Gene Therapy (2007) 14, 173–179
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ORIGINAL ARTICLE
Third strand-mediated psoralen-induced correction
of the sickle cell mutation on a plasmid transfected
into COS-7 cells
Y Varganov, O Amosova and JR Fresco
Department of Molecular Biology, Princeton University, Princeton, NJ, USA
A significant level of correction of the mutation responsible
for sickle cell anemia has been achieved in monkey COS-7
cells on a plasmid containing a b-globin gene fragment. The
plasmid was treated in vitro with a nucleic acid ‘third strand’
bearing a terminal photoreactive psoralen moiety that binds
immediately adjacent to the mutant base pair. Following
covalent attachment of the psoralen by monoadduct or
diadduct formation to the mutant T-residue on the coding
strand, the treated plasmid was transfected into the cells,
which were then incubated for 48 h to allow the cellular DNA
repair mechanisms to remove the photoadducts. Upon reisolation and amplification of the transfected plasmid, sickle
cell mutation correction, as determined by sequence analysis
of both complementary strands, was established in a full 1%.
This result encourages extension of the approach to correct
the mutation directly on the chromosome.
Gene Therapy (2007) 14, 173–179. doi:10.1038/sj.gt.3302850;
published online 31 August 2006
Keywords: sickle cell anemia; b-globin gene; psoralen-mediated gene repair; COS-7; sickle cell mutation
Introduction
The prevailing approach to gene therapy for diseases due
to point mutations has been to try to add the wild-type
antecedent of the mutant gene using a viral vector that
integrates into the genome.1–3 While this type of scheme
is intrinsically reasonable, the results of such efforts have
been fraught with problems because of the difficulty in
assuring integration of the replacement gene at the
correct chromosomal site and its long term expression.
This is particularly the case when the regulation of a
gene is complex. Hence, the prospect for correcting a
point mutation directly in the endogenous gene on the
chromosome is very attractive. Previous attempts to do
so have been made using ‘chimeraplasty’, that takes
advantage of cellular mismatch repair coupled to
homologous recombination using chimeric RNA–DNA
strands.4–7 However, the resultant frequency of mutation
correction has been highly variable;8,9 more consistent
results were obtained when deoxyoligonucleotides
instead of chimeric strands were used to induce sitespecific base changes.10
We have been exploring an alternative approach to
correct a mutant base pair directly on the chromosome.
This approach uses selective chemical modification of the
mutant base pair to trigger error prone DNA repair
mechanisms that tend in some cases to insert the desired
This is paper IV in the series ‘Repair of the Sickle Cell Mutation’, of
which the last is reference 27.
Correspondence: Professor JR Fresco, Department of Molecular
Biology, Princeton University, Princeton, NJ 08544, USA.
E-mail: [email protected]
Received 16 February 2006; revised 20 July 2006; accepted 20 July
2006; published online 31 August 2006
wild-type base pair.11–15 The demonstration target we
selected for this approach is the mutation responsible for
sickle cell anemia, a heritable hematological disease
which results from an A T-T A transversion in the
sixth codon of the b-globin gene. This transversion
changes a GAG codon to GTG, resulting in a single
amino-acid change from Glu to Val.16 The resultant
enhanced hydrophobicity on the protein surface leads to
hemoglobin polymerization that alters red cell shape,
with many pathological consequences including chronic
hemolytic anemia and painful vasoocclusive events. As
the presence of only one sickle cell allele does not cause
severe disease pathology, yet confers resistance to
malaria, the mutation has become widespread wherever
malaria is prevalent.17
In order to revert this mutation, we designed a
methodology for selectively modifying the mutant T
residue with psoralen delivered to the mutation site by a
deoxyoligonucleotide which binds with extremely high
specificity immediately downstream from the mutation
site by a combination of third strand-binding via triple
helix formation and strand invasion via complementary
base pairing.18,19 With an upstream 30 -end which is
covalently linked by a deliberately short linker to a
photoreactive intercalating psoralen moiety, the third
strand binds via triplex formation to the first seven
purine residues of the target duplex in the antiparallel
G/T motif;20 it then switches to the parallel C/T motif21
to bind to the purine-rich residue sequence in the other
strand of the target duplex; finally, it forms six
complementary base pairs with one of the two duplex
strands, displacing the other by strand invasion.22
(Figure 1). The uniqueness of the b-globin gene target
in the human genome was confirmed by a careful
Sickle Cell mutation repair on a plasmid in COS-7
Y Varganov et al
174
Figure 1 Interaction of the psoralen delivery strand (solid dashes connect its residues) with the target sequence immediately downstream of
the sickle cell mutation in the b-globin gene. The psoralen moiety that modifies the mutant T residue is linked to the 30 end of the delivery
strand. The dots indicate the hydrogen bonds of the duplex base pairs, while the slanted dashes represent hydrogen bonds between the
delivery strand bases and the target bases in the duplex.
sequence search. Moreover, the specificity of the psoralen
delivery strand for that target is consistent with what is
known of the inherent great specificity of third strandbinding.23–25 Upon irradiation, the photoreactive psoralen moiety forms a photoadduct to the mutant T residue
in the coding strand (Figure 2).18,19,26,27 The psoralen
photoadduct is recognized as DNA damage and is
removed by the DNA repair mechanisms of the cell,
which often replace the psoralen-T adduct with an A
residue, resulting in the desired mutation correction.12,14
To explore the feasibility of the triplex-mediated
approach for the desired mutation correction, experiments were undertaken under tractable circumstances
with a plasmid bearing a fragment of the sickle cell
b-globin gene sequence (Figure 3). A plasmid photomodified in vitro and purified from nonmodified plasmid
before transfection was used for this purpose. Thus, only
photomodified plasmid was transfected into cells. Consequently, only plasmid whose psoralen photoadducts
were removed by the DNA repair machinery could be
replicated. In this way, the maximum repair potential
of directed psoralen modification was evaluated. The
presence of any nonphotomodified plasmid would
obscure the true repair potential inherent in the cells.
In what follows, we describe the binding of the psoralendelivery strand to the plasmid in vitro, and the significant
level of mutation correction that occurs in vivo when the
treated plasmid is transfected into COS-7 cells.
Figure 2 Major photoproducts formed by psoralen upon irradiation of the bound delivery strand. The principal psoralen monoadduct (MA) that forms both upon UVA and visible light irradiation
is indicated by a rectangle, while the only crosslink (XL, only
formed upon UVA irradiation) observed is shown by the shaded
ellipse.
Results
Plasmid target design
The sickle cell b-globin target duplex (Figure 1) was
inserted into a shuttle vector pEGFP-N1 (Clontech, Palo
Alto, CA, USA) capable of replication in both mammalian and bacterial cells. To enable separation of plasmid
with covalently bound delivery strand from unmodified
plasmid, a unique BaeI recognition site overlapping the
delivery strand target sequence was created by inserting
an extra six base pair sequence upstream of the b-globin
sickle cell gene target. BaeI can cleave both strands of the
plasmid DNA upstream and downstream of its recognition
sequence [(10/15)ACNNNNGTAYC(12/7)]28 (Figure 1).
The delivery strand directs the psoralen moiety to form
a photoadduct (either monoadduct or a crosslink with
the sickle cell mutant T residue (Figure 2). The UVA
(365 nm) and visible light (419 nm) irradiation protocols
Gene Therapy
Figure 3 The plasmid with the sickle cell b-globin insert showing
the DdeI, BaeI and BanI restriction sites. The sickle cell target is
indicated by the diagonals. The bound psoralen delivery strand,
indicated in gray, blocks the BaeI recognition site, preventing
linearization of the plasmid by this restriction enzyme. An
additional DdeI site is generated when the sickle cell mutation is
corrected.
employed in this work27 were designed and tested on a
linear model system,18,19 and the photoproduct yield and
variety were similar for the plasmid system. Thus, the
UV irradiation protocol leads predominantly to formation of both monoadducts to the mutant T residue (35%
Sickle Cell mutation repair on a plasmid in COS-7
Y Varganov et al
of the total photoproducts) and crosslinks between this T
residue and one of the C residues on the opposite strand
(45% of the total photoproducts); minor quantities of
other photoproducts, at a T residue of the previous codon
(o5%) and at C residues on the opposite strand (o5%)
also form.19 In contrast, visible light irradiation results
in monoadduct formation exclusively; no crosslinks are
formed.27 The mutant T residue is in the first base pair of
the unique BaeI recognition site; covalent binding of
the delivery strand to this residue and downstream
of the BaeI recognition site prevents BaeI endonuclease
from cleaving the plasmid (Figure 3). Hence, plasmids
with covalently bound delivery strand cannot be
linearized by BaeI and remain supercoiled; they are
separated from unmodified plasmids by agarose gel
electrophoresis as they migrate faster than the linearized
plasmid. In fact, the supercoiled plasmid resistant to
BaeI cleavage was shown by bandshift assay to be
almost fully (98% in different experiments) photomodified (Figure 4). This plasmid population was used
for transfection.
Delivery strand binding to the plasmid and
photoproduct yield
Delivery strand binding and psoralen photoproduct
formation was monitored by bandshift of a 252 bp BanI
restriction fragment containing the target (see Figure 4).
To enable quantitation, this fragment was labeled with
DNA polymerase, which fills the recessed (sticky) end
of the duplex without modifying the exposed 50 -end
of the delivery strand. Kd for delivery strand binding to
the plasmid is B300 nM. Polyethyleneglycol (PEG) was
added to the plasmid/delivery strand mixture to mimic
intracellular macromolecular crowding,29 resulting in
80% photomodified plasmids compared to B55% without PEG.
Figure 4 PAGE analysis showing bandshift of the target-containing BanI fragment upon covalent binding of the psoralen delivery
strand to the plasmid and enrichment of the delivery strand-bound
fraction after BaeI restriction. (A) No delivery strand; (B) covalently
bound delivery strand; (C) BaeI-protected plasmid fraction. Band 1:
shift of the 252 bp BanI fragment upon delivery strand binding;
band 2: 252 bp BanI fragment; band 3: 154 bp BanI–BaeI fragment
containing target sequence. Note that the gel-purified supercoiled
fraction of the plasmid after BaeI (C) virtually eliminates band 3,
which corresponds to the target-containing BaeI–BanI fragment
without attached delivery strand.
175
Transfection of the photomodified plasmid into Monkey
COS-7 cells
Monkey COS-7 cells were selected for these experiments
because of the extensive knowledge of their ability to
process third strand-directed psoralen photoadducts.12–14
Thus, nucleotide excision repair has been shown to be
responsible for repairing bulky DNA adducts. Psoraleninduced mutagenesis has also been found to depend on
whether the photoproduct is a monoadduct or a crosslink and even on the size of the psoralen-delivery
strand.11,12,30,31 Bound but not covalently attached third
strands are also recognized as DNA damage and are
mutagenic in these cells.32 Particularly appropriate for
our effort to correct the sickle cell A T-T A transversion, psoralen adducts at T residues preferentially induce
T A-A T transversions.13,14 It is important to note,
however, that the foregoing observations were made
using a simple homopurine strand with the psoralenlinked to its 50 -end. In the present work with the sickle
cell target, we designed a complex delivery strand that
binds to alternate strands of the target duplex in two
different motifs (first G/T antiparallel and then pyrimidine/parallel), followed by a strand-invading element;
in this case the psoralen is linked to the 30 -terminus.18
Transfection conditions and plasmid expression
COS-7 cells were transfected with DNA photoproducts
using Tfx20 transfection reagent (Promega) and the
Promega protocol for transfection of adherent cells.
Based upon the amount of plasmid DNA, the plasmid
transfection efficiency determined by flow cytometry,
and the number of cells used for transfection, it is
estimated that there are B105 plasmid copies per cell,
which is consistent with other data on transfection of
mammalian cells with GFP-expressing vectors.33
The same plasmid subjected to the same irradiation
and purification steps in the absence of the psoralendelivery strand, was used as a control in all experiments.
It is noteworthy that delivery strand attachment
results in a significant delay in GFP expression (Figure
5). In addition, the eventual number of green cells was
significantly less for the photomodified plasmid than for
the control, which was transfected with the same amount
of plasmid (Figure 5). It seems unlikely that covalent
binding of the delivery strand to the supercoiled plasmid
should affect transfection efficiency, since delivery strand
Figure 5 Photomicrographs at the same magnification of GFP
fluorescence of COS-7 cells 46 h after transfection. The fields shown
represent the same cell density treated with the same amount of
transfecting plasmid. (a) Plasmid with the psoralen delivery strand
covalently linked by irradiation with 419 nm light. (b) Control
plasmid irradiated with 419 nm light in the absence of the psoralen
delivery strand.
Gene Therapy
Sickle Cell mutation repair on a plasmid in COS-7
Y Varganov et al
176
binding does not change plasmid conformation or alter
its interaction with the transfection reagents. It is more
reasonable to think that expression of plasmid-encoded
GFP is delayed until psoralen damage is repaired.
Screening for correction of the sickle cell mutation
After replication in COS-7 cells, plasmid DNA was
purified and used to transform Escherichia coli. Previously,12,14 DpnI digestion was used to eliminate
unreplicated plasmid molecules prior to E. coli transformation. To evaluate the input from the residual unreplicated plasmid in our experiments, we transformed
the photomodified plasmid into K562 cells, in which the
pEGFP-N1 vector cannot be replicated. We, therefore,
compared the number of E. coli transformants obtained
after standard plasmid purification from transfected
COS-7 and K-562 cells after 48 h of incubation. The
plasmids reisolated from K562 cells yielded less than five
clones, whereas the same quantity of plasmid transfected
into COS-7 cells and reisolated generally gave 200–500
E. coli transformants. For this reason, the potential input
from unreplicated plasmids was considered negligible,
and the DpnI digestion step was omitted.
As correction of the sickle cell mutation results in
a new DdeI restriction site (Figure 6a), a DdeI restriction
Figure 6 Screening assay for mutation correction and other
mutational events based on DdeI digestion. (a) Mutation correction
results in an additional DdeI restriction site and leads to shortening
of the corresponding restriction fragment from 679 to 651 bp, and
therefore an increase in its mobility. (b) DdeI digestion patterns
observed for the sickle cell mutation (SC), after its correction (W)
and for other mutational events (M). Other restriction enzyme
digests (BanI, HaeIII – not shown) display mutant restriction
patterns only in the same clones as DdeI, suggesting that DdeI
screening is comprehensive.
analysis was used as an initial screening for mutation
correction. This assay was employed after plasmid DNA
was replicated in the COS-7 cells, amplified in E. coli and
then purified from bacterial clones. The assay depends
upon a reduction in the size of one of the existing
fragments. As DdeI has another restriction site 28 bp
upstream from the mutation site, the same assay also
serves to indicate whether any large deletions or
translocations have occurred in the region surrounding
the mutation (Figure 6b).
Frequency of psoralen adduct-induced correction
of the sickle cell mutation
In each experiment, we first screened plasmid DNA from
B200 clones using the DdeI restriction assay. Clones
displaying faster mobility of the target DdeI fragment
(indicative of sickle cell mutation correction) were
further analyzed by sequencing in both directions using
primers at residues 426 on one strand and 822 on the
other (the target is located between residues 634 and
670). It is important to note that in addition to the desired
T-A transversion at the sickle cell mutation site, other
changes in the DdeI restriction patterns were sometimes
observed (Figure 6b), presumably indicative of additional mutagenesis as a consequence of covalent attachment of the delivery strand.
The cumulative results of two types of experiments,
one with UV irradiation and the other with visible light
irradiation, are given in Table 1. Plasmid without an
attached delivery strand that otherwise underwent the
same experimental steps (irradiation, supercoiled plasmid purification by gel-electrophoresis and transfection)
served as one control. When the control plasmid was UVirradiated for the required 20 min, a 3.4% background
rate of nonspecific mutagenesis was observed. In contrast, after the required 4 h of visible light irradiation, no
mutations were detected. This difference is consistent
with the known mutagenicity of UV irradiation of DNA,
which results in error-prone repair of T–T dimers of
various types.34
As an additional control, plasmids with covalently
attached delivery strand were transformed directly in
E. coli in order to establish that the E. coli amplification
step does not contribute to the overall mutagenesis
or mutation correction. In fact, no mutation correction
or any other sequence changes were detected. This lack
of mutagenesis in the E. coli control is consistent with
what is known about the processing of psoralen adducts
in bacterial cells, that is, that activation of SOS-repair
by UV-irradiation is crucial for psoralen photoadduct
Table 1 Sickle cell mutation correction and nonspecific mutations in COS-7 cells after third strand delivered psoralen photoproduct
formation on a plasmid in vitro
Host cells
Psoralen delivery
strand
Type of
irradiation
Number of
clones analyzed
Number of
corrected clones
Number of other
mutant clones
COS-7
COS-7
+
+
UV
VIS
273
328
3 (1.1%)
3 (0.9%)
27 (9.9%)
51 (15.5%)
CONTROLS
COS-7
COS-7
E. coli
+
UV
VIS
UV
208
465
100
0
0
0
7 (3.4%)
0
0
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Y Varganov et al
removal.35 As the E. coli were not irradiated, the clones
obtained after E. coli transformation with photoadductcontaining plasmid most probably result from the trace
amount of unmodified plasmid. More important, this
result shows that all the observed mutation correction
occurs in the COS-7 cells.
Against these controls, a total of six clones were found
in which the sickle cell mutation was corrected without
any other changes in the plasmid sequence, that is, 1%;
three in 273 clones after UV-irradiation and three in 328
after irradiation with visible light. Covalent binding of
the delivery strand by irradiation also leads to other
types of mutations, including deletions around the
mutation site and long-range translocations. In all, 9.9%
of such mutations were observed after UV, and 15.5%
after visible light irradiation. This level of cumulative
mutation induction is much higher than the rate
observed for psoralen-triggered triplex-mediated mutagenesis in COS-7 cells previously reported,12,14,32 o1%
and B5% for visible light and UV irradiation, respectively. It is also B1000-fold higher than the rate of
mutation induced by noncovalently attached triplexes.32
Interestingly, whereas mutation correction was essentially the same by both irradiation protocols, the fraction
of undirected mutations was surprisingly greater with
visible than with UV light. Both monoadducts and
crosslinks greatly stabilize the triplex (by converting
the duplex-third strand interaction from a bimolecular
into a unimolecular one). However, while monoadduct
formation should not prevent duplex strand separation,
crosslink formation does; therefore, it is possible that
different DNA repair mechanisms are activated to deal
with the two types of damage, and the degree to which
they are error-prone may differ.
There are several possible explanations for the
differences between the results presented here and those
reported by others. One explanation for our generally
higher mutation rate, including mutation correction, may
be our careful purification of the plasmids bearing the
covalently attached delivery strand. In fact, the numbers
reported here represent a direct measure of the frequency
of errors introduced by the DNA repair system when
correcting DNA damage in the form of psoralen
photoproducts. Obviously, the presence of unmodified
plasmids would reduce the true proportion of induced
mutations. Alternatively, overall higher mutagenesis that
includes many long-range mutational events could be
the result of our delivery strand design in which its third
strand binding component hydrogen bonds first to one,
then to the other strand of the duplex and so serves as
a ‘clamp’ that prevents the strand unwinding essential
for adequate functioning of the DNA repair enzymes. In
contrast, most previous studies used third strands that
are directed to residues on only one strand of the target
duplex. In addition, whereas our screen recognizes
mutations throughout the plasmid, the screen employed
by Glazer et al., for example, was limited to changes in
the Sup F gene component of their construct.12,14,32
Discussion
We here have demonstrated relatively efficient correction
of the sickle cell mutation using the endogenous cellular
DNA repair machinery activated by site-specific DNA
damage inflicted on the mutant T residue in a plasmid
by psoralen brought there by a highly sequence-specific
deoxyoligonucleotide delivery strand. The specificity of
that delivery is determined by an uncommon combination of third strand-binding elements and a strandinvading one, which together fix the site of psoralen
delivery. Photoadducts of the delivery strand were
formed on the plasmid in vitro, and the resultant
modified plasmid was transfected into cells so that the
efficiency of such photomodification in vivo would not be
a factor in determining the rate of mutation correction.
In this way, the resultant correction could be unambiguously linked to the photoadduct formation. The 1% rate
of correction achieved, as confirmed by sequencing, is
much higher than in previous attempts to use psoralen
photoproduct formation as a mutagen in the Sup F gene
within a plasmid.12,14 It is also worth noting that the
nonspecific mutagenesis we have found need not be a
concern for the therapeutic application of our approach,
since a selection step can be used to eliminate them. A
level of 1% mutation correction coupled to such selection
may therefore be considered of ‘therapeutic efficiency’.
This result encourages us to use the same delivery strand
to achieve correction of the sickle cell mutation directly
on the chromosome.
177
Materials and methods
Delivery strand binding to plasmid
Psoralen-delivery strand binding to the plasmid was
performed in vitro in 0.1 M sodium acetate-pH 5.0/0.01 M
magnesium acetate and 20% PEG 1000. Pre-warmed
(651C, 5 min) delivery strand stock solution was added to
plasmid maintained at 231C; final concentrations were
200 nM plasmid and 10 mM delivery strand, well above
the Kd value. Complexes were incubated overnight at
231C and then at 41C for 3 h, before irradiation either
with UVA light (365 nm, 30 min) to mainly produce
crosslinks or with visible light (419 nm, 4 h) to produce
monoadducts exclusively.27 The resultant covalent complexes were purified from free delivery strands using
a Qiagen PCR purification kit.
Isolation of plasmid with covalently bound third strand
In order to separate photoproduct-containing DNA from
unmodified plasmid, the irradiated mixture was digested by BaeI for 1 h at 201C, conditions that maximize
digestion of unprotected restriction site while minimizing cleavage of photoproducts. Following restriction, the
DNA was fractionated on 1% agarose gel (TBE, 75 V, 3 h).
The band containing supercoiled photo-modified plasmid was excised from the gel, and a QIAquick Gel
Purification kit (QIAGEN) was used to purify the
plasmid.
Electrophoretic bandshift analysis of BanI digestion
fragments showed that the DNA so isolated contained
o2% of unmodified plasmid and was overwhelmingly
supercoiled and therefore suitable for subsequent transfection into mammalian cells.
Transfection of COS-7 cells
Typically, the COS-7 cells were plated 24 h before
transfection on six-well cell culture plates (Falcon),
B2.5 105 cells/well. At this density, cells are B80%
Gene Therapy
Sickle Cell mutation repair on a plasmid in COS-7
Y Varganov et al
178
confluent on the day of transfection. Tfx20 (1.6 mg) was
resuspended in 400 ml of nuclease-free water the day
before transfection. For each well, 2–3 mg of plasmid
DNA in o10 ml H2O was added to prewarmed to 371C
DMEM (ATCC) Serum-free medium (0.9 ml/well) in a
sterile tube and vortexed. Tfx20 reagent (4.5 ml for each
mg DNA) was added, and the mixture was immediately
vortexed. The reagent/DNA mixture was incubated for
10–15 min at 231C. The media was then removed from
the cells and 0.9 ml/well of the reagent/DNA mixture
in serum-free media was added.
Plates were incubated at 371C for 1 h, and then the
cells were gently overlaid with complete cell culture
medium containing FBS (2 ml/well) and returned to the
371C incubator. After 3–4 h, half the medium in the wells
was carefully replaced with fresh medium to reduce the
toxicity of the Tfx reagent. The next day, plates were
checked for appearance of GFP in the cells. Cells were
harvested 48 h after transfection. For harvesting, media
was removed from the wells, cells were gently rinsed
with sterile PBS, trypsinized (0.25% trypsin with EDTA
(Invitrogen) 371C, 10 min) and collected in a sterile tube.
After centrifugation, the cell pellet was washed 3 times
at 221C with sterile PBS, and then frozen at 201C in
preparation for plasmid extraction.
Plasmid DNA recovery
Replicated plasmid was recovered from COS-7 cells
using a Qiagen DNA purification kit and transformed by
electroporation into E. coli DHa5 cells for clonal analysis.
Acknowledgements
This work was supported by NIH Grants RO1 HL063888
and 5R33 CA088547. We are grateful to Dimitri Klimov
for technical assistance.
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