity to the resulting

polycations The reaction conditions determine the MWs of the

fi nal products These polycations exhibited great variations in their

transfection activity and cytotoxicity levels Polymers with moderate

MW gave the best overall performance both in transfection effi ciency

and cytotoxicity, while polymers with high MWs had high toxicity

and limited useful dosages for transfection Polymers with increased

linear charge density from oligoamines did not increase the overall

transfection effi ciency vs a polymer of lesser density from a simple

diamine, but required lower polymer to DNA ratios for peak level

of transfection The cross-linkers with different side chain lengths

drastically affected the transfection and toxicity of the polymers,

with medium length being the optimal structure Finally, inclusion

of reducible disulfi de bonds to the polymer backbone increased

transfection activity and reduced cytotoxicity Overall, our studies

reveal some structure-activity relationships of these new polycations

in gene delivery

487 Microarray Analysis of Intracellular

Signaling Pathways in Nonviral Gene Transfer

Gina Boanca,1 Angela K Pannier.1

1 Biological Systems Engineering, University of Nebraska-Lincoln,

Lincoln, NE.

The use of gene delivery in therapeutic applications, including gene

therapy to treat genetic defi ciencies or tissue engineering matrices

for the treatment of organ loss and failure, has been limited due to

challenges with current delivery systems Nonviral vectors, which

typically involve electrostatic complexation of cationic polymers or

lipids with DNA, are signifi cantly less effi cient than viral vectors,

but offer advantages of low toxicity and immunogenicity, lack

pathogenicity, and are easy to produce with greater control and

fl exibility, making these vectors attractive alternatives to viruses

To date, most efforts to understand and improve the effi ciency of

nonviral delivery have focused on altering the physicochemical

properties of delivery systems and developing new delivery strategies

However, the exact mechanisms involved in gene delivery are poorly

defi ned, including the intracellular signaling pathways activated in

response to gene transfer, which govern traffi cking of the DNA The

importance of cell signaling in achieving successful nonviral gene

transfer has not been thoroughly examined, though it clearly plays

a role in regulation of cellular responses and may affect the ability

of cells to become transfected We have used microarray analysis to

identify signaling pathways that are modulated during nonviral gene

transfer Nonviral DNA complexes (composed of cationic lipids or

polymers complexed with plasmid DNA encoding for GFP) were

delivered to HEK293T human embryonic kidney epithelial cells, a

widely used cell line in transfection experiments Flow cytometry

was then used to sort GFP-positive cells, allowing isolation of a

population of transfected cells These cells were then lysed and their mRNA collected and purifi ed using standard techniques The RNA samples were then hybridized to Affymetrix GeneChip expression arrays Control and experimental samples were hybridized to separate chips, in triplicate Expression patterns were compared between transfected and nontransfected samples, which revealed several key differential gene expression profi les regulated by nonviral gene transfer With a greater understanding of key signaling pathways involved in gene delivery, we hope to understand the mechanisms that render cells responsive to DNA transfer to develop more effi cient nonviral delivery schemes

siRNA to Adherent and Suspension Cancer Cells

by Sendai Virosomes

Jom Ee Baek,1 Jung Seok Kim,1 Yeon Kyung Lee,1 Sang Il Park,1

Hwa Yon Jung,1 Keun Sik Kim,2 Yong Serk Park.1

1 Biomedical Laboratory Science, Yonsei University, Wonju, Gangwon, Korea, Republic of; 2 Biomedical Sciences, Youngdong University, Woungdong, Chungbuk, Korea, Republic of.

Previously, we have shown that the Sendai F/HN virosomes

are an effective gene delivery system for in vitro and in vivo

transgene expression The Sendai virosomes consist of two different glycoproteins, hemagglutininneuraminidase (HN) and fusion protein (F) which are required for binding to cell surface and cell fusion, respectively In this study, we constructed two different types of virosomes, the so-called cationic Sendai F/HN virosomes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN virosomes (PCSVs) The plasmid DNA or siRNA was complexed with the CSVs

or PCSVs, and then transferred to adherent cancer cells (293 and HeLa) and suspension cancer cells (Jurkat) Generally, the PCSVs were more effective in delivery of pDNA to the cultured cancer cells than the CSVs and conventional cationic lipoplexes Meanwhile, according to the FACS analysis the CSVs exhibited more effective delivery of siRNA to Jurkat cells, one of the toughest cells for transfection, than the PCSVs The effective intracellular uptake and endosomal escape of pDNA (or siRNA) transferred with the PCSVs and CSVs were confi rmed by confocal-microscopic analysis From these experimental results, it can be concluded that the PCSVs and CSVs would be widely utilized as an alternative gene (or siRNA) delivery system for various types of cells including suspension cancer cells

Manufacture of Non-Viral Gene Therapy Formulations

Lee A Davies,1,4 Graciela A Nunez-Alonso,1,4 Henry L Hebel,2

Ron K Scheule,3 Seng H Cheng,3 Deborah R Gill,1,4 Stephen C Hyde.1,4

1 Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom; 2 VGXI Inc., The Woodlands, TX; 3 Genzyme Corporation, Framingham, MA; 4 UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh/London/Oxford, United Kingdom.

The generation of non-viral gene therapy formulations requires the complexation of negatively charged plasmid DNA (pDNA) with cationic gene transfer agents (GTAs) such as lipids, polymers and peptides Within the laboratory, small volumes of reagent are often prepared by stepwise addition of one reagent to the other However, this technique is inappropriate for the production of larger amounts

of material required for clinical applications because incomplete or variable mixing associated with larger volumes can signifi cantly

affect both the physical characteristics and the in vivo performance

of the complexes We have developed a pneumatic mixing device that allows the reliable and reproducible mixing of large volumes

Molecular Therapy Volume 17, Supplement 1, May 2009

of GTAs and have investigated its suitability for the production of

two non-viral gene therapy formulations of interest for treatment of

cystic fi brosis lung disease The LMD2 pneumatic mixer consists

of a compressed gas driven system, designed for the controlled

mixing of reagents, packaged side-by-side in a dual-lumen syringe

attached to an 8-element HDPE static mixer The rates of mixing

and extrusion are fully adjustable allowing practical liquid mixing

rates in the range 0.2-20ml/s Video analysis of the device in action

demonstrated the linearity and reproducibility of extrusion rate

over the full range of mixing rates even when using formulations

with viscosities in excess of 20cP The LMD2 was utilised to form

complexes between the 5.6 kb luciferase expression plasmid pCIKLux

and the cationic lipid GL67A (Genzyme) (0.8 mM pDNA: 0.6 mM

lipid), or 25kDa polyethylenimine (0.6mM pDNA, N:P 10:1) A total

of 10 ml of each formulation was prepared at mixing rates from 1 -

20 ml/s and the physical characteristics of the resultant complexes

compared with those prepared by standard small volume mixing

(<500µl) Irrespective of the mixing technique, the measurements of

particle size and zeta potential were similar for complexes of pDNA/

GL67A (Range 314.83 - 278.63nm; 3.37 - 4.10mV), or pDNA/PEI

(Range 79.95 - 112.17 nm; 24.33 - 27.70mV) at the mixing rates

tested Importantly, agarose gel analysis of dissociated complexes

revealed no shear degradation of pDNA To confi rm the biological

effi cacy of complexes prepared using the LMD2, 10ml of pCIKLux/

GL67A (8 mM: 6 mM) or pCIKLux/PEI (0.6mM pDNA, N:P 10:1)

were aersolised to the lungs of BALB/c mice using a whole body

exposure chamber Luciferase expression was analysed 24 hr later

and equivalent gene expression was observed in mice exposed to

aerosols prepared using the LMD2 and those prepared using small

volume mixing, for both pCIKLux/GL67A and pCIKLux/PEI

These data demonstrate that this novel mixing device is suitable

for large-scale production of functional gene therapy reagents in

a standardised and reproducible manner essential for reproducible

clinical administration

490 Indole-Modifi ed Self-Assembled

Monolayers Enable Host Inclusion Complex

Formation with α-CD Modifi ed Polyethylenimine

Polyplexes for Substrate-Mediated Gene Delivery

Chung-Huei K Wang,1 Shaoyi Jiang,2 Suzie H Pun.1

1 Bioengineering, University of Washington, Seattle, WA; 2 Chemical

Engineering, University of Washington, Seattle, WA.

Substrate-mediated gene delivery has been shown to enhance

gene transfer due to increased DNA concentration at the cell surface

Various methods have been employed for delivery of nucleic acids

from solid surfaces, including physical adsorption, nonspecifi c

charge interaction, biotin/streptavidin interaction, as well as

chemical conjugation We have previously demonstrated specifi c

polyplex immobilization on surfaces mediated by inclusion complex

interaction between β-cyclodextrin host molecules conjugated to

polyethylenimine (PEI) polyplexes and adamantane guest molecules

on self-assembled monolayer surfaces In this work,

α-cyclodextrin-modifi ed PEI was synthesized and used to form polyplexes with

plasmid DNA Control PEI and α-cyclodextrin-PEI (α-CD-PEI)

polyplexes were assessed for interactions with indole-modifi ed

self-assembled monolayers by surface plasmon resonance Polyplexes

formulated from α-CD-PEI bound specifi cally to self-assembled

indole-monolayers compared to control PEI polyplexes By patterning

indole- and adamantane-modifi ed self-assembled monolayers, two

different polyplex formulations can potentially be immobilized

with spatial specifi city using both this second host-guest interaction

between α-cyclodextrin and indole, as well as our previously

developed β-cyclodextrin and adamantane interaction Keywords:

substrate-mediated gene delivery, self-assembled monolayer, host

inclusion complex, α-cyclodextrin, indole

Head Group for DNA Delivery

Xiao-xiang Zhang,1 Carla A H Prata,1 Dan Luo,2 Thomas J McIntosh,3 Mark W Grinstaff.1

1 Biomedical Engineering and Chemistry, Boston University, Boston, MA; 2 Biological and Environmental Engineering, Cornell University, Ithaca, NY; 3 Cell Biology, Duke University Medical Center, Durham, NC.

Cationic lipids are one of the most used synthetic vectors for gene delivery These vectors offer advantages such as low toxicity, nonimmunogenicity, large nucleic acid payloads, and ease of synthesis compared to viral vectors, but suffer from low transfection activities The majority of cationic lipids studied to date complex DNA through electrostatic interactions In nature, the recognition of nucleic acids by proteins involves electrostatic, hydrogen bonding and π-stacking interactions In order to mimic these interactions we designed peptide-based amphiphiles These peptidic based lipids were synthesized and characterized The complexation of the amphiphile

to the DNA was monitored using an ethidium bromide displacement assay DNA transfection effi ciency of this new class of amphiphiles was comparable to LipofectamineTM2000 in CHO and NIH 3T3 cells The supramolecular amphiphile/DNA complexes were also characterized by DLS and X-ray diffraction The data show that those amphiphile possessing high transfection activity shared similar structure characteristics: the cationic charges were separated from the head group by three amino acids and a large repeat period present in the lipoplexes lamellar structures

DNA Nanocarriers; In-Vitro Evaluation

Nicolas Duceppe, Marinella G Sandros, Maryam Tabrizian

Biomedical Engineering, McGill University, Montréal, QC, Canada.

Introduction: In the last several years, the fi eld of gene delivery

has focused a great deal of attention on the development of non-viral vectors made with biocompatible polymers to circumvent the hurdles found with the use of viral vectors Chitosan-based nanoparticles have been shown to possess the properties needed to deliver DNA or siRNA both in-vitro and in-vivo Efforts are now made to produce smart materials able to control DNA release spatio-temporally by altering the polymer properties in response to changes in the environment The approach presented by our group consists of the functionalization

of chitosan with photo-cleavable (PC) molecules, which will allow the release of DNA in response to light stimuli Data presented here focuses on toxicity, uptake, DNA localization, and transfection with

PC-chitosan/hyaluronic acid nanoparticles Material & methods:

Hyaluronic acid 64 kD (Lifecore Biomedical, USA) and chitosan 5

kD (Medipol, SA) were used All other reagents come from Sigma Aldrich, Fisher and Acros Organics Confocal and fluorescent microscopes (Nikon, USA) were used to localize DNA in the cells Viability, uptake and transfection tests were carried out on the HEK-293T cell line using fl ow cytometry (FACScalibur, BD Biosciences, CA) with appropriate procedures Flow cytometry data was analyzed

with Flowjo software (Treestar; USA) Results & discussion:

Previous work showed that unmodifi ed chitosan/hyaluronic acid nanoparticles are effi cient DNA carriers for cell transfection In this work, we have applied viability tests using the nanoparticles to confi rm that the chitosan functionalization with the PC molecules did not alter the biocompatibility of chitosan Flow cytometry analyses were used to confi rm the effi ciency of the light induced release of the DNA Also, uptake and transfection effi ciency of nanoparticles assembled with PC-chitosan were compared to nanoparticles assembled with unmodifi ed chitosan Confocal microscopy was used to localize nanoparticles inside the cells, using fl uorescent dye

to track the DNA Taken together, these preliminary results show