Gene targeting is a powerful technique to introduce genetic change into the genome of

eukaryotic cells. It is widely used to create defined mutations in murine embryonic stem

cells and theoretically could be used to create or repair mutations in somatic cells. In this

way gene targeting could be a powerful form of gene correction type gene therapy. Despite

its potential, gene targeting has not been widely used in somatic cells because of its low

efficiency. We report on a system based on the correction of a mutated GFP gene that allows

the efficient study of gene targeting in somatic cells. Using this system we show that gene

targeting is stimulated over 2000-fold by the introduction of a DNA double-stranded break

in the target locus (DSB-GT). We find that the rate of DSB-GT can be increased by increasing

the amount of repair substrate, the amount of homology between the gene target and repair

substrate, and by increasing the frequency of double-stranded break creation. When we

optimize conditions for DSB-GT we obtain targeting rates of 3-5%. Finally, we show that

chimeric nucleases, protein fusions between zinc finger DNA binding domains and the

endonuclease domain of the Fokl restriction enzyme, can stimulate gene targeting in the

genome of human somatic cells by several-thousand fold. Our data provides a paradigm for

the use of gene targeting as a form of gene therapy for monogenic diseases

Baltimore, David

Porteus, Matthew

Correction of gene defects in human somatic cells by targeting as has been used in murine embryonic stem cells (1, 2) has been precluded by the low spontaneous rate of gene targeting (3). However, creation of a DNA double-stranded break (DSB) in the genomic target (DSB-GT) can stimulate homologous recombination by over 1000-fold (4). 



We can rapidly and quantitatively measure gene targeting by correcting a mutation in a green fluorescent protein (GFP) gene that has been stably integrated into the genome (5) (fig. S1). With an optimized GFP gene targeting system, the introduction of a DSB by I–Sce I (Sce) stimulated GT >40,000-fold and the absolute rate of gene targeting reached 3 to 5% (fig. S2). Such a system, however, depends on the prior introduction of a Sce binding site into the target gene and cannot be used for endogenous genes. Chimeric nucleases (CNs) have the potential to create sequence-specific DSBs (6). CNs—fusions between zinc finger binding DNA binding domains and the endonuclease domain of Fok I—can sitespecifically cleave naked DNA in vitro (6), extrachromosomal DNA in Xenopus oocytes (7), and chromosomal DNA in Drosophila (8). CNs work as dimers, and their efficiency depends on the spacing and orientation of the zinc finger binding sites with respect to the length of the amino acid linker between the DNA binding and endonuclease domains (7, 9). 



QQR is an artificial zinc finger DNA binding domain that recognizes the sequence 5′-GGGGAAGAA-3′ with nanomolar affinity (10). We modified QQR chimeric nucleases (QQR-CNs) (7, 9) (Fig. 1A) and tested whether they stimulated gene targeting (Fig. 1B). The background rate of gene targeting was 0.71 events per million transfected cells (fig. S1C). QQRL18-CN stimulated gene targeting 17-fold on target QQR6 and 260-fold on target QQR8 (Fig. 1B). QQRL0-CN did not stimulate gene targeting on target QQR8, but it was as efficient as Sce in stimulating gene targeting by over 2000-fold on target QQR6 (Fig. 1B). QQRL18-CN showed some preference for an 8–base pair (bp) spacing between binding sites whereas QQRL0-CN preferred 6-bp spacing. Thus, removing the linker between the zinc spacfinger and the nuclease domains increased the activity and specificity of the fusion protein in mammalian cells. As controls, we showed that the CNs did not stimulate gene targeting if (i) they lacked a nuclear localization signal, (ii) there was a single binding site rather than an inverted repeat binding site in the target, and (iii) the cognate binding site was changed. Thus, homodimers of CNs are potent stimulators of gene targeting in human somatic cells

Porteus, Matthew H.

Caltech Authors - Main

Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
GFP Gene Targeting System
The components of the system we used to study gene targeting are depicted in
Fig. S1.  It is based on the correction by homologous recombination of a mutated green
fluorescent protein (GFP) gene (“GFP gene targeting system”).  A single copy of an
artificial gene target (A658) is stably integrated into the genome of 293 cells, creating
cell line “293/A658.”  The genomic target consists of a GFP gene that is mutated by the
insertion of an in-frame stop codon followed by the recognition site for the I-SceI
endonuclease (“Sce”) at basepair (bp) 327 of the GFP coding region. 293/A658 cells
remain GFP negative (data not shown).  We measured gene targeting by transfecting
293/A658 with a “substrate” construct that was promoter-less and missing the first 37
nucleotides, including the initiation codon, of the GFP gene ("RS2100" for repair
substrate with 2100 bp of homology). After transfection we measured the number of GFP
positive cells by flow cytometry and determined the rate of gene targeting by normalizing
the percentage of GFP positive cells to the transfection efficiency.  When we transfected
RS2100 into 293 cells without a gene target (293-0 cells) there were no GFP positive
cells (data not shown).  When we transfected supercoiled RS2100 alone into 293/A658,
the gene targeting rate was 7.1 x10-7, or 0.71 events per million transfected cells
(“spontaneous gene targeting or “S-GT”) (Fig. S1, B and C). We found that the frequency
of S-GT increased as the insertional mutation size in the GFP gene decreased (Fig. S1C).
We measured the double-stranded break induced gene targeting (DSB-GT) rate by
co-transfecting RS2100 with a Sce expression plasmid (see Fig. S1B for a representative
flow cytometry plot). Creating a DSB in the target GFP gene increased the rate of gene
targeting over 2000-fold (Fig. S1C).  Transfection of the Sce expression plasmid without
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
the repair substrate RS2100 generated no GFP positive cells (Fig. S1B for a
representative flow cytometry plot).  Transfection of the Sce expression plasmid with the
repair substrate RS2100 into 293-0 cells did not generate GFP positive cells (data not
shown).  Unlike spontaneous gene targeting, the DSB-GT did not decrease as the
insertional mutation size increased (Fig. S1C).  We determined the time course of DSB-
GT by serially measuring the number of GFP positive cells after transfection and found
that the maximum number of GFP positive cells occurred 2.5-3 days after transfection
and remained stable for at least two weeks (Fig. S1D).  The gene targeting reaction,
therefore, occurred relatively quickly after transfection and created stable genetic change.
Finally, we purified single GFP positive cells by fluorescence activated cell sorting and
determined the sequence of the GFP gene after gene targeting. In the 9 GFP positive cells
tested, the sequence of the GFP gene was wild-type, demonstrating that gene targeting
had occurred (data not shown).  The DSB-GT rate in murine 3T3 and human SaOS-2
cells was similar to that in 293 cells (data not shown).   Further, the DSB-GT rate was
similar whether a pool of cells or a clonal cell line  with single insertion sites for A658
was examined (data not shown).  Thus, our findings were not cell type or integration site
dependent.
Using the GFP gene targeting system we quantitatively explored several variables
that regulate the rate of DSB-GT.   We found that increasing the amount of substrate
(RS2100) transfected increased the rate of DSB-GT until a plateau is reached (Fig. S2A).
This result demonstrated that gene targeting is dependent on the amount of repair
substrate available.  We found that increasing the length of homology between the repair
substrate and the target linearly increased the rate of DSB-GT (Fig. S2B).  In these
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
experiments we kept the amount of 5’ homology constant at 290 basepairs (bp) and
varied the amount of 3’ homology from 500 bp to 3700 bp.  This result suggests that
while spontaneous gene targeting is logarithmically dependent on homology length (S1),
DSB-GT is linearly dependent on homology length.  In either case, increasing the length
of homology between the target and the repair substrate increased the frequency with
which the cell undergoes gene targeting.  Figure S2C shows that the DSB-GT rate was
linearly dependent on the amount of PGK-Sce transfected.  The DSB-GT rate reached a
plateau, however, when higher amounts of CBA-Sce were transfected (Fig. S2C).  This
data suggests that DSB-GT is dependent on the creation of a DSB to initiate gene
targeting but eventually becomes saturated for DSB creation.  We found that
manipulating the transcriptional status of the repair substrate can affect the rate of gene
targeting.  Transcribing the truncated repair substrate with a CMV promoter (CMV-
RS2100) increased the rate of DSB-GT by 50% (Fig. S2D).  Just as with RS2100,
transfecting CMV-RS2100 into 293-0 cells did not generate GFP positive cells (data not
shown).  The rate of DSB-GT was highest when Sce expression is driven by the CBA
promoter, intermediate with the CMV promoter, and lowest with the PGK promoter (Fig.
S2E).  This result probably reflects the different levels of Sce expression from each
promoter.  Figure 1E also demonstrates that the rate of DSB-GT can be increased by
placing the repair substrate on the same plasmid as the Sce expression cassette rather than
co-transfecting two plasmids.  The stimulation was lost when the DSB-GT process was at
saturation, as when the CBA promoter was used to express Sce.  When we optimized the
above parameters we achieved  gene targeting rates of 3-5% which is a  20-fold increase
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
over the DSB-GT rate obtained without optimization and gives an overall stimulation of
gene targeting by a DSB of over 40,000-fold.
A
C
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B
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P
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IR
E
S
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8
P
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K
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E
O
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e T
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et (A
658):
R
ep
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u
b
strate (R
S
2100):
W
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E
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lem
en
tary F
ig
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re 1
BD
0
.0
0
.5
1
.0
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.5
2
.0
0
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
1
3
1
4
1
5
Relative Rate of
Gene Targeting
S
to
p
-S
ce site
37
C
tru
n
cG
F
P
IR
E
S
C
D
8
R
ep
air S
u
b
strate (R
S
2700):
37
tru
n
cG
F
P
IR
E
S
C
D
8
W
P
R
EOran
g
e
R
2
293/A
658 w
ith
 S
ce A
lo
n
e
Green
O
ran
g
e
Green
D
S
B
-G
T
G
en
e T
arg
et
S
ce
E
ven
ts/ 10
6 cells
R
ate
1b
p
 in
sertio
n
N
o
29.5 +
/- 13.0
2.9 x 10
-5      
7 b
p
 in
sertio
n
N
o
1.5 +
/- 1.1
1.5 x 10
-6
35 b
p
 in
sertio
n
 (A
658)
N
o
0.71 +
/- 0.27
7.1 x 10
-7
35 b
p
 in
sertio
n
 (A
658)
Y
es
1600 +
/- 500
1.6 x 10
-3
F
o
ld
 S
tim
u
latio
n
B
y S
ce In
d
u
ced
 D
S
B
>
2000
Green
S
-G
T
O
ran
g
e
66 b
p
 in
sertio
n
 (Q
Q
R
8)
Y
es
1900 +
/- 250
1.9 x 10
-3
D
ay A
fter T
ran
sfectio
n
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
Fig. S1. GFP gene targeting system. A) Gene Targeting System.  The artificial gene
target (A658) consisted of a GFP gene mutated by a 35 basepair insertion which includes
a stop codon and a recognition site for the I-SceI endonuclease (Sce) (5’
TAGGGATAACAGGGTAAT  3’) at basepair 327 of the coding sequence.  The GFP
gene was driven by a hybrid cytomegalovirus enhancer/chicken β-actin promoter
(“CMV/CBA" or “CBA”).  The GFP gene was part of bicistronic transcript in which an
internal ribosomal entry site ("IRES") allowed translation of the human CD8α gene
(“CD8”).  The bicistronic message contained a Woodchuck post-trancriptional regulatory
element (“WPRE”) to increase messenger RNA levels (S2).  Finally, the locus contained
a gene with the phosphoglycerate kinase promoter (“PGK”) driving the neomycin
phosphotransferase gene (NEO) to allow selection by the antibiotic G418.  The repair
substrates RS2100 and RS2700 are also depicted.  They consisted of a GFP gene that has
been truncated at basepair 37 of the coding sequence and thus were missing the initiation
codon (“truncGFP”).  The truncated GFP gene was followed by the IRES-CD8 for
RS2100 or IRES-CD8-WPRE for RS2700 as in A658.  The A658 target gene was
introduced into 293 cells by electroporating 2 x 106 cells with 10 µg of supercoiled A658
plasmid DNA.  Cells were selected in 500 µg/ml G418 for two weeks.  Monoclonal cell
lines were made by picking individual colonies and identifying those with high surface
CD8 expression by staining with phycoerythrin-conjugated anti-CD8 antibody (BD
Biosciences, San Jose, CA) (293 cells normally do not express CD8).  Polyclonal cell
lines were made by purifying a population of cells using Miltenyi anti-CD8 microbeads
and a MACS mini-column (Miltenyi Biotec, Auburn, CA).  Gene targeting was measured
by transfecting 293/A658 cells with RS2100 with or without  a Sce expression plasmid
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
along with  a control plasmid (pON405) to determine the transfection efficiency.  We
used three different promoters to drive Sce expression: PGK, cytomegalovirus (“CMV”),
and CBA.  The cells were then incubated for 3 days and the percentage of GFP positive
cells measured by flow cytometry using a FACScan (BD Biosciences, San Jose, CA).
The gene targeting rate was determined by normalizing the measured percentage of GFP
positive cells to the transfection efficiency.  B)  Representative Flow Cytometry Plots of
Gene Targeting.  GFP positive cells were quantitated in region “R2” as depicted in the
right flow plot.  The left plot, “S-GT,” shows 293/A658 cells after transfection with
RS2100 alone.  The two GFP positive cells are circled and represent spontaneous gene
targeting events.   The middle plot, “DSB-GT,” shows 293/A658 cells after co-
transfection with RS2100 and CBA-Sce.  In this plot there are numerous GFP positive
cells in region R2.  The right plot shows 293/A658 cells after transfection with Sce
expression plasmid alone.  There are no GFP positive cells.  C)  Gene Targeting Rates in
293 Cells.  The results are shown as both the number of gene targeting events per million
transfected cells ("Events/106 cells") plus/minus one standard deviation and as an overall
rate.   The results are shown for four different gene targets.    In the "1bp mutation"
target, a nonsense mutation was created in the GFP gene at bp 321 of the coding region
that abrogates functional GFP expression.  For the “7 bp insertion” target, a 7 bp
sequence was inserted at bp 327 of the GFP coding region.  The gene target for the “35
bp insertion” was A658 and the target for the “66 bp insertion” was QQR8 (schematized
in Figure 1B).  The row labeled "Sce" shows whether Sce was co-transfected or not.
The column labeled "Fold Stimulation by Sce Induced DSB" was the stimulation of the
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
gene targeting rate on target A658 induced by expression of Sce.  D) Time Course of
Gene Targeting.  The relative rate of DSB-GT was normalized to day 3.
S
u
p
p
lem
en
tary F
ig
u
re 2
0
10,000
20,000
30,000
40,000
50,000
60,000
1
2
3
4
5
6
GFP Positive Cells Per
Million Transfected Cells
     2        1       2       1       2       1
  P
G
K
-S
ce    C
M
V
-S
ce   C
B
A
-S
ce
0.0
0.5
1.0
1.5
2.0
2.5
1
2
U
n
tran
scrib
ed
T
ran
scrib
ed
N
an
o
g
ram
s o
f R
S
2100
A
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0
100
200
300
400
500
N
an
o
g
ram
s o
f S
ce E
xp
ressio
n
 P
lasm
id
0.0
1.0
2.0
3.0
0
1000
2000
3000
4000
5000
H
o
m
o
lo
g
y L
en
g
th
 in
 B
asep
airs
B
E C
D
0.0
0.5
1.0
1.5
2.0
2.5
0
200
400
600
Relative Gene
Targeting Rate
Relative Gene
Targeting Rate
Relative Gene
Targeting Rate
Relative Gene
Targeting Rate
C
B
A
-S
ce
P
G
K
-S
ce
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
Fig. S2. Parameters regulating the rate of DSB-induced gene targeting. In these
experiments, transfections were performed by the calcium phosphate technique in 24-
well plates. In plots A-D, the rates of gene targeting were normalized to the standard
conditions of using 200 nanograms (ng) of RS2100 and 200 ng of PGK-Sce.  In
experiments where the amount of a transfected component was varied, the total DNA
amount was kept constant by adding pBSK(-) plasmid (Stratagene, La Jolla, CA).  A)
Gene Targeting Rate vs. Substrate Amount.  The results were normalized to the rate of
gene targeting obtained after transfecting 200 ng of RS2100.  B) Gene Targeting Rate vs.
Homology Length.  The results were normalized to the rate obtained with plasmid
RS2100.  The plasmid with 800 bp of homology (RS800) was missing the IRES-CD8
component of RS2100.  The plasmid with 2700 bp of homology (RS2700) is depicted in
Fig. S1A.  The plasmid with 4200 bp of homology (RS4200) had the addition of both the
WPRE and the PGK-NEO components to the 3’ end of RS2100.  A constant amount (200
ng) of each repair substrate was transfected but the relative rate of DSB-GT was
normalized to the molar amount transfected.  C) Gene Targeting Rate vs. Amount of Sce
Expression Plasmid Transfected.  The results were normalized to the rate of gene
targeting obtained when 200 ng of PGK-Sce was transfected. D)  Gene Targeting Rate vs.
Transcriptional Status of Repair Substrate.  “Untranscribed” was the rate of DSB-GT
using RS2100.  “Transcribed” was the rate of DSB-GT when the sense strand of RS2100
was transcribed using the CMV promoter (CMV-RS2100).  The rates were normalized to
the rate of gene targeting obtained using RS2100.  E) Optimization of Gene Targeting.
Columns labeled “1” are when Sce and RS2100 are on the same plasmid and columns
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
labeled “2” are when Sce and RS2100 are on separate plasmids.  30,000 GFP positive
cells per million transfected cells is equivalent to a gene targeting rate of 3%.
Abbreviations:  CBA, cytomegalovirus enhancer/chicken beta-actin promoter; PGK,
phosphoglycerate kinase promoter; Sce, I-SceI endonuclease, CMV, human
cytomegalovirus early enhancer/promoter.
Porteus and Baltimore, Chimeric Nucleases and Gene Targeting
References
S1. C. Deng, M. R. Capecchi, Molecular and Cellular Biology 12, 3365-3371 (1992).
S2. R. Zufferey, J. E. Donello, D. Trono, T. J. Hope, J Virol 73, 2886-92. (1999).


Chimeric nucleases stimulate gene targeting in human cells

NEW TECHNOLOGIES IN GENE AND CELL THERAPY 
transduced cells and therefore we hypothesize that the latent presence of CMV antigens in 
vivo will maintain the long-term presence of these T cells. These results demonstrate that 
redirection of virus specific T cells towards anti-leukemic reactivity can be an attractive 
strategy to use for clinical purposes. 
Abstract# 821 
Chimeric Nucleases Stimulate Gene Targeting in Human Cells. 
Matthew Porteus, David Baltimore*. Biology, California Institute of 
Technology, Pasadena, CA, USA. 
Gene targeting is a powerful technique to introduce genetic change into the genome of 
eukaryotic cells. It is widely used to create defined mutations in murine embryonic stem 
cells and theoretically could be used to create or repair mutations in somatic cells. In this 
way gene targeting could be a powerful form of gene correction type gene therapy. Despite 
its potential, gene targeting has not been widely used in somatic cells because of its low 
efficiency. We report on a system based on the correction of a mutated GFP gene that allows 
the efficient study of gene targeting in somatic cells. Using this system we show that gene 
targeting is stimulated over 2000-fold by the introduction of a DNA double-stranded break 
in the target locus (DSB-GT). We find that the rate ofDSB-GTcan be increased by increasing 
the amount of repair substrate, the amount of homology between the gene target and repair 
substrate, and by increasing the frequency of double-stranded break creation. When we 
optimize conditions for DSB-GT we obtain targeting rates of 3-5%. Finally, we show that 
chimeric nucleases, protein fusions between zinc finger DNA binding domains and the 
endonuclease domain of the Fokl restriction enzyme, can stimulate gene targeting in the 
genome of human somatic cells by several-thousand fold. Our data provides a paradigm for 
the use of gene targeting as a form of gene therapy for monogenic diseases. 
Abstract# 822 
Direct Delivery of Adenoviral Vectors to Malignant Cells Using 
Tumor-Specific CTL. Patricia N. Yotnda, Nicolas Charlet-Berguerand*, 
Barbara Salvodo*, Clio M. Rooney*, Malcolm K. Brenner. Center for Cell 
and Gene Therapy, Baylor College of Medicine, Houston, TX, USA. 
A major barrier to successful gene therapy of disseminated malignancy is our inability 
to generate targeted vectors that can cross multiple tissue boundaries to reach all tumor 
deposits. Specific cytotoxic T lymphocytes (CTL<), however, can be generated against 
tumor antigens, and in animal models and some human settings (Rooney et al Blood. 92: 1549-
55 l 998) these CTLs are able to cross tissue barriers and to specifically recognize tumor 
cells. We have attempted to combine the mobility and targeting functions ofT cells with the 
capacity of adenoviral vectors to efficiently transduce tumor cells and deliver oncolytic 
genes. As proof of principle, we genetically modified Epstein Barr Virus (EBV)specific-
CTLs (currently used for the treatment of EBY associated malignancies) to make them able 
to locally produce a therapeutic or lytic adenovirus when specifically activated by their 
target cells. The intent was to see whether these CTL would produce vector only on activation 
once they had encountered their tumor target, and to discover if the vector they produced 
would have functional activity. EBY specific CTLs were transduced with a Moloney based 
retrovirus encoding a CD40L promoter driving the El adenovirus gene. The CD40L 
promoter is strictly regulated by T cell activation and is functional only for a brief period 
during the T cell activation process. The CTL were also transduced by an El deficient 
chimeric Ad5F35 vector that we have previously shown is effective at transducing 
hemopoietic lineage cells that lack the conventional coxsackie adenovirus receptor (Yotnda 
et al, Hum Gene Ther.12:659-70 2001). We used Ad5/f35-GFP or Ad5/f35-TK to transduce 
the E l-CTLs (I 03 viral particles per cells). Twenty-four hours after transduction, 40% of the 
El-CTL expressed the Ad transgene. When these CTLs were co-cultured with A549 cells, 
which are highly sensitive to adenoviral transduction, less than 5% were transgene positive, 
indicating low baseline production of active adenovector by CTL "producer" cells. When 
autologous, but not allogeneic, EBY-infected B cells (EBV-LCL) were added to the cultures. 
>95% of A549 target indicator cells were transduced by day 4 of culture, indicating increased 
vector production following T cell activation. Of the autologous EBV-LCL in the culture, 
38% were transduced, even though these target cells are much more resistant than A549 
cells to adenoviral vector transduction. Vector production decreased over 2-3 days, falling 
to 5-10% of peak values by day 4, but could be increased upon restimulation of the CTLs 
by fresh autologous tumor cells (EBV-LCL). The virus production decreased to 5-10% 4 
days after the last stimulation. Directed delivery of adenoviral vectors to malignant cells 
using tumor-specific CTL may be a useful addition to cellular-based cancer immunotherapy. 
Abstract# 823 
Host Chromosomal Effects of Recombinant Adeno-Associated Virus 
Vector Integration In Vivo. Hiroyuki Nakai,' Eugenio Montini*, 2 Sally 
Fuess*,' Theresa A. Storm*,' Markus Grompe*, 2 Mark A. Kay*.' 
1Departments of Pediatrics and Genetics, Stanford University School of 
Medicine, Stanford, CA, USA; 'Departments of Moleuc/ar and Medical 
Genetics and Pediatrics, Oregon Health Science University, Portland, OR, 
USA. 
Recombinant adeno-associated virus (rAAV)° vector is an attractive viral vector to treat 
genetic diseases. Although extrachromosomal vector genomes are the most predominant 
form of vector DNA, we and others have established that rAAV vector does integrate into 
host chromosomes of hepatocytes in vivo. Although the integration efficiency is normally 
very low even when very high doses are used (Nakai et al., J. Virol. 75: 6969-6976, 2001. 
and in press), rAAV vector integration has raised the concern of insertional mutagenesis 
since: l) development of hepatic tumors was reported in rAAV-treated 
mucopolysaccharidosis type VII mice (Donsante et al., Gene Ther. 17:1343-1346, 2001) 
and 2) frequent chromosomal deletions were observed at rAAV integration sites in in vitro 
cultured cells (Miller et al.. Nat. Genet. 30: 147-148. 2002). However, the alteration of host 
219a 
chromosomal DNA as a result of vector integration in vivo is not known. As a first step 
toward elucidating the host chromosomal effects of integration in vivo, we attempted to 
isolate the whole rAAV proviral genomes together with flanking host chromosomal 
sequences using a hereditary tyrosinemia type I (HT!) mouse model with fumarylacetoacetate 
hydrolase (FAH) deficiency. In the absence of NTBC drug administration, these mice 
accumulate toxic tyrosine metabolites that result in a cell autonomous lethal phenotype. 
Thus. genetically corrected hepatocytes have a selective advantage and can repopulate the 
liver. We constructed an FAH-expressing rAAV shuttle vector whose vector sequences are 
then retrieved in bacteria. HT! mice were injected via the portal vein with 3x 10 11 particles 
of AAV-FAH, and in vivo selection (removal of NTBC) was started 6 weeks post-injection. 
Hepatocytes were isolated after an 8-week in vivo se1ectiun, transplanted into HTI mouse 
recipients ( l x l O' hepatocytes per mouse), selected again in vivo for 7 months, and then the 
livers were harvested for DNA extraction. The transplantation and second in vivo selection 
facilitated dilution of extrachromosomal genomes present in the liver samples. The whole 
proviral genomes were isolated from the liver samples together with adjoining cellular 
DNA with a standard plasmid rescue technique, and sequenced. To date, 12 proviral genomes 
were characterized and sequenced. The vector genomes were frequently deleted at both 
terminals and AAV-ITRs were completely deleted in some cases. rAAV integration commonly 
accompanied a host chromosomal DNA deletion (by 2, 4, 13, 21, 27, 44, 58, 119, 216, 274 
and 2107 base pairs). A small insertion of unknown origin of up to 4 bp at junctions was 
sometimes observed. There was no significant homology between vector and cellular DNA 
sequences, while there was some microhomology of up to 4 bp between them. Integration 
sites were randomly distributed in chromosomes (chr. #I, 2, 4, 8, 9, 11, 15, 19) and there 
appeared to be no hot spot for vector integration. Five out of 12 integrations targeted within 
predicted or known mouse genes. Because our study relied on in vivo selection and 
ultimately transgene expression, we cannot establish if these integration were representative 
of all r AAV integration events. However, for the purposes of gene therapy, elucidating the 
structures of proviral genomes and the sites where the expressible transgene inserts is an 
important parameter. This study is the first we are aware of that establishes the imperfect 
nature of rAAV vector integration in vivo. 
Abstract# 824 
Targeting CD19 with Genetically Modified EBV-Specific Human T 
Lymphocytes: The Role of the Antigen-Presenting Cell in Chimeric 
Receptor-Mediated T Cell Responses. Claudia Rossig*, 1 Sibylle 
Pscherer*, 1 Annette Baer*,' Josef Vormoor, 1 Cliona M. Rooney*,2 Malcolm 
K. Brenner, 2 Heribert Juergens* .1 / Department of Pediatric Hematology 
and Oncology, University Children's Hospital Muenster, Muenster, Germany; 
'Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, 
TX, USA. 
Primary T cells expressing chimeric receptors specific for tumor antigens have 
considerable therapeutic potential. Their successful therapeutic use has been limited because 
most tumor cells lack the costimulatory molecules essential for the induction and maintenance 
of a T cell response. B cells latently infected with Epstein Barr virus (EBV-LCL) are known 
to be excellent antigen-presenting cells that are rich in costimulatory molecule expression. 
Cell surface expression ofCDl 9 is shared between EBV-LCLand many B cell malignancies, 
including B cell precursor acute lymphoblastic leukemia (ALL). To investigate the role of 
costimulation for chimeric receptor-mediated T cell functionality, we generated EBY-specific 
cytotoxic T lymphocytes (CTLJ from four healthy seropositive donors and transduced them 
with a retroviral vector encoding the CD 19-specific chimeric receptor CD l 9-zeta. We then 
compared their capacity to respond to CD19+ tumor cells and to EBY-transformed CDl 9+ 
B cells. While nontransduced CTL were EBY-specific and HLA-restricted, lysing 36-78% 
autologous EBV-LCL in the absence of significant cytotoxicity against mismatched EBY 
or tumor targets, CD l 9-transduced CTL lysed both autologous (29-74%) and mismatched 
allogeneic EBV-LCL (46-73%) as well as the CDl9+ Reh leukemia cell line (37-44%) and 
primary ALL blasts (30-47% ), but not CD! 9-negative K-562 tumor cells (0-19% ). Whereas 
lysis of mismatched LCL and Reh cells was not significantly inhibited by HLA class 1 and 
II antibodies, up to 58% (Reh) and 49% (LCL) inhibition of lysis was obtained by 
preincubation with anti-CD19 monoclonal antibody, confirming a chimeric receptor-
mediated, non-MHC-restricted mechanism of recognition. Incubation of a clonal population 
of CD l 9-transduced CTL with CD l 9+ tumor targets failed to elicit a proliferative response, 
as demonstrated by [3H]thymidine incorporation. However, following coincubation with 
CD19+ mismatched allogeneic LCL, [3H]thymidine uptake was 54-80% of that obtained 
by native receptor stimulation using autologous LCL. In contrast, the failure of tumor cells 
to induce T cell proliferation could not be overcome by CD28 costimulation provided by 
antibody crosslinking. Hence, CD! 9-zeta-expressing CTL could not be maintained in 
culture for longer than three weeks when stimulated with CD l 9+ tumor cells even in the 
presence of immobilized anti-CD28 antibody. By contrast, co-culture with irradiated 
mismatched allogeneic LCL promoted expansion not significantly different from that 
observed with autologous LCL. Thus, chimeric receptor-mediated T cell proliferation and 
expansion are significantly enhanced when the target antigen is expressed in the cellular 
microenvironment of a professional antigen-presenting cell. By delivering abundant and 
diverse costimulatory signals along with the chimeric receptor signal, adoptively transferred 
CD 19-zeta-expressing EBV-CfL may induce long-lasting immune control of B cell precursor 
leukemia. As the modified cells maintain their native specificity for EBY, this strategy may 
be exceptionally useful for patients recovering from allogeneic stem cell transplantation, 
thus preventing both EB V-associated lymphoproliferative disease and leukemia relapse. 


https://authors.library.caltech.edu/51855/7/Porteus.SOM.pdf

Chimeric nucleases stimulate gene targeting in human cells

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