Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead

Abstract

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Molecular Therapy vol. 18 no. 6 june 2010 1061
© The American Society of Gene & Cell Therapy commentary
1Department of Medicine, Stanford University
School of Medicine, Stanford, California, USA;
2Department of Medicine, University of Califor-
nia San Diego School of Medicine, La Jolla, Cali-
fornia, USA; 3Department of Radiology, Stanford
University School of Medicine, Stanford,
California, USA; 4Institute for Stem Cell Biology
and Regenerative Medicine, Stanford University
School of Medicine, Stanford, California, USA
Correspondence: Joseph C. Wu, 300 Pasteur
Drive, Grant S140, Stanford, California
94305, USA.
E-mail: joewu@stanford.edu
See page 1192
Gene Correction in Human Embryonic
and Induced Pluripotent Stem Cells:
Promises and Challenges Ahead
Kazim H Narsinh1,2,3 and Joseph C Wu1,3,4
doi:10.1038/mt.2010.92
The sequencing of the human genome
has greatly facilitated our ability to iden-
tify the gene candidates that are critical for
developmental regulation and disease pro-
gression. However, investigators seeking to
use such data to gain a better understanding
of the developmental biology and thera-
peutic potential of pluripotent stem cell
types may be frustrated by the limitations
of current genetic engineering techniques.
Targeted genomic manipulation of mam-
malian cells is inecient, and unintended
eects of such manipulation on the cells’
developmental and proliferative potential
remain uninvestigated. To this end, the
recent demonstration of targeted gene cor-
rection in human induced pluripotent stem
cells (hiPSCs) before and aer reprogram-
ming, as reported in this issue of Molecular
erapy, warrants special mention.1
e successful isolation of human em-
bryonic stem cells (hESCs) has enabled the
implementation of renewable generation
of a wide variety of cell types in regenera-
tive medicine approaches.2 e prospect of
implementing cell replacement therapies
for diverse pathologies, including neuro-
degeneration, cardiovascular disease, and
diabetes, is now conceivable owing to the
ability to generate large quantities of func-
tional dierentiated cell types. However,
the use of hESCs remains controversial, and
cell transplantation therapy as a eld still
faces major obstacles such as acute donor
cell death, low engrament rates, immune
rejection, and tumorigenicity,3,4 which have
impeded eorts toward clinical translation.
e recent development of hiPSCs has
oered exciting solutions to allay the ethical
concerns and mitigate the possibility of im-
mune rejection by providing a mechanism
for the creation of “patient-specic” pluri-
potent stem cells from somatic cell types.5,6
It is possible to generate hiPSCs without de-
stroying human embryos, and autologous
hiPSC gras have the potential benet of
being less likely to provoke an immune re-
sponse. Nevertheless, challenges such as low
derivation eciency and exposure to viral
factors during the reprogramming process
remain to be solved, but new advances have
recently been made.7
Although the utility of gene target-
ing in hESCs has been conrmed by sev-
eral reports,8,9 these techniques remain
unimplemented on a large scale. By con-
trast, relatively facile reprogramming of so-
matic cell types from virtually any genetic
background into hiPSCs has enabled the
generation of myriad cell lines containing
disease-conferring mutations.10–12 e use
of these hiPSCs and their derivatives for
disease modeling is expected to signicantly
enhance investigators’ ability to probe the
mechanics of a multitude of pathologies. As
the repertoire of genetically diverse hiPSC
lines expands, the ability to site-specically
modify the genome in order to alter
genes of interest will become increasingly
important. To realize the full potential of
hESCs and hiPSCs, ecient methods for
genetic modications are needed.
Techniques for precise targeting of de-
ned modications to the genome rely on
activation of the cell’s homologous recom-
bination (HR) machinery (Figure 1). In the
presence of a donor DNA sequence sub-
stituting for the sister chromatid, HR can
yield targeted clones with an appropriate
experimental system. However, the donor
DNA oen enters other pathways of DNA
repair, most notably undergoing nonho-
mologous end joining at a higher frequency
than HR. erefore, targeted HR is a rela-
tively inecient process, occurring at a
frequency between 10−5 and 10−7 in treated
mammalian cells.13 is process may be en-
hanced by some orders of magnitude with
the introduction of a DNA double-strand
break at the genomic locus of interest.14
Other strategies for enhancing HR involve
augmenting the activity of nucleo lament
proteins RAD51 and RAD52, either by
transient overexpression or via small mol-
ecules,15 although some of these studies
have yielded conicting results. Further in-
vestigation into the cellular determinants of
the choice between the intended (i.e., HR)
versus unintended (e.g., nonhomologous
end joining, single-strand annealing,
microhomology-mediated end joining,
and random integration) target is required
to yield satisfying insight into potential
methods for improving the relative
frequency of HR.
Gene-correction eciency may vary
considerably, depending on the genomic
context of the target locus, so comparing the
results of various studies is challenging. For-
tunately, the most frequently targeted gene
in hESC studies is the HPRT1 locus encod-
ing hypoxanthine-guanine phosphoribosyl
transferase (HPRT). HPRT is a key enzyme
in the purine salvage pathway, the decien-
cy of which is manifested as Lesch–Nyhan
syndrome in patients. Also, because HPRT1
is located on the X chromosome, a single
gene targeting event can lead to a complete
loss of function and 6-thioguanine resis-
tance in XY cells, providing a convenient
culture system for screening correctly tar-
geted cells from random integrants. Initial
attempts at HR in hESCs at the HPRT1
and POU5F1 loci yielded successful gene

1062 www.moleculartherapy.org vol. 18 no. 6 june 2010
© The American Society of Gene & Cell Therapy
commentary
targeting at frequencies on the order of
10–6. e percentage of selection marker–
resistant clones that actually contained the
intended gene correction as conrmed by
Southern blotting, referred to as the target-
ing eciency, varied between 27% and 40%,
depending on the targeting construct used.8
Several studies have now validated the
use of zinc-nger nucleases (ZFNs) to in-
duce a chromosomal double-strand break at
the site of the targeted gene, which greatly
enhances the frequency of the homology-
directed repair.14 is approach requires the
engineering of zinc-nger DNA-binding
domains to recognize a DNA sequence
of interest. e DNA-binding domains
are then fused to the nuclease domain of
the FokI endonuclease to enable cleavage
at a specic genomic locus. However, the
potential remains for o-target cleavage
events at chromosomal sequences bearing a
similarity to the ZFN target site. One group
has demonstrated the use of ZFNs in con-
junction with integrase-defective lentiviral
(IDLV) vectors to achieve high gene target-
ing eciencies. Increasing concentrations
of IDLVs delivering ZFNs that target the
CCR5 locus resulted in 0.5–5.3% of trans-
duced cells expressing the green uorescent
protein (GFP) transgene. However, this par-
ticular study lacked Southern blot analysis,
which would have conrmed whether gene
targeting had generated a single-copy GFP
insertion without any random integration
elsewhere. Also, the increase in viral titer
concomitantly increased the frequency of
mismatch mutations from 4% to 28% in the
GFP+ clones.16
Subsequent studies have demonstrated
the use of ZFNs to successfully target the
POU5F1, PIGA, PITX3, and AAVS1 loci in
hESCs and hiPSCs.17,18 Because techniques
employing ZFNs require concomitant deliv-
ery of three DNA constructs (two to encode
the ZFN pair and one donor DNA sequence),
reported eciencies may be lower for these
studies, which employed electroporation in
place of viral vectors. Using a nonviral elec-
troporation system, these studies demon-
strated successful gene targeting for a variety
of loci in hESC and hiPSC lines on the order
of 10−5. Targeting eciency varied from 8%
to 100%, depending on the ZFN pair used
and genomic locus targeted. Notably, these
studies utilized ZFNs designed by dierent
groups using entirely dierent methods of
validation. However, a ZFN-based gene-
correction approach requires extensive
design and validation of new zinc-nger
binding domains for each target locus. In
addition to verifying proper targeting when
using ZFNs, it is of paramount importance
to limit cells’ exposure to ZFN-expressing
plasmids to prevent o-target cleavage and
even potential integration of ZFN-express-
ing DNA into the genome.
Viral vector delivery systems have also
been implemented in order to overcome the
low transient transfection eciencies ob-
served using electroporation in hESCs. Ad-
enoviral and lentiviral vector gene delivery
systems are well established in their ability
to transduce a broad range of cell types at
high eciency, although safety concerns re-
garding random chromosomal integration
have prevented their clinical use.19 Modi-
ed viral vectors that have been developed
to allay such concerns include AAV, helper-
dependent adenovirus, and IDLV. Helper-
dependent adenoviruses, for example, have
been modied by deletion of all viral genes
from the vector genome, resulting in re-
duced cytotoxicity and an expanded clon-
ing capacity to allow for insertion of larger
targeting constructs. ese properties have
been exploited by one group to achieve suc-
cessful gene correction at the HPRT1 locus
in hESCs at a frequency of ~10–6, with a tar-
geting eciency of ~40% (ref. 20).
It is within this context that Khan et al.,1
in this issue of Molecular erapy, make a
signicant step forward. Remarkably, the
investigators observed successful insertion
of a neomycin-resistance cassette into the
HPRT1 locus in ~10–5 of hESCs and hiPSCs
aer transduction with AAV gene target-
ing vectors. Importantly, their work is also
the rst demonstration of successful gene
targeting in dierentiated somatic cells
(broblasts) before their reprogramming
into hiPSCs. A 4–base pair (bp) segment
was successfully inserted into the HPRT1
locus of transduced broblasts, and one
of these broblast cell lines was then suc-
cessfully reprogrammed into hiPSCs using
lentiviral vectors, albeit at a reduced deriva-
tion eciency when compared with an un-
modied broblast cell line. Once derived,
these genetically modied hiPSCs were also
subsequently corrected to delete the origi-
nal 4-bp insertion. Despite these multiple
genomic modications, karyotypic abnor-
malities were observed only rarely (i.e., in
one hiPSC line at later passages, as is com-
monly observed in hESC culture).
However, unintended mutations are a
common result of gene correction attempts
using any approach, including the one used
in the current article. Improving the propor-
tion of site-specic versus nonspecic inte-
gration events has remained a major chal-
lenge to the gene therapy eld. Although in
vitro culture allows for the careful selection
and clonal expansion of the targeted cell
population of interest, clinical protocols may
Figure 1 Methods for gene correction in human embryonic and induced pluripotent stem
cells. (a) A double-strand break (DSB) induced by ionizing radiation or a zinc-finger nuclease
signals recruitment of DNA repair machinery. DNA repair then proceeds by a variety of pathways
depending on the cell cycle stage and extent of 3′ end resection. Homology-directed repair results
in successful gene targeting, whereas nonhomologous end joining (NHEJ), single-strand anneal-
ing (SSA), or microhomology-mediated end joining (MMEJ) will result in nonspecific mutations.
(b) A recombinant adeno-associated virus (AAV) expression vector consists of single-stranded DNA
(ssDNA) flanked by palindromic inverted terminal repeats. After removal of the viral capsid, DNA
repair machinery components coat the ssDNA to form a nucleofilament structure. Resolution of
vector-chromosomal DNA intermediates results in introduction of a targeted modification at the
homologous chromosomal locus.

Molecular Therapy vol. 18 no. 6 june 2010 1063
© The American Society of Gene & Cell Therapy commentary
involve transduction of hundreds of millions
of cells. Currently, the approach demon-
strated by Khan et al. remains limited to use
in cultured cells that can subsequently be ex-
panded to achieve the cell numbers required
for downstream applications. Also, despite
potential uniformity of HLA subtypes across
hiPSC derivatives and their recipients, it re-
mains to be seen whether the short exposure
of transplanted cells to viral capsids is suf-
cient to induce an immune response in the
transplanted host. It is remarkable, however,
that dierentiated broblasts, having under-
gone AAV transduction, retain the prolifera-
tive capacity for subsequent reprogramming
into hiPSCs. Dierentiated somatic cells se-
nesce over time, and senescence is known
to inhibit successful reprogramming.21 e
low derivation eciency of hiPSCs is al-
ready a well-recognized and prominent
impediment in the reprogramming eld.
e further reduction in eciency observed
when reprogramming genetically modied
broblasts raises further questions. Will ad-
ditional increases in the degree and number
of genomic modications in broblasts lead
to further reductions in reprogramming ef-
ciency? Or is the reduction in reprogram-
ming eciency correlated only with the
increased number of plating and passaging
steps required for broblast culture prior to
transduction with reprogramming factors?
Khan et al. have convincingly dem-
onstrated the feasibility of AAV-mediated
gene correction in hESCs and hiPSCs.
Overall, their results open many new
avenues for investigators to site-specically
modify candidate genes before and af-
ter the derivation of pluripotent stem cell
types. Aer perturbing a gene of interest,
the derived iPSC lines can be dierentiated
into the relevant cell population to measure
the gene’s eect on cell function. Impor-
tantly, the ability to site-specically modify
the genome mitigates the risk of malignant
transformation induced by random retrovi-
ral or lentiviral transgene insertion.22 Safe,
reliable genomic modication would great-
ly assist the therapeutic cell transplantation
eld to overcome this signicant obstacle
to clinical realization. In any case, these ad-
vances have signicantly streamlined the
genetic manipulation of pluripotent stem
cells, and have brought a small step closer
the realization of the full potential of pluri-
potent stem cells for investigating disease
and treating patients.
ACKNOWLEDGMENTS
We acknowledge funding support from the
National Institutes of Health (DP2OD004437
and R01AI085575), the Edward Mallinck-
rodt Jr. Foundation (J.C.W.), and the Howard
Hughes Medical Institute (K.H.N.).
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1Department of Pediatrics, Division of Cellular
and Molecular Therapy and Powell Gene
Therapy Center, University of Florida, Gaines-
ville, Florida, USA
Correspondence: Roland W Herzog, University
of Florida, Cancer and Genetics Research Com-
plex, 2033 Mowry Road, Room 203, Gaines-
ville, Florida 32610, USA.
E-mail: rherzog@ufl.edu
Hepatic AAV Gene Transfer and the
Immune System: Friends or Foes?
Roland W Herzog1
doi:10.1038/mt.2010.96
Two ongoing clinical trials utilize dif-
ferent adeno-associated viral (AAV)
vectors for liver-directed factor IX (F.IX)
gene transfer with the goal of sustained
therapy in patients with severe hemophilia
B. Although preclinical studies have docu-
mented immune tolerance and long-term
expression of F.IX in animals, the single
prior clinical trial of this approach achieved
only transient therapeutic gene expres-
sion and exposed preexisting immunity
to the AAV vector as a major obstacle for
therapy.1 Although accumulating preclini-
cal data continue to fuel a debate over the
potential impact of immune responses on