381 A New Cell Line for Clinical Grade Production of E1 Deleted Adenovirus Encoding Toxic Proteins Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell T[.]
Trang 1Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S148
CELL PROCESSING AND VECTOR PRODUCTION
for the Treatment of SCID-X1 Using a Stable
Producer Cell Line
Tim Lockey,1 Susan Sleep,1 Robert Throm,2 Mike Greene,2 Paolo
Fagone,2 Harry Malech,3 Brian Sorrentino,2 John T Gray.2
1 Children’s GMP, LLC, St Jude Children’s Research Hospital,
Memphis, TN; 2 Hematology, St Jude Children’s Research
Hospital, Memphis, TN; 3 Laboratory of Host Defenses, NIAID,
NIH, Bethesda, MD.
We describe here our process and large scale production results for
GMP grade production of a clinical lentiviral vector for the treatment
of SCID-X1 using a stable, HIV based producer cell line (described
in Throm, et al Blood, v113 n21 p5104) Process optimization was
performed with cells from our master cell bank at 1 and 5 liter scale
prior to pre-clinical production at 5 liter scale and clinical production
at 25 liter scale The fi nal process consists of cell expansion in fl asks
prior to seeding into a WAVE bioreactor bag containing Fibracel
disks for expansion to fi nal production scale When daily glucose
consumption exceeds 1 – 1.5 g/L, the media (DMEM+10%FCS+1
ng/ml Doxycycline) is replaced with Doxycycline free media to
induce vector production On Days 3 through 8 post induction (p.i.)
supernatant is harvested and held at 4C On days 4, 6, and 8 two
harvests are pooled, adjusted to pH 8.0 and 300 mM NaCl, and
fl owed through Mustang Q cartridges to capture vector particles
Following elution with 1.5 M NaCl, vector is diluted with buffer
to reduce salt concentration and held at 4C until the fi nal harvest
has been similarly processed The three anion exchange eluates are
then pooled, desalted, and concentrated by using tangential fl ow
fi ltration with a 500 kDa cutoff membrane The fi nal concentrated
vector is formulated in PBS containing 0.5% human serum albumin
Production yield and QC assay results for the pre-clinical and clinical
batches were similar and results for the clinical product are presented
here Infectious titer of the vector product was determined on ED7R
cells, a T-cell line that does not signifi cantly express the γc gene Peak
supernatant titer occurred on day 3 p.i at 1.25x107 tu/ml, and the
average of all 6 harvests was 5.5x106 tu/ml, giving a total transducing
unit yield of 8x1011 tu p24 similarly peaked on day 3 p.i at 5.2 µg/
ml, and averaged 2 µg/ml for all 6 harvests After purifi cation, yield
was 2.3x1011 total tu, or ∼30%, predominantly due to losses at the
Mustang Q step Final volume was 500 mls and titer was 4.5x108
tu/ml, with an infectivity of ∼5000 tu/ng p24, which is comparable
to that described for the transient transfection product used for the
successful lentiviral treatment of adrenoleukodystrophy (Cartier et al,
Science, v326 n5954 p818) The product has been extensively tested
for adventitious agents including replication competent lentivirus and
confi rmed negative Critical potency measurements were performed
by transducing mobilized adult human peripheral blood CD34+ cells
at 108 tu/ml CFU-C derived from those transductions were typically
>20% positive by PCR depending on the donor Final FDA review of
this clinical grade product is pending, with anticipated initiation of
clinical trial immediately following approval This represents to our
knowledge the fi rst clinical grade lentiviral vector produced from a
stable cell line system
Producer Cell Lines
John Martin,1 Amy Frederick,1 Yuxia Luo,1 Robert Jackson,1 Dianna Ambach,2 Christopher Renzi,2 Lisa Budzinski,2 Lois Horton,2 Angela Johnsen,2 Simon Godwin,2 Donna Armentano,1 Sam Wadsworth,1 Karen Vincent.1
1 Molecular Biology, Genzyme Corporation, Framingham, MA;
2 Gene Therapy Development, Genzyme Corporation, Framingham, MA.
AAV producer cell lines represent an effective method for large-scale production of AAV vectors for clinical applications In the system described here, a single plasmid containing three components:
the vector sequence, the AAV rep and cap genes and a selectable
marker gene is stably transfected into HeLa S3 cells Following transfection, candidates (referred to as masterwells or MW) are initially selected on the basis of a relative production screen where vector yields are estimated and used to rank MW relative to each other High-producing MW are then subjected to a specifi c production screen
in which yield (of DNase-resistant particles; DRP) is determined on
a per cell basis A transfection to generate an AAV2/SEAP producer cell line has been performed The relative production screen revealed that 6.4% of the MW yielded greater than 1 x1010 DRP/ml Several of these showed vector yields in the 5 x 104 to 2.0 x 105 DRP/cell range when assessed for specifi c production Analysis of genomic DNA in the high-producing MW showed plasmid copies integrated in a
head-to-tail confi guration Upon infection with adenovirus, rep/cap copy number was amplifi ed approximately 100-fold Rep expression in the
producer cell line was fi rst detected a few hours post-infection and was
followed closely by cap; expression of both genes reached a peak at
48 hr This pattern of gene expression correlated with the appearance
of packaged vector at the 24 hr time point Vector production reached
a maximum at 48 hr and was maintained through the 96 hr (fi nal) time point AAV2/SEAP was produced in a small-scale (1L) shaker culture and purifi ed via affi nity chromatography to provide vector for assessment of vector potency Results of experiments comparing AAV2/SEAP generated by the producer cell line with vector produced via the triple transfection method will be presented
381 A New Cell Line for Clinical Grade Production of E1-Deleted Adenovirus Encoding Toxic Proteins
Rénald Gilbert,1,2 Claire Guilbault,1 David Gagnon,1 Danielle Jacob,3 Lucie Bourget,1 Amine Kamen,3 Bernard Massie.1,4
1 Genomics & Gene Therapy Vectors, Biotechnology Research
Institute, Montreal, QC, Canada; 2 Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montreal, QC, Canada; 3 Animal Cell Technology, Biotechnology Research Institute, Montreal, QC, Canada; 4 Département de Microbiologie et Immunologie, Université de Montréal, Montreal,
QC, Canada.
First generation (E1-deleted) adenovirus vectors (FGAd) are important vectors for therapeutic applications such as cancer and vaccination An ideal cell line for clinical grade production of FGAd should produce high viral titers in suspension culture for scale-up and
in serum-free medium for regulatory compliance Most importantly,
it should not produce replication competent adenovirus (RCA) Some gene products carried by FGAd, such as those employed for cancer therapy, are cytotoxic For this reason, they should not be synthesized during the propagation of FGAd, because their toxicity reduces the vector yield The present study describes the construction
of a cell line (Sf-BMAd-R) that possesses such properties The cell line was generated by stable transfection into A549 cells of the E1A and E1B regions of adenovirus and of the repressor of the cumate-switch (CymR) A clone producing elevated amounts of FGAd was
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RNA VIRUS VECTORS II
isolated and adapted to serum-free suspension culture CymR binds
to the cumate operator (CuO) and prevents transcription when CuO
is inserted downstream of a strong enhancer/promoter such as CMV
Expression of a transgene regulated by the CMVCuO promoter is
repressed when the FGAd carrying the transgene are propagated on
Sf-BMAd-R cells The yield of FGAd expressing GFP regulated by
CMVCuO (Ad-CMVCuOGFP) was 1000 transducing units (TU)
per cell, a value comparable to the yield obtained with our 293SF
cell line previously characterized Production of Ad-CMVCuOGFP
and repression of GFP by CymR were stable for at least three
months of culture in the absence of selective agent Production of
Ad-CMVCuOGFP to a level of 1000 TU/cell was also validated in
a 2.5 liter bioreactor in protein-free chemically defi ned medium We
also demonstrated that the yield of FGAd encoding a suicide gene
(Cytosine deaminase::uracil phosphoribosyltransferase) regulated by
CMVCuO was 6 times higher with our Sf-BMAd-R in comparison
to our standard 293SF cells In summary, because of its interesting
characteristics, the Sf-BMAd-R cell line should become a useful tool
to produce clinical grade preparations of FGAd for gene therapy and
vaccination
RNA Virus Vectors II
Retroviral Vectors Using a Beta-Lactamase Protein
Fragment Complementation Assay
Wu Ou,1 Connie Lu,2 Jakob Reiser.1
1 Division of Cellular and Gene Therapies, FDA/CBER, Bethesda,
MD; 2 University of Maryland, College Park, MD.
We have developed a rapid assay to titrate lentiviral and
gamma retroviral vectors The assay is based on protein fragment
complementation involving the N-terminal (Bla1) and C-terminal
the yeast FRB (the FKBP-rapamycin binding domain of TOR) and
FKBP (FK506 binding protein) domains, respectively In addition,
a 15-amino acid membrane anchoring sequence (S15) derived from
c-Src was fused to the N-terminus of FRB-Bla1 For vector production
a plasmid encoding S15-FRB-Bla1 was co-transfected with lentiviral
packaging and vector plasmids into HEK 293T cells, resulting
in the incorporation of S15-FRB-Bla1 in the vector’s envelope
Transduction of HEK 293 cells stably expressing FKBP-Bla2 using
vector particles containing S15-FRB-Bla1 resulted in BLAK fragment
complementation, mediated by FRB and FKBP and rapamycin
Enzymatically active BLAK could be detected by the conversion
of a green fl uorescent substrate CCF2/AM into a blue fl uorescent
product and BLAK activity could be easily quantifi ed in that way
using FACS The enzymatic conversion of CCF2/AM was found
to be directly related to vector entry since a neutralizing antibody
completely blocked the conversion The titers obtained using this rapid
assay correlated well with titers measured by functional transduction
assays The whole assay can be fi nished within 8 hours This time
frame is considerably shorter compared to other transduction-based
titration assays for lentiviral and gamma retroviral vectors This assay
may also be useful for rapid titration of other enveloped viruses and
for studying virus entry processes
383 Insulated Lentiviral Vectors towards Safer Gene Transfer to Stem Cells
Caroline Duros,1 Alexandre Artus,1 Armelle Gaussin,2 Scholtz Simone,3 Daniela Cesana,4 Eugenio Montini,4 Manfred Schmidt,3 Christof von Kalle,3 Nicolas Mermod,2 Odile Y D Cohen-Haguenauer,1 Odile Y D Cohen-Haguenauer.5
1 LBPA and CLINIGENE, Ecole Normale Superieure, Cachan, France; 2 Laboratory of Molecular Biotechnology, University
of Lausanne (UNIL), Lausanne, Switzerland; 3 3Laboratory of Translational Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; 4 FCSR-TIGET, San Rafffaele, Milan, Italy; 5 Medical Oncology and Oncogenetics, Hôpital Saint-Louis and University Paris-Diderot, Paris, France.
The need for better gene transfer systems towards improved risk/benefi t balance for patients remains a major challenge in the clinical translation of gene therapy (GT) We have investigated the improvement of integrating vectors safety in designing new short synthetic genetic insulator elements (GIE) In otherwise successful GT-trials in SCID, CGD and WASp patients, insertional mutagenesis has resulted in leukemia from transduced cells The identifi cation
of new GIEs which would prevent such activation effects is a main challenge since attempts e.g with the chicken β-globin HS4, have met with poor effi cacy and genetic instability We have constructed SIN-insulated retrovectors with two candidate GIEs and compared them to native and SIN-LTRs With each constructs two internal promoters have been tested: either the strong Fr-MuLV-U3 or the housekeeping hPGK We could identify a specifi c combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) In target cells a dramatic shift of expression is observed with an homogenous profi le the level of which is strictly conditioned
by the promoter strength These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective
of the multiplicity of infection (MOI) In comparison, control vectors show heterogeneous expression profi les with levels which depend
on the MOI and prove unstable We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing Data indicate reduced clonality developed over time In addition, utmost recent data in cancer-prone mice do not show statistically different from mock-untransduced mice We have developed new lentivectors (and gammaretrovirus) which might provide improved versatile tools toward improvement of integrating gene therapy vectors
Pluripotent Stem Cells Using Polycistronic Foamy Virus Vectors
Iram F Khan,1 Jordan Kho,3 David R Deyle,1 Yi Li,1 David W Russell.1,2
1 Medicine, University of Washington, Seattle, WA; 2 Biochemistry, University of Washington, Seattle, WA; 3 Program in Developmental Biology, Baylor College of Medicine, Houston, TX.
Induced pluripotent stem cells (iPSCs) generated by ectopic expression of transcription factors are an ideal source of patient-specifi c cells given their capacity to differentiate into different cell types However, multiple insertions of retroviral vectors at random sites within the genome can alter endogenous gene expression and render these cells unsuitable for clinical applications Expressing all transcription factors from a single vector can minimize the number
of viral integrations Here we describe excisable, polycistronic Foamy Virus (FV) vectors for reprogramming human somatic cells into pluripotent stem cells Wild type FV is non-pathogenic and FV vectors exhibit no preference for integration within genes,