308 Titer and Long Term Stability of Retroviral Titers Produced in Murine Based Producer Cell Lines gate into immobile clusters The results suggest that the epitopes are useful to assay the quality of[.]
Trang 1gate into immobile clusters The results suggestthat the epitopes
are useful to assay thequality ofhMSCs cultures for use in animal
models and clinical trials
CELL PROCESSINGVECTOR PRODUCTION
306 Testing Large-Scale Lentiviral Vector
Preparations for Replication Competent
Lentivirus
LisaDuffy,' Sue Koop,' lingYao,' RobertGetty,' Scott Cross,'
Lakshmi Sastry,1Kenneth Cornctta.'
'Medical and Molecular Genetics /National Gene Vector
Labora-to/yo Indiana University School ofMedicine, Indianapolis IN.
As lentiviral vectors move into the clinical arena, the need to
screen forreplication competent lentivirus (RCL) will be an
im-portant step in certifying vector supernatant We have previously
reportedan analysisofRCL testing methods(Sastryetal Molecular
Therapy 8:830-9, 2003) and now report our initial experience with
RCL screening In brief, the two-phase assay utilizes an
amplifica-tion phase where the T cell line C8166 is exposed to the test article
After 4 hour incubation the cells are passaged for 3 weeks and cell
free media is used to inoculate naive C8166 cells which are then
passagedfor I week (indicatorphase), RCLis presentwhen p24-gag
I-IIV-Iantigen (by ELISA)and/or psi-gagrecombinant(by PCR)are
detected in indicator phase cells.An attenuated HIV-I virus (R8.71)
is used as a positive control.The assay was validated by performing
infection of C8166 cells according to the protocol using R8.71 at
the TCID50 (0.5 infectious unit/culture).Three cultures were tested
per assay and three independent assays were performed Seven of
nine cultures tested positive at the indicator phase for both p24
antigen and psi-gag recombinants, which validated the assay and
confirmed the limits of detection was approximately I infectious
unit.Sincevalidation,theassay hasbeenuscd totestlarge scale
vectorpreparationsand cells in 9independent assays.For each assay,
three positive controls were inoculated at the estimated TCID50 at
the start of the amplification phaseand were subsequently carried
through the indicatorphase In addition,fiveculturesof naive C8166
were inoculated with virus at the TCID50 at the start ofthe indicator
phase In reviewing the positive control portion ofthe amplification
phase, 24 of 27 cultures at thc end of the amplificationphase had
elevatedp24 antigen levelsand after passageintothe indicatorphase,
27 of27 had detectable virus by p24 and by psi-gag recombination
PCR.For the 5 positive controls set up at the start of the indicator
phase,41 of 45 were positive for p24 and 36 of 45 were positive
for psi-gag recombinants at the end of the week of culture These
findings suggest the 3 week amplification does increase sensitivity
compared to a 1 week amplification In terms of test articles, 7 of
the RCLassays were used to screen 4 largescale vector productions
that had a combined pre-concentration volume of over 100 liters,
of which 5% of the total final product was tested.In addition,10'
post-productioncells were also screened from each of the 4
produc-tions and 2 assays were performed on cells transduced with clinical
grade vector To date,no RCL has been detected in any of the test
articles submitted for analysis This analysis providesencouraging
news forlentiviralvector development, although continued
refine-ment of the RCL assay is warranted to maximize sensitivity while
streamlining the methodology
S1I6
307 Generation of Lentivirus Vectors Using Recombinant Baculoviruses
Hanna P Lesch.!' Sanna Turpeinen,':'Einari A Niskanen,' Anssi
1.Mahonen,':'Kari 1.Airenne,ISeppoYla-Herttuala.v-'
I Department ofBiotechnology and Molecular Medicine A.I Vlr-tanen Institute University ofKuopio, Kuopio, Finland;2 Depart-ment ofMedicine and Gene Therapy Unit A./ Virtanen Institute University ofKuopio, Kuopio, Finland;jKuopio University Hos-pital Kuopio University HosHos-pital Kuopto, Finland; "Department ofBiological and E nvironmental Science NanoScience Center; University of'Jyvaskyla Jyviiskyld , Finland ; sArk Therapeutics
qgArk Therap euticsqg Kuopio, Finland
The production ofreplication defective lentiviralvectors in clini-cal sclini-cale is challenging The four plasmid method by conventional transienttransfectionto producethirdgeneration lentiviralvectorsis tedious,time consuming and suffers from batch-to-batch variation Some induciblestableproduction cell lines are available but the toxicity of several viral proteins prohibits constitutive expression Baculovirus technology offers an attractive possibility to a scalable virus production as a result of ease production and concentration
ofbaculoviruses,efficient transduction of suspension mammalian cells in serum free conditions and safety of the baculoviruses,As a firststep towards scalable lentiviralproductionsystem we havecon-structed four recombinantbaculoviruscs,BAC-transfer, BAC-gag-pol, BAC-VSVg and BAC-rcv,expressingall c1cmcnts required for safe lentivirusvectorgeneration After 293T cell transduction with recombinantbaculovirusesfunctional Ientiviruses were produced Different baculovirusconcentrationswere used to findoptimal bacu-lovirusconcentrationfor lentivirusproduction.The un-concentrated lentiviral titers in cell culture mediums were on avarege 1,21x 106 TUlml which are comparable to titers ofthelentivirusproduced by the conventional four plasmid method Lentivirus transduced Hela cells were grown forthreemonths without loosingtheGFP exprcs-sion Our results show for the first time that baculoviruses can be
usedfor the production of lcntiviruscs in mammalian cells
308 Titer and Long Term Stability of Retroviral Titers Produced in Murine Based Producer Cell Lines
Lisa Duffy,' Sue Koop,'ling Yao,'Kenneth Cornctta.'
'Medical and Molecular Genetics /National Gene l-ector Labora-tory Indiana University School ofMedicine Indianapolis IN.
While the spectrum of integratingvectors under considerationfor clinical use continues to expand,vectors based on gamma retrovi-ruses continue to beutilized and improved.Tobetter understand factors involved with vector production,we studied the impact of genome size,temperature, harvest timing, and long-termstorage on
vector titer.Tostudy genomesize we prospectivelydesigneda series ofdeletions in the transgene portion of the GcSAM vector (kindly provided by Richard Morgan, NIH) that contains non-expressing neomycinphosphotransferase(neo) and beta galactosidases genes Eight constructs were evaluated with genome sizes ranging from
3258 to 6970 bascpairs Titer was assessed using real-time PCR for detection of vector RNA using a probe and primer sct designed within the packaging sequence Titer for vector genornes between
4559 and 6346 basepairs had similar titers, with a decrease outside this range.The effect of temperature and harvest intervals was also evaluated Data from 27 PG13and 5 GP+envAM12derived Master Cell Banks(MCB)generatedin our facilitywascompiled,evaluating titer of vector produced at 32 or 37°C and harvest intervals of 8,
12 or 24 hours Thc majority of PG13based ccll Iincs (13/27) had the highest titers whcn harvested at 24 hours and 32°C,although 6/27 wcre optimal at 37°C, 12 hour harvest;4/27 wcrc optimal at 37°C,24 hour harvest; and 4/27 were optimal at 32 DC, 12 hour
Molecul ar The 4lpy Volume 15.Suppl ement t• • \br 2007
Co pyright © " 111e Ameri can S OI;ic;ty o f Gene
Trang 2Thcrapj-harvest All 5 GP+envAMI2 cell lines were optimal at 32 °C, but
optimal harvest time varied The data suggests harvest conditions
should be optimized for each MCB generated Also,those MCBs
with an optimal titer at 24 hoursoften have a 12 hour titer that
ex-ceeds 50% of the 24 hour titer Therefore, more frequent harvests at
shorter intervals can increase the total yield of vector particles and
may be preferred when the material can be concentrated Finally,
the stability of vector stored was measured over a 5 year period
Aliquots of the LNL6 vector (expressing neo) and the Ampho/GFP
and GALVIGFPvectors (expressing enhance greenf1uorescent
pro-tein,MGF-eGFP) were maintained between -70 and -80 °C and the
number oftransducing units was periodically measured using G418
selection and flow cytometry,respectively In all three vectors the
titer appeared to be stable during the observation period suggesting
an expiration date of5 years would be appropriate for unconcentrated
vectors The information obtained serves as a baseline for assessing
new packaging cell lines, including those designed to generate novel
integrating vector systems such as HIV and foamy viruses
309 Plasmid DNA Production in a Wave
Bioreactor under cGMP Conditions
Kenneth A Laderman,'Jonathan M Anderson,'Ivy V Derecho,'
Nina T Dunphy,' RossJ.Mclvlahon,' Valerie R Quezada,' David
Hsu,' Larry A.Couture.'
'Center for Biomedicine and G enetics Centerfor Applied
Technology Development Beckman Research Institute ofCity of
Hope Duarte CA.
Plasmid DNA production is an essential step in the generation
of genetic therapies, either as the therapeutic agent or as reagents
for the production of viral vectors A robust process for the
genera-tion of plasmid bearingE colibiomass is necessary as it is the rate
limiting step for plasmid production and it is most variable as there
are construct specific factors that affect the plasmid yield There
are a number of factors that make the transition ofthe fermentation
process from a traditional stirred tank reactor to a Wave bioreactor
desirable Foremost among these are the disposable culture vessel in
the form of the wave bag The disposable culture vessel minimizes
the possibility of prior product contamination in a multi-product
facility and decreased production cost while increasing
through-put due to the ease of set up and tear down.Twolentivirus helper
plasmids,which had previously been produced in the facility using
a cGMP stirred tank procedure, were used to develop the wave
fer-mentation process Ferfer-mentations were completed using media and
fermentation parameters adapted from the stirred tank fermentation
process To mimic the aeration and agitation of the stirred tank the
wave angle and rate were set to their practicalmaxima.The mean
specific yield (mg of plasmid Ig wet weight) obtained from the
wave bioreactor was found to range from equivalent to 3 fold higher
than that obtained from the same bacterial master cell bank in the
stirred tank fermentor while the bacterial yield (g wet weightIliter
of culture) from the wave bioreactor was approximately equivalent
to that obtained from the stirred tank The wave fermentation has
proven to be scalable with minor increases in specific yield observed
as the culture volume is increased to 25 Iitcrs Limited optimization
of temperature and pH conditions have established conditions that
increase the specific yield approximately 100% from the default
fermemation conditions used for the original feasibility
assess-ment The resulting improvement in specific yield ofthe optimized
process is approximately 600% greater than that obtained with the
stirred tank system
Molecular Therapy Volume15 SupplementI ~ br 2 007
C opyright © T he American So ci etyo r G ene "1l1f :r:lpy
310 A Rapid, Accurate and Precise Assay System To Analyze the Quality and the Quantity of Clinical Grade HSV Vector Stocks
Ali Ozucr,' Steve K Wendell,2 Darren Wolfe; WilliamF.Goins; David M Krisky,"Mohammad M.Ataai,'Joseph C Glorioso.'
f Molecular Genetics and Biochemistry University ofPittsburgh
Pittsburgh PA;]Dental Medicine, University ofPittsburgh
Pitts-burgh, PA; 'Chemical and Petroleum Engineering Universityof Pittsburgh Pittsburgh, PA; "Department ofPathology ; University ofPittsburgh Pittsburgh PA.
As the number of clinical and research applications of herpes-virus-based vectors increase,it becomes critical to develop rapid, accurate and precise assay systems to analyze the quality and the quantity of clinical grade vector stocks.Our assay system contains two independent and complementary DNA and protein quantification methods: a real-time quantitative PCR system and two micro-plate assays using PicoGreen and NanoOrange reagents for the quanti-fication of total DNA and protein respectively This assay system
is being optimized to quantify the amount of infectious versus defective HSV particles, and the purity ofHSV vector stocks Our real-timequantitative PCR assay employs two independent primer sets The first set,speeific for ICP47 sequences present in all HSV genomes, allows for the quantification of total viral particles and for defective DNA containing particles when compared with the number of plaque forming units generated by standard virologi-cal plaque assay A second primer set,specific for the human 18S gene, enables the estimation of purity ofgene therapy vector stocks
in combination with NanoOrange protein assay system Our real time PCR assays are linear from 4 to 4x 103HSV genome copies per reaction, and 0.005 to 50 ng host cell genomic DNA per reaction
In contrast to our PCR method that quantifies the viral and host cellular DNA concentration,we developed an independent micro-plate assay measuring the total DNA and protein concentrations of vector stocks.Our micro-plate assays are fast and accurate, with detection limit as low as 0.5ng of DNA and 0.4 mg/mL protein The resultant combination of real-time PCR and micro-plate DNA and protein quantitation assays represents a standard in the field of HSV vector quality assessment
311 Development of a High Cell Density Process for cGMP Production of VRX496 Modified HIV Infected CD4+ T Lymphocytes
Andrew Worden,'TonyEncinas.'Yclena Skripchcnko.! James Rogers,'ReubenCohen,'VladimirSlepushkin,' Laurent Hu-meau.'
f Researchand Development VIRxSYS Corporation Gaithers-burg AID; l Operations VIRxSYS Corporation Gaithersburg, MD.
VIIb:SYS Corporation has developed an autologous CD4+ T cell therapy for the treatment ofI-IlV infection, which uses a patient's own CD4+ T cells that have been genetically modified by a lenti-viral vector, VRX496, carrying a 937-base antisense targeting the HIV envelope.Aftera patient's white blood cells are harvested, the cells are then purified to obtain the CD4+ T cells, transduced with VRX496,expanded, and then rcinfused into the patient We reported the successful completion of our Phase I clinical trial where we demonstrated the safety and tolerability ofa single dose consisting ofapproximately 10 billion autologous I-IIV infected CD4+T cells transduced with VRX496, (Levine and Humeau et al., PNAS 2006)
We immediately initiated a Phase II clinical trial testing the safety and tolerability ofsingle and repeated infusions ofVRX496-modifed CD4+ T cells with the aim to determine the optimaltherapeutic dose A large scale cGMP cell process was developed and has been presented (Humeau et aI.,ASGT 2006) Briefly, the positively
se-SI17