176 efficient transfection of porcine kidney cells (in vitro) and kidney papilla (rx vivo) by holmium laser

176 Efficient Transfection of Porcine Kidney Cells (In Vitro) and Kidney Papilla (Rx Vivo) by Holmium Laser Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����������� �!���[.]

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

integrating systems such as retroviruses SB integration efficiencies

have been reported to be approximately 5% However, since these

values were obtained from systems which scored integration events

that relied on transgene expression from sequences contained within

the transposon, we hypothesize that using a new strategy with an

autonomous SB transposon system will show that the actual SB

integration efficiency is higher than previously thought To establish

a novel integration assay that does not rely on transgene expression,

we constructed a single plasmid containing (i) a luciferase expressing

transposon, and (ii) a separate bicistronic expression cassette

containing transposase (SB)-internal ribosomal entry site

(IRES)-green fluorescent protein (GFP) After transfection with this plasmid,

FACs sorting for GFP will result in a population of cells that have

received the plasmid vector, after which clonal cell lines will be

created and screened using PCR to detect the presence of integrated

transposon sequences The results from this screen will then be

used to determine an accurate integration efficiency for this system

PCR positive lines will be further characterized by genomic Southern

analysis to confirm the number of integration events in each clone

Concurrently, cell line expression profiles will be determined by

quantifying luciferase expression, which will then be used to

determine what percentage of integrated sequences are silenced

Having established an integration efficiency, the exact locations of

the integrations will be determined through sequencing, genomic

database analyses, and then correlated with transgene expression

data in an effort to compare between site selection and expression

These results will be compared to other systems such as retroviral

integration studies, which have shown that expression of integrated

sequences are affected by their location in the host genome and

regulated through a variety of mechanisms, including methylation

and acetylation Through this new strategy we will determine a

more accurate integration efficiency for the Sleeping Beauty

transposon system and we hope to establish a connection between

site selection and expression, which may even allow further

investigation into the causes of how integrating vectors are silenced

after integration

175 Are There Significant Differences between

the Penetration of Naked Plasmid DNA and

Oligonucleotides into Bladder Tissue?

Axel Schaaf,1 Sreedhar Sagi,1 Michael Siegsmud,1 Peter Alken,1

Maurice S Michel.1

1 University Hospital Mannheim, Urological Department,

Mannheim, Germany.

Introduction

A lot of new strategies for the treatment of bladder cancer or

genetic disorders deal with plasmid DNA or antisense

oligonucleotides It is of great interest for future clinical trials, to

what extent both of these methods are capable not only to effect

suspended cells or monolayered cell cultures in vitro but how they

take effect on deeper cell layers of solid organs For this purpose the

penetration in cultured cells and the depth of penetration into an ex

vivo porcine bladder of plasmid DNA and oligonucleotides were

compared in this study

Material and Methods

RT 112, HT 1197 and UM-UC 3 human bladder carcinoma cell

lines were treated with the pEGFP-N1 plasmid (4.7kb) encoding

for the enhanced green fluorescent protein or with a nonsense

FITC-labeled oligonucleotide Porcine bladders were 1hour after removal

instillated with plasmid or oligonucleotid containing solutions with/

without transfecting agents (Lipofectamine™ 2000) After

incubation, the bladders were cryo-sectioned Detection of the

effected cells was performed with the help of fluorescense

microscopy

Results

The oligonucleotide treatment of cell cultures with and without transfecting agents resulted in transfection rates of almost 100% in every case The plasmid transfection of the cell lines without transfecting agents rates effected significant below 1% of the cells The treatment of the cell lines with lipofectamine during plasmid transfection resulted in transfection rates from 36.61% (RT 112) to 88.69% (UM-UC 3)

With the treatment of the porcine bladder with the pEGFP-N1 plasmid only a transfection of cells of the superficial layer could be achieved In contrast, the treatment with oligonucleotides resulted

in a transfection of deeper cell layers, particularly when transfecting agents were used

Discussion

For future bladder cancer treatment strategies it has to be considered, that even malignant cells in deeper layers of the tissue have to be affected This work points out, that not all of the strategies for future treatments are capable to fulfil this task: plasmid-DNA in contrast to oligonucleotides is not able to penetrate deeper cell layers, probably because of its larger size Currently further studies are underway to determine the potential therapeutic effect of intravesical oligonucleotide application

176 Efficient Transfection of Porcine Kidney Cells (In Vitro) and Kidney Papilla (Rx Vivo) by Holmium Laser

Thomas Knoll,1 Sreedhar Sagi,1 Axel Schaaf,1 Lutz Trojan,1 Peter Alken,1 Maurice S Michel.1

1 Department of Urology, University Hospital Mannheim, Mannheim, Baden-Wuerttemberg, Germany.

Introduction and objectives: Cystinuria is the cause of 1-2% of

stones observed in adults and about 10% of those occurring in children Urinary stone disease is the only clinical presentation in patients with cystinuria Recurrent stone formation and multiple operations cause considerable morbidity Gene therapy might offer new perspectives in the treatment of cystinuria We are investigating the transfection efficiency of naked plasmid DNA in porcine kidney

cells by applying holmium laser (Ho:YAG) in vitro as well as ex

vivo in a porcine kidney papilla model.

Materials and methods: In vitro: LLC-PK1 cells were suspended

in a serum free DMEM medium (3.5 Mio/ml) They were treated with naked plasmid DNA (pEGFP-N1 plasmid, 4.7 kb) at 300 μg/

ml of cell suspension Ho:YAG Laser was applied to the cell suspension in pulses of 50, 200 and 500 with repetition rate of 10

Hz and pulse energy of 2000 mJ Positive (Lipofectamine) and negative controls were used to ascertain the efficiency of the method The cells were later incubated for 24 hours in complete DMEM medium The transfection efficiency was measured by the expression

of EGFP reporter gene in the cells by FACScan analysis

Ex vivo: Papilla from a porcine kidney was excised with

surrounding tissue Naked plasmid DNA at 200 μg/ml of cell suspension was added to the tissue in a serum free DMEM medium This tissue was subjected to 200 impulses of the laser with repetition rate of 10 Hz and pulse energy of 2000 mJ The tissue was later incubated for 48 hours in complete DMEM medium After incubation the tissue was Cryosectioned The transfection efficiency was studied by fluorescence microscopy

Results: LLC-PK1 cells were susceptible for transfection by the

holmium laser The efficiency of transfection significantly improved with the increase in impulses; p=0.01, Kruskal-Wallis test Transfection at 50 impulses was averaged 0.87(± 0.16)%, at 200 impulses 28.29(± 7.72)% and at 500 impulses 36.04(± 3.07)% Cell mortality rate also increased with the higher pulse rate up to 70%

The fluorescence microscopic pictures of ex vivo trials showed

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

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transfection in various regions of the tissue There is high transfection

in the peripheral layers of the papilla Transfection was also seen

throughout the tissue

Conclusions: Our study indicates that the transfection of benign

kidney cells by holmium laser which is routinely used in endourology

is a promising new gene transfer strategy The ex vivo trials showed

that different tissues of the kidney are susceptible for transfection

by Ho:YAG laser warranting further studies to optimize transfection

This could prove to be a useful technique in delivering gene therapy

to the kidney with minimal invasiveness

177 A Putative Nuclease Associated with

Renal Brush Border Membranes Is Required for

Transport of Double Stranded DNA by the Nucleic

Acid Conducting Channel

Avelino Teixeira,1 Edgar Leal-Pinto,1 Basil Hanss,1 Paul E

Klotman.1

1 Nephrology/Medicine, Mt Sinai School of Medicine, New York,

NY, United States.

We have previously described a nucleic acid conducting channel

complex (NACh) found in rat proximal tubule brush border

membranes (BBM) The BBM were isolated on a 15% Percoll

gradient followed by solubilization with CHAPS and the complex

purified by a series of chromatography steps On reconstituting

purified NACh in a planar lipid bilayer in a symmetrical buffered

solution no current was observed On addition of 5uM

oligonucleotide (ODN, 20-mer deoxythymidine) to each side of the

bilayer, current was observed as revealed by clear transitions between

closed and opened states (gating) and the channel has been shown to

conduct ODN However, the purified NACh complex does not

conduct double stranded DNA (dsDNA) Furthermore dsDNA has

been shown to block the activity of the purified NACh actively

gating in the presence of ODN

By fusing washed BBM from a 15% Percoll gradient to planar

lipid bilayers we have demonstrated that NACh is present in the

native membranes and that NACh in native membranes interacts

with ODN in a manner indistinguishable from the purified channel

complex In this work we describe the presence of a Mg2+ dependent

nuclease associated with washed Percoll purified BBM as observed

in an incubation assay Furthermore the nucleic acid channel activity

of Percoll purified BBM reconstituted in a bilayer and actively

gating in the presence of ODN is not blocked by dsDNA in the

presence of Mg2+ We hypothesized that one strand of the double

strand was being degraded by the nuclease activity allowing the

remaining strand to be transported as a single strand To address this

question, a 130 bp double stranded DNA of known sequence was

added to the trans (ground) chamber of the bilayer apparatus in

which reconstituted BBM were actively gating in the presence of

ODN At intervals samples were taken from the opposite chamber

and the removed volume replaced with fresh buffer Samples were

also taken at the start of the experiment prior to addition of BBM

and prior to addition of the dsDNA Samples were desalted and

concentrated to be used in their entirety in a PCR reaction using

primers homologous to the ends of the 130bp dsDNA DNA of the

correct size was recovered from the PCR indicating that at least one

of the strands had been conducted by the channel in this period The

PCR of the control samples taken prior to addition of dsDNA were

negative The presence of this nuclease we believe has important

implications for the in vivo functioning of this channel and may

provide a mechanism for translocation of double stranded DNA

across plasma membranes

178 Purification of Supercoiled Plasmid DNA for Gene Therapy

Joachim Stadler,1 Raf Lemmens,2 Yamuna Dasarathy.3

1 Amersham Biosciences Europe GmbH, Freiburg, Germany;

2 R&D Separations, Amersham Biosciences AB, Uppsala, Sweden;

3 Amersham Biosciences, Piscataway, NJ, United States.

Purification of supercoiled plasmid DNA from other isoforms continues to be a challenge in the field of gene therapy and vaccines

We have formulated a three step chromatographic protocol that will purify supercoiled plasmid DNA from other isoforms We also have demonstrated that our procedure eliminates contaminants such

as genomic DNA, RNA, endotoxins and proteins This protocol does not involve the addition of RNase and the purity of the end product - supercoiled plasmid DNA - makes it ideal for gene therapy

In the first step, the initial clarified bacterial lysate (concentrated using hollow fiber ultra filtration membranes, in the case of low copy plasmids) is pumped onto a Sepharose® 6 Fast Flow column

to remove most of the RNA by group separation In the second step, the plasmid DNA collected from Sepharose 6 Fast Flow column

is pumped onto a PlasmidSelect® column Under specific buffer conditions, this column purifies selectively supercoiled plasmid DNA by thiophilic aromatic adsorption and removes proteins, endotoxins and the remaining RNA In the third step, the supercoiled plasmid DNA eluted from the PlasmidSelect column is further purified and concentrated by SOURCE® 30Q column

In conclusion, the process described has the following features:

- by using group separation, contaminating RNA can be removed without the use of RNase

- by using a thiolic aromatic ligand, complete separation of open circular from supercoiled plasmid DNA can be accomplished

- by using an anion exchange medium, the levels of endotoxins and traces of RNA and genomic DNA can be further decreased

- by combining three chromatographic steps based on different selectivity and mechanism, an efficient reduction in the levels of contaminants can be achieved

- fully scalable for easy transfer to GMP compliant production

- Regulatory support package facilitates easy GMP compliance The protocol is made attractive by its flexibility to be generic, scalable, GMP compliant and can be automated using an ÄKTAexplorer 100 system

179 Large Scale GMP Production, Formulation, and Fill/Finish

Magda Marquet,1 Roy Musil,1 Rick Hancock.1

1 Plasmid Production, Althea Technologies, Inc., San Diego, CA, United States.

Large-scale GMP production of plasmid DNA has emerged as one of the challenges industry faces as the clinical aspects of gene therapy continue to grow While the production quantities for clinical phase I and phase II material can still be met by employing modified laboratory scale methods, phase III and commercial manufacturing require truly scaleable methods Another trend we are witnessing as products proceed in the clinical pipeline is the tightening of specifications For example, as doses increase in clinical trials, endotoxin levels must be lowered accordingly In the same vein, thorough plasmid characterization becomes a crucial aspect of the manufacturing development process An often overlooked part of the entire development process is the detailed characterization of the formulation and final fill parameters Numerous clinical formulations now include plasmid DNA with several excipients and adjuvants to assure greater efficacy Further adding to the complexity

of final formulation is discerning the interaction of the drug product with the final filling container A number of different raw materials may be included in the composition of a final container (boro-silicate