Transient transfection vs' Permanent: cloned genes

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Transient transfection vs.
Permanent cloned genes
Unintegrated DNA
Chromosomally integrated Unnatural?
Position effects ? (so analyze a pool of
many to Super-physiological expression?
average levels (per transfected
cell) Transient -gt 10-90 transfection
efficiency (stain) Permanents more like 0.001
transfectants per µg DNA per cell (high).
i.e., 106 -gt 1000 colonies could be much less
for certain types of cells
2
One the most dramatic first applications of gene
transfection from total DNA Gen transfer of
the growth-transformed phenotype the ability to
grow in multilayers or in suspension in soft agar
(Weinberg, Wigler) DNA from tumor transfected
into growth-controlled mouse 3T3 cells. Look
for foci (one focus). Make a library from a
growth-transformed transfectant. Screen for human
Alu repeat as a human-specific marker. (300 bp,
1 million copies per haploid genome, or 1 per 3
kb). Verify cloned DNA yields high frequency of
focus-forming transfectants. Isolate cDNA by
hybridization to the cloned genomic
DNA. Sequence. Identify gene a dominant
oncogene. Ras, a signaling protein in a
transducing pathway for sensing growth factors
Transformed Mouse 3T3 cells transfected with an
EGFreceptor gene
Mouse 3T3 cells
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Recombination of transfected genes gene
targeting Mitotic recombination between
homologous chromosomes known to take place (even
if rarely), so the enzymatic machinery for
homologous recombination is present.
Recombination of transfecting genes homologous
ecombiantion is rare Non-homologous
illegitimate recombination is much more common).
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Gene knockouts via homologous recombination
Killer gene
ES cells and transgenic mice. Selection for
homologous recombinants via the loss of HSV TK
genes (Capecchi) tk homol. region YFG
homol. region tk (YFG your favorite gene)
Gene X in figure at right. Non-homologous
recombination favors ends tk is inserted,
conferring sensitivity to the drug gancyclovir
(HSV-TK specific, not a substrate for human
TK) Still have to screen lots of
drug-resistants.. Most work has been in ES cells
? mice ? homozygosis via F1 breeding. Little
work in cultured lines Allele replacements in
cultured cell lines (e.g., APRT). Myc double
sequential K.O. viable, sick (J.
Sedivy) Splicing factor (ASF) double K.O. - see
next graphic. APRT adenine phosphoribosyltransfe
rase ASF alternative splicing factor
Resistant to gancyclovir
Die in gancyclovir
HSV-TK gene is removed during homologous
recombination, but remains joined during
non-homologous recombination. Unlike mammalian
TK, HSVTk converts gancyclovir to a toxic product
HSV Herpes simplex virus tk thymidine
kinase gene FIAU equivalent to gancyclovir,
today
M. Capecchi, Nature Medicine  7, 1086 - 1090
(2001) Generating mice with targeted mutations
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neo
Double knockout of the ASF gene, a vital gene, by
homologous recombination
Chicken DT40 cells high recombination
frequencies
One ASF gene allele disruted by homologous
recombination

hol
ASF-
hol
neo
neo
Tet-off promoter
Screen by Southern blotting
pur
Hol histidinol resistance pur puromycin
resistance Drug resistance genes here chosen for
illustration.
Both alleles have been disrupted in some neoR,
holR cells
neo
ASF-
neo
tet
pur
ASF-
pur
Screen by Southern blotting
X
Wang, Takagaki, and Manley, Targeted disruption
of an essential vertebrate gene ASF/SF2 is
required for cell viability. Genes Dev. 1996 Oct
1510(20)2588-99.
Cell dies without ASF(follow events
biochemically)
Cells remain viable(covered by human ASF gene)
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Histidinol dehydrogenase detoxifies histidinol,
confers histidinol resistance
protein synthesis
inhibits protein synthesis charged to tRNA but
cannot be transferred to growing peptide so
truncates
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Gene amplification for high level production in
CHO dhfr- cells.
DHFR system (dihydrofolate reductase)
Step-wise selection for resistance to marginal
levels of methotrexate
DHFR
DHFR
Folate
tetrahydrofolate
dihydrofolate
Glycine Purine nucleotides (AMP and
GMP) Thymidylic acid (TMP)
FH4
FH2
Resistance can occur via 3 different
mechanaisms 1) Methotrexate permeation mutants
(incl. MDR, increased efflux)) 2) Altered DHFR
with lower MTX binding affinity (smarter
enzyme) 3) Overproduction of DHFR protein (most
common in cultured cell lines)
MDR multiple drug resistance gene
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• Gene amplification dhfr
• Historically methotrexate resistance
• MTX inhibits dihydrofolate reductase (DHFR)
• MTX-resistant cells have (in order of discovery,
  1970s)
• High DHFR enzyme activity
• High DHFR protein
• High protein synthetic rate
• High translatable mRNA
• High mRNA level (by hybridization)
• High DNA level.
• Homogeneously staining, expanded chromosomal
  regions (HSRs)
• HSRs are the location of the high number of dhfr
  genes.
• Double minute chromosomes are an occasional
  alternative form.
• Amplicons (distance between repeated genes) are
  large (300 KB).
• (dhfr gene 25 kb)

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Reduction of folate to tetrahydrofolate
MTX
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Methotrexate
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Gene amplification
HSR Homogenously staining region
Nunberg et al. PNAS 1980
(Schimke, Sci. Amer.)
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Gene amplification
Homogeneously staining region FISH, here
C. Chen?
FISH fluorescent in situ hybridization
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Original locus?
HSR ? dmin upon DS break induced by a homing
endonuclease (I-SceI).
Restriction-type enzyme with a very long
recognition sequence ( 20 bp)
HSR homogeneously staining region Dmin double
minute chromosomes
Arnaud Coquelle, Lorène Rozier, Bernard
Dutrillaux and Michelle Debatisse ONCOGENE,
2002, 21 7671-7679 Induction of multiple
double-strand breaks within an hsr by
meganuclease I-SceI expression or fragile site
activation leads to formation of double minutes
and other chromosomal rearrangements
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Some other amplifiable genes
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Ampification models over-replication, unequal
sister chromatid exchange, breakage and fusion
(Tanaka et al.). Map dhfr amplicons (Schimke,
Hamlin) 300 kb , but wide range Gene
amplification is rare in normal cells (Wahl,
Tslty). p53- mutation allows it. In nature
rDNA in oocytes Drosophila chorion genes. In
medicine chemotherapy resistance (MDR,
P-glycoprotein, efflux pump) cancer (myc,
ras) In biotechnologyhigh level recombinant
protein production in mammalian cells
MDR multiple drug resistance
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2X
2X
C Ma, S Martin, B Trask, et al. Sister chromatid
fusion initiates amplification of the
dihydrofolate reductase gene in Chinese hamster
cells. Genes Dev. 1993 7 605-620
17
Gene amplification for high level recombinant
protein production in mammalian cells.
Principal system dhfr- CHO cells Facilitated
by the availability of DHFR-deficient mutant CHO
cells
CHO dhfr- cells vector with dhfr minigene
YFG
-GHT medium Most cells die. Transfectants live.
gradually increasing concentrations of MTX
Cells with gradually amplified dhfr transgenes
survive. YFG is co-amplified along with the dhfr
minigene.
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DHFR- cells require G,H,T
and are resistant to tritiated deoxyuridine
X
Blue nutrients required by DHFR- cells
- DHFR- cells selected by their resistance to
radioactive 3H-deoxyuridine 3HdU ? 3HdUMP ?
3H-TMP ? 3H DNA ? death from radioactive decay. -
DHFR- cells require glycine, hypoxanthine and
thymidine (GHT). - In GHT-free medium CHO dhfr-
cells die, but transfectants that have received a
dhfr gene survive. Urlaub, G. and
Chasin, LA., Proc Natl Acad Sci., 1980,
774216-20.
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A different major system for high level Mab
production glutamine synthetase in NS0
cells Mouse myeloma cells, high IgG producers ?
IgG- variants NS0 No endogenous IgG, but cell
is a natural IgG secretor. And it lacks
glutamine synthetase (GS) glutamate NH3
ATP ? glutamine ADP Pi Vector MAb genes
driven by strong promoters (H-chain, L-chain)
GS cDNA gene (Bebbington) Select on
glutamine-free medium Inhibit GS with methionine
sulfoximine (gln analog) Select for GS
overproducers ---gt--gt (gene amplification does
not seem to be operating in this system of the GS
cDNA gene and linked Mab genes) Proprietary
(Lonza Biologics)
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Resting T- or B-cell)
Mature T-cell (effector T-cell)
Plasma cell (effector B-cell)
Extensive ER
2002 Molecular Biology of the Cell by Bruce
Alberts, Alexander Johnson, Julian Lewis, Martin
Raff, Keith Roberts, and Peter Walter.
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Transfection strategies

• YFG (Your Favorite Gene) linked to a dhfr
  minigene on a single plasmid
• A. Insures co-integration
• B. Insures co-amplification
• YFG and dhfr on separate plasmids
• A. Allows a high ratio of YFG to dhfr to start

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Linked amp
CHO cells
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Co-amp1
(co-amplification first demonstrated)
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Co-amp3
Idea
(with or without pre-ligation)
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kaufman
(ribosome read-through)
Also, later, better dhfr translation using an
IRES, Internal ribosome intiation site, Mostly
viral but also in some cellular genes In theory,
not an advantage.
Y.F.G.
DHFR
IRES
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Amplification protocol
Note Process is lengthy and tedious.
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Some marketed recombinant proteins Erythropoietin
(Epogen, Procrit) JJ, Amgen Tissue
plasminogen activator (TPA) Genentech Growth
Hormone (Genentech) Insulin (Genentech) Beta-in
terferon (Avonex) Biogen-IDEC Alpha-interferon
(IntronA) Schering-Plough Neupogen
(Amgen) Etanercept (Enbrel) soluble TNF
receptor Monoclonal antibodies (mAbs) several
As of 2006 (Walsh, G., Nature Biotech. 24
769) AvastinErbituxRaptiva Xolair
HumiraRemicadeZenapax Simulect
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Examples of FDA approved therapeutic monoclonal
antibodies Antibody , Brand name, Approval date,
Type, Target, Approved treatment(s)
Abciximab, ReoPro, 1994, chimeric, inhibition of
glycoprotein IIb/IIIa, Cardiovascular
disease Adalimumab, Humira, 2002, human,
inhibition of TNF-a signaling, Several
auto-immune disorders Alemtuzumab, Campath, 2001,
humanized, CD52, Chronic lymphocytic
leukemia Basiliximab, Simulect, 1998, chimeric,
IL-2Ra receptor (CD25), Transplant
rejection Bevacizumab, Avastin, 2004, humanized,
Vascular endothelial growth factor (VEGF),
Colorectal cancer Certolizumab pegol, Cimzia,
2008, humanized, inhibition of TNF-a signaling,
Crohn's disease Cetuximab, Erbitux, 2004,
chimeric, epidermal growth factor receptor,
Colorectal cancer, Head and neck
cancer Daclizumab, Zenapax, 1997, humanized,
IL-2Ra receptor (CD25), Transplant
rejection Eculizumab, Soliris, 2007, humanized,
Complement system protein C5, Paroxysmal
nocturnal hemoglobinuria Efalizumab, Raptiva,
2002, humanized, CD11a, Psoriasis Gemtuzumab,
Mylotarg, 2000, humanized, CD33, Acute
myelogenous leukemia (with calicheamicin) Ibritumo
mab tiuxetan, Zevalin, 2002, murine, CD20,
Non-Hodgkin lymphoma (with yttrium-90 or
indium-111) Infliximab, Remicade, 1998, chimeric,
inhibition of TNF-a signaling, Several autoimmune
disorders Muromonab-CD3, Orthoclone OKT3, 1986,
murine, T cell CD3 Receptor, Transplant
rejection Natalizumab, Tysabri, 2006, humanized,
alpha-4 (a4) integrin,, Multiple sclerosis and
Crohn's disease Omalizumab, Xolair, 2004,
humanized, immunoglobulin E (IgE), mainly
allergy-related asthma Palivizumab, Synagis,
1998, humanized, an epitope of the RSV F protein,
Respiratory Syncytial Virus Panitumumab,
Vectibix, 2006, human, epidermal growth factor
receptor, Colorectal cancer Ranibizumab,
Lucentis, 2006, humanized, Vascular endothelial
growth factor A (VEGF-A), Macular
degeneration Rituximab, Rituxan, Mabthera, 1997,
chimeric, CD20, Non-Hodgkin lymphoma Tositumomab,
Bexxar, 2003, murine, CD20, Non-Hodgkin
lymphoma Trastuzumab, Herceptin, 1998, humanized,
ErbB2, Breast cancer
  
 
 
http//en.wikipedia.org/wiki/Monoclonal_antibody_t
herapy
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Ways to increase production and/or lessen
development time Mitchell Reff (IDEC patent)
Screen for a high production genomic position.
Integrate YFG into it by homologous
recombination, selecting for reconstitution of a
split dhfr minigene, then amplify. Mitsubishi
(T. Shibou, Mitsubishi Pharma Corporation.
European Patent Application. Vol. EP001293564A1,
PCT/JP01/04801) Same, but use a lox site and
site-specific recombination to integrate
YFG. Add chromatin remodeling sequences to
vector to open chromatin. Add insulator
sequences to vector to block postion specific
repression. Search for even better promoters
(current CMV, EIFalpha, actin) Or even
synthetic promoters (E. coli Stephanopolos, MIT)
Engineer cells with advantageous glycosylation
patterns Engineer cell to eliminate or defer
apoptosis (for longer productinon runs) Etc.
(including Chasin lab project)
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2005 Web site presentation
2004 claim 2.8 g/L
(1990)
Antibody grams/liter
Millions of cells per liter

• Note improvement include
• Higher cell density
• Longer times
• Higher output per cell

(22 d.)
(Old values from
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20,000 liter fermentor
20,000 liter mammalian cell fermentor - Lonza
Biologics - Portsmouth, NH
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High level production in mammalian cells Do the
math

• Reff patent (IDEC) 55 pg/cell/day
• Max cell density 107/ml ?
• So 1010 cells/L
• Therefore 55 x 10-12 g/cell/day x 1010 cells/L
  
• 55 x 10-2 g/L/day
• 0.55 g/L/day 11 g/L/20 days, calculated
• Lonza (contract manufacturer) claims (2005) 5
  g/L yield
• Same ballpark.
• 30,000 L reactor (largest)
• 30,000 L. X 5 g/L. 150 kg in 20 days, or say
  one month
• x 12 months 1800 kg/year 1,800,000 g/year
• One MAb dose 500 mg 0.5 g
• 1,800,000/0.5 3.6 million doses per reactor per
  year.
• 6 doses per patient per year?
• 3,600,000/6 600,000 patients per year per
  reactor.
• At 15,000 per patient per year ? 9B in sales
  /per 30 kL reactor

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