486 rapid deployment plasmid production process, combining inducible high yield fed batch fermentation process with novel autolytic plasmid DNA purification

486 Rapid Deployment Plasmid Production Process, Combining Inducible High Yield Fed Batch Fermentation Process with Novel Autolytic Plasmid DNA Purification 484 Safety of q>C31 Integrase Expression in[.]

484 Safety of q>C31 Integrase Expression in

Mouse Liver Following Hydrodynamic Injection

ChristopherL.Chavez, Lauren E Woodard,Annahita Keravala,

Vanessa Gabrovsky

'Genetics , Stanford Stanford C A.

tpC3l intcgrasc, a site-specific integrase from the bacteriophage

tpC31,catalyzes chromosomal integration of plasmid DNA at

'pseudo' attP sites in mammalian genomes tpC3l integrase has

been used to achieve integration and long-term,robust expression

of therapeutic genes in many tissues,including mouse liver No

immune reaction or tumors have been observed in these studies

We are undertaking additional studies to examine more thoroughly

the safety of using tpC31 integrasc in gene therapy of the liver The

predicted short duration of integrase expression would be a safety

feature by limiting exposure of cells to the reeombinase In order

to test this hypothesis,we are determining the longevity ofintegrase

protein afler hydrodynamic injection of an integrase expression

vector in the mouse liver.The longevity of integrase expression

will be determined by removing the liver at various time points,

isolating total protein for Western blotting from half,and

section-ing the remainsection-ing half for immunohistochemistry with an antibody

detecting <pC31 integrase Based on the observed lifelong expression

oftherapeutic genes integrated with phiC31 integrase,it appears that

integrase is not highly immunogenic To specifically ascertain if

tpC31 integrase elicits a humoral immune response within the mouse,

we will perform ELlSAs on plasma from treated mice Liver

histol-ogy will also be examined for evidence of infiltration of immune

cells over a time course To evaluate the oncogenic potential ofthe

tpC31 integrase system for gene therapy, we will be performing the

gene therapy in mice that arc predisposed to liver cancer The mice

are double transgenic for LAP-tTA and TRE-MYC,so the oncogene

c-MYC is overexpressed specifically in the liver upon removal of

doxycycline from the drinking water For this experiment, MYC

expression in the liver will be induced at 7 weeks of age One week

later,mice will be treated with hydrodynamic tail vein injection,

with or without DNA By evaluating the tumor latency of various

treatments versus positive and negative controls,we will determine

the saftey of both hydrodynamic delivery to the liver and the tpC31

integrase system for gene therapy

485 Analysis of the Transfection Behaviour of

Supercoiled Plasmid DNA Concatemers In Vitro

Christof'Maucksch.PFlorian Hoffmann,' Martin Schleef,'

ManishK.Aneja,'Markus Elflnger;'?Dominic Hartl,' Carsten

Rudolph.l-'

'Department ofPediatrics, Ludwig-Ma;rimilians-University.

Munich, Germany; lDepartment ofPharmacy, Free University

ofBerlin, Berlin Germany; "Plasmidliactory GmbH & Co KG.

Plasmidliactory GmbH & Co KG Bielefeld Germany.

Naked supercoiled plasmid DNA is widely used in nonviral gene

deliveryin vitro and in vivo However, it has not yet been

systemati-cally examined, ifthe arrangement ofconcatemers may be beneficial

for gene expression ofnaked supereoiled plasmid DNA In this study

we compared a supercoiled 4.7kb pEGFP monomer and its 9.4 kb

dimerie coneatemer,which carries two repeats of identical pEGFP

monomer molecules linked to each other head-to-tail Naked pDNA

was transfected into Jurkat T cells by electroporation The number

and mean fluorescent intensity of EGFP expressingcells was

ana-lyzed by flow cytometry (FACS) 24 hrs after electroporation In

ad-dition,we determined the relativeamounts ofpDNA delivered to the

nucleus by using fluorescently TOTO-I labeled pDNA to establish

a correlation between intranuclear pDNA and gene expression The

meanaverage diameter ofboth plasm ids was measured by dynamic

light scattering Transfections were performed using either equal

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C opyright © TheAmerican Society of GeneTherapy

numbers of EGFP gene copies or plasmid molecules The nuclei

of these cells were isolated and the relative intranuclear amounts ofpDNA expressed as the TOTO-I MFI were measured by FACS Transfection ofthe 80 nm pEGFP-monomer resulted in significantly 1.54-fold higher number of EGFP expressing cells compared to the

150 nm dimeric pEGFP,when equimolar numbers of EGFP gene copies were transfcctcd, Although the EGFP-MFI was not different, relatively less EGFP gene copies entered the nucleus when EGFP-monomer compared with dimer was transfected as indicated by the 1'01'0-1 MFI ofnuclei The relative gene copy expression efficiency indicated by the ratioof the EGFP MFI of the transfected cells to 1'01'0-1 MFI per nucleus was 1.5-fold greater for pEGFP-dimer than for pEGFP-monomer Analogous observations were made when equal numbers of plasmid molecule were transfcctcd, These obser-vations suggest that the eoncatemer arrangement increases relative gene expression efficiency,whereas plasmid size is important for cell and nucleus entry afler e1ectroporation We suggest using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors for nonviral gene therapy

486 Rapid Deployment Plasmid Production Process, Combining Inducible High Yield Fed-Batch Fermentation Process with Novel Autolytic Plasmid DNA Purification

Aaron Carnes,1James Williams,' Jeremy Luke; Sarah Langtry,' Clague Hodgson.'

[Research and Development Nature Technology Corporation Lincoln NE.

DNA vaccines and gene medicines, derived from bacterial plas-mids, are emerging as an important new elass of pharmaceuticals They may allow the manufacturer to bypass years of development for the production of efficacious vaccines,and literally create new vaccine entities in days and mass producevaccines in 2-3 weeks for rapid deployment against new biological agents However,the challenges of producing plasmid DNA at an industrial scale are well known: low bioreactor yields, scaling up alkaline lysis, clear-ance of host RNA and chromosomal DNA, and avoiding the use

of animal sourced products.This limits their utility to meet cost and capacity needs for existing plasmid applications, or to rapidly produce kilograms of plasmid DNA for pandemic vaccination The development of an inducible fed-batch fermentation process that dramatically increases volumetric yield and specific plasmid yield, while maintaining or enhancing plasmid integrity has been the first achievement toward these goals This inducible process utilizes com-mercially available media that we designed specifically for plasmid production The process consists ofan initial biomass accumulation phase,followed by a plasmid accumulation phase The plasmid is stably maintained at low levels during a period ofnutrient restricted growth and reduced temperature (30°C), and then the temperature

is increased (37-42°C) to induce plasmid amplification.Typically, the specific plasmid yield increases over a period of up to 15 hours following temperature up-shill Volumetric yields exceeding 2 I g plasmid DNA/L with specific yields of 43 mg/gm dry cell weight have been achieved with this process, and this process has been successfully scaled up to the 300 L scale for GMP production

To address downstream purification challenges,we have success-fully demonstrated feasibility of a cost effective,streamlined,and simple purification process that eliminates costly alkaline or heat lysis steps and the associated toxic waste streams We developed autolyticE coli host strains with integrated chimeric nucleases In

this purification process,autolysis is performed at moderate tem-peratures, without alkaline lysis or the addition of lysozyme The endogenously produced chimeric nuclease then selectively degrades host nucleic acids Purification of the plasmid is completed in a chromatography-free process, with high final product recoveries

Sl87

pSV2 cZ !loo~ g / 200 ~ IIN=2)pSV2 , 100 " 111200 " I (N=2)

Molecular Therapy Volume15 S upplement 1 ~tJ )' 2007

C opyright © ' J11c Ame rican S ociety o f Gen e Therapy

Various Growth Factors during the Cutaneous Wound Healing of Streptozotocin Diabetic Mice Provoked by Non-Viral VEGF165 Gene Therapy Via Sonoporation

'Molecular Therapy Lab, Paik Memorial Institute for Clinical

Chronic un-healing foot wound is a serious problem in

complicat-ed diabetic patients To accelerate the healing of diabetic cutaneous wounds, various kinds of growth factors have been employed with limited success The short halflife ofadministered growth factors in

To overcome this, growth factor gene therapy might be an attractive

beds The normal wound healing requires the harmonious actions of

less immunogenic but it has been shown to be a less effective vec-tor to transfect heart in vivo A novel efficient transfection method, which is based on ultrasound and hydrodynamics, was developed here to transfect the heart with plasmid DNA Methods:ln anesthe-tized rats an ultrasound probe was aimed at the heart for 30 seconds

and a PBS solution containing pSVLacZ was quickly injected into the left ventricle The animal was maintained in this condition for

20 seconds, and then the clamp was opened and the needle was removed Rats were monitored with electrocardiogram (I, 7 and 28 days) and creatine phosphokinase-MB (CPK-MB) was determined after 24 h One group of animals was euthanized for staining with X-gal after 48 h and another group after 4 weeks for Hematoxy-lin-Eosin (HE) or Masson Trichrome (MT) staining Results: The electrocardiograms and CPK-MB level of the gene treated animals showed mild or no sign of ischemia Visual evaluation of pieces of

of blue cells and those that received only PBS had no blue cells

The base and epicardium of the hearts had many more blue cells than the rest of pieces Histology with HE or MT showed similar morphology between control and transfected groups Conclusions: Gene delivery by plasmid vector in association with ultrasound and hydrodynamics was highly effective in transfecting rat heart

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487 Amplified IL-15 Expression Vector for

Cancer Immuno-Gene Therapy

Xianghui He, Weidong Li, Na Zhao, Yujie Qiu, Liwei Zhu

Interleukin-15 (lL-15) is a pleiotropic cytokine that plays a key

role in regulating both innate and adaptive immune responses It

promotes the survival, proliferation, activation and maintenance

of natural killer (NK) cells and CD8+ T cells, also stimulates the

IL-15 could be a potential cytokinc for cancer immune therapy

The therapeutic effect of cytokines could relate to their

expres-sion levels Here we report an amplified IL-15 expresexpres-sion plasmid

vector pHi-IL-15 In pHi-IL-15, the IL-15 expression is droved by

1-I1V2 LTR which then transactivated by CMV promoter controlled

the expression of tat In addition, the native IL- 15 signal peptide is

replaced by IL-2 signal peptide to enhance its secretion Compared

high IL-15 expression is achieved when transfected into tumor cells

in vitro IL-15-expressing tumor cells promote the proliferation of

human peripheral blood mononuclear cells (PBMC) when

co-cul-tured in transwell In addition, data of targeting IL-15 expression

Transfection of Heart with Plasmid DNA

'Interdisciplinary Center for Gene Therapy Federal University oj

-sity ofSao Paulo, Sao Paulo, Sf, Brazil; 'Biophysics, Federal

diseases has a high therapeutic potential Plasmid is a safe vector and

SI88

DNA vaccine plasmid A plasmid yield of 2.1 gm/L was

obtained

The overall result is a rapid deployment plasmid production system

linking autolytic plasmid purification with a high productivity

fer-mentation platform to facilitate immediate production of a variety

transferability to existing manufacturing facilities in developed and

emerging countries