570 development of a MDCK cell line for the manufacturing of canine adenovirus type 2 (CAV 2) vectors

570 Development of a MDCK Cell Line for the Manufacturing of Canine Adenovirus Type 2 (CAV 2) Vectors Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cel[.]

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CELL PROCESSING AND VECTOR PRODUCTION II

longer time period (PEI and PEI-OA in particular) The maximum

(∼50%) [Fig 1] This lead to a 3-fold increase in drug accumulation

in cells ( owcytometry) and a 30% increase in cytotoxicity (MTT

Assay) Conclusions : The fact that a higher amount of drug is able

to enter the WT cell line compared to the MDR cell line, con rms

the presence of MDR in the cells This fact is further proven by the

MTT assay where WT cells show more toxicity The amount of siRNA

uptake into cells is considerably high which is a positive factor for

therapeutic applications Speci c siRNA delivery showed ef cient

P-gp suppression on the MDR cell line which lead to signi cant

increase in drug accumulation and cytotoxicity Our in-vitro results

indicated that siRNA pre-treatment of cells for P-gp suppression leads

to a higher ef ciency of chemotherapy on MDR cells

Fig 1 P-gp expression of cells treated by siRNA/Carrier

complexes

569 Gene Editing Induces Double-Stranded

Breakage in Mammalian Cells

Melissa A Bonner, Eric B Kmiec

Marshall Institute for Interdisciplinary Research, Marshall

University, Huntington, WV.

The process of gene editing has advanced in recent years with

the successful repair of genes in vivo Using mouse models, genes

at the center of both retinal and neurodegenerative diseases have

been corrected, demonstrating the wide applicability of gene

repair technology We are interested in utilizing single-stranded

oligonucleotides (ODNs) to elicit targeted nucleotide exchange

(TNE), the mechanism of gene editing, because in an in vivo setting,

ODNs offer many advantages that viral-based gene correction systems

cannot Understanding the mechanism by which ODNs elicit TNE is

imperative to achieving optimal correction ef ciencies and medical

applicability Previously, introduction of an ODN into cells has been

shown to activate DNA damage response pathways, but no evaluation

of the status of the DNA during this reaction has been undertaken The

activation of H2AX, a hallmark protein of DNA breakage, suggests

that a double-strand break (DSB) could be occurring during the

gene editing reaction Using the human transgenic HCT116 cell line

with a single integrated mutant eGFP gene as our model system, we

demonstrate that the DNA strand breakage occurs when a speci c

ODN, designed to direct TNE, is transfected into the cells Both

single and double-stranded DNA cleavage is observed dependent

on the level of ODN added to the reaction and its speci city for the

reporter gene; notably the modi cation state of the ODN does not

in uence the level of breakage Importantly, the DNA breaks repair

over time with minimal effects on cell viability and replication status

We are interested in the role of the DNA helicase, Bloom syndrome

helicase (BLM), in the TNE reaction, speci cally with relation to

DSB formation BLM has been shown to be involved in replication

fork restart, partially by mediating the interaction of the replication

fork with an endonuclease that creates a DSB as part of the fork repair mechanism BLM is also involved in both homologous and homeologous recombination, stimulating the latter Our investigation into the role of BLM and other proteins involved in homeologous recombination and replication fork restart will allow us to decipher the role of DSB formation in TNE and identify proteins that have an effect on correction ef ciency levels in our model system, ultimately

leading to an increase in in vivo correction levels.

Cell Processing and Vector Production II

570 Development of a MDCK Cell Line for the Manufacturing of Canine Adenovirus Type 2 (CAV-2) Vectors

Virgínia Santiago,1 Eric Kremer,2,3 Paula M Alves,1 Ana S

Coroadinha.1

1 IBET/ITQB-UNL (Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica – Universidade Nova de Lisboa, Oeiras, Portugal; 2 Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France; 3 Universitiés Montpellier I & II, Montpellier, France.

Background: Human adenoviruses are one of the most ef cient

vehicles for delivering nucleic acids, however they are ubiquitous

in all population, posing memory immunity responses obstacles for their vectors during clinical gene transfer To avoid this drawback, vectors derived from nonhuman adenovirus like CAV-2 are being developed Several studies have shown they circumvent the memory humoral, cellular, and innate immune response in humans

E1-deleted (∆E1) and helper-dependent (HD) CAV-2 vectors are promising tools for understanding and treatment of cognitive traits and neurodegenerative disorders In vivo gene transfer of rodent, dog and nonhuman primate CNS led to global and long-term expression without immune suppression However, the production of nonhuman adenovirus can require the use of restricted host cell lines for their ampli cation For the replication of CAV-2 a canine cell line is required The manufacturing of bioproducts for human therapeutics demand stringent quality control and for that reason the use of an

“FDA pre-approved” cell line is preferred The objective of this work was to establish a transcomplementing Madin-Darby Canine Kidney (MDCK) cell line for the production of high titer and high

quality ∆E1 CAV-2 vectors Methodology: Currently the production

system for CAV-2 propagation uses a dog kidney (DK) cell line In search of an alternative well-established safer and scalable culture system for the propagation of ∆E1 CAV-2 the MDCK cells were chosen as a transcomplementing cell line The MDCK cells were stably transfected with a plasmid harbouring the CAV-2 E1A and E1B genes The gene expression was analysed by RT-PCR More than one hundred clones were screened for the ampli cation of ∆E1 CAV-2 The stability of E1 expression and ∆E1 CAV-2 production after several passages was analysed To asses viral quality total and

infectious viral vectors were quanti ed Results: Several MDCK-E1

transcomplementing cell clones were successfully established

However, only 10% of the clones were able to ef ciently amplify

∆E1 CAV-2 Ten clones were selected for further analysis three of them showing production yields similar to DK-E1 cells and therefore con rming MDCK cells as an alternative for ∆E1 CAV-2 vector propagation The clones were stable over > 3 months in culture and even after adaptation to serum-free conditions where production of infective virus was ef ciently achieved Due to the high  exibility

of these cells in terms of culture system and media, much further enhancement in the viral yield can be obtained The ability of these cells to produce HD CAV-2 vectors is also being assessed

by stably expressing Cre recombinase Conclusions: MDCK-E1

transcomplementing clones have demonstrated to be ef cient host cells for ∆E1 CAV-2 propagation presenting the additional advantage

of deriving from a well-known cell line, with well-established culture systems and already approved by the regulatory agencies for the production of viral products

571 Production of a rAAV1-CB-hAAT Human Clinical Trial Lot by rHSV Co-Infection of Suspension BHK Cells

David R Knop,1 Brad Grimm,2 Lijun Wang,1 Darby L Thomas,1 Guo-jie Ye.1

1 AGTC, Alachua, FL; 2 SAFC Pharma, Carlsbad, CA.

The excellent safety pro le, long-term expression, and recent clinical data generated by human application of adeno-associated virus (AAV) vectors have made them the vehicle of choice for gene delivery studies rAAV manufacturing via transient transfection for human clinical application is slowly being supplanted by alternate production methods which have increased  exibility, are higher yielding, and are substantially easier to scale-up for diseases with large patient populations and higher dosing protocols One such production method is herpes helper vector-assisted rAAV manufacturing Here

we report the manufacture of rAAV under cGMP conditions using

a recombinant herpes simplex virus type 1 (rHSV) co-infection of suspension Baby Hamster Kidney (sBHK) cells The clinical trial material (CTM) will be employed in a Phase II human study for the treatment of alpha-1-antitrypsin (AAT) de ciency The rAAV1-CB-hAAT, a serotype 1 rAAV vector encapsidating the chicken-b-actin promoter-driven human AAT gene, was produced by co-infection of

sBHK cells with two hybrid rHSV/rAAV vectors to provide all cis and trans-acting rAAV elements, and helper virus functions, in 25 L

disposable Wave bioreactors The 100 L CTM generation consisted of four, batch-campaigned 25 L cell culture and viral vector production runs The resulting rAAV1-CB-hAAT was recovered from individual

25 L Wave bioreactor runs via detergent lysis and Benzonase exposure

of producer cells, primary clari cation, concentration and buffer exchange, anion exchange capture chromatography, and af nity chromatography polishing steps Af nity chromatography elution peaks were tested and pooled to create Drug Substance (DS) which was concentrated, buffer exchanged into the  nal formulation, sterile

 ltered, and  lled into the  nal product container to generate Drug Product (DP) for human injection The entire production campaign generated greater than 1 × 1016 DNase resistant particles (DRP) at the harvest step Additionally, 3.9 × 1015 DRP were recovered at the af nity chromatography intermediate and were available for subsequent processing to DS and DP The rHSV-based production

of rAAV was highly ef cient and generated material in suf cient quantity and purity for application in a human gene therapy clinical study for treatment of alpha-1-antityrpsin de ciency

572 RNA Based Plasmid Selection System for Antibiotic-Free Plasmid DNA Vector Production

Aaron E Carnes, Jeremy M Luke, Justin M Vincent, Clague P

Hodgson, James A Williams

R&D, Nature Technology Corporation, Lincoln, NE.

Antibiotic resistance markers, typically kanamycin resistance

(kanR), allow selective retention of plasmid DNA during bacterial

fermentation and are the most commonly utilized selectable markers However, to ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to: 1) the potential transfer of antibiotic resistance

to endogenous microbial  ora; and 2) the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome Here, we describe the development and application of a novel antibiotic-free (AF) selection system

Vectors with this selection system incorporate and express a 150 bp RNA-OUT antisense RNA RNA-OUT represses expression of a

counter-selectable marker (SacB) from the host chromosome (Fig 1a) SacB encodes a levansucrase, which is toxic in the presence of

sucrose Sucrose selectable mammalian expression vectors (Fig 1b)

combine antibiotic-free selection with highly productive fermentation

manufacturing (>1 g/L plasmid DNA yields), while improving in vivo

expression of encoded proteins The RNA-OUT selectable marker

can be used to retro t existing kanR DNA vaccine plasmids into

antibiotic-free vectors Interestingly, a minimum vector size for high yield plasmid production was identi ed; strategies to ensure high yield production of small plasmids are reported These vectors are safer, more potent, alternatives for DNA therapy or vaccination

573 Robust Recombinase-Free System Production of Helper-Dependent Adenoviral Vectors with Negligible Contamination by Delayed Packaging Process

Dan Cots,1 Raul Alba,1 Patrick Hearing,2 Eric J Kremer,3 Maria Mercedes Segura,1 Assumpció Bosch,1 Miguel Chillón.4

1 Departament de Bioquímica i Biologia Molecular, Centre de Biotecnologia Animal i Teràpia Gènica, Bellaterra, Catalunya, Spain; 2 Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY; 3 Institut Génétique Moléculaire de Montpellier, Montpellier, Languedoc-Roussillon, France; 4 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalunya, Spain.

Helper dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and retain only minimal viral cis-acting sequences required for vector propagation The Cre/loxP system is the classical and most widely method for producing HDAds In this system, the helper adenoviral packaging signal is excised rendering the helper virus unpackageable but able to trans-complement HDAd propagation However high activity of the Cre recombinase needed for low Ad helper contamination is associated to high toxicity To better address this issue, we have cloned attB/attP-ϕC31 sequences

 anking the packaging signal of both, human and canine adenovirus (Ad-5 and CAV-2) vectors to develop a new system based on delayed packaging of Helper-Ad respect to HDAds Surprisingly, the mechanisms causing this delay were not related to the presence

of the recombinase Subsequent band-shift studies suggested that

an unknown cell factor (in both, permissive human 293 and canine

DK cells) interacts with the attB sequence and affects the correct functioning of the packaging complex Viral cycle studies indicated

a clear delay in both canine and human attB-Ad, showing its potential

in different adenoviral serotypes

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