26 Current Gene Therapy, 2009, 9, 26-32

A Possible Approach for Stem Cell Gene Therapy of Fanconi Anemia

Liting Song*

Department of Chemistry, University of Waterloo, Waterloo, ON, Canada

Abstract: Fanconi anemia (FA) is an inherited chromosomal recessive syndrome characterized by cellular hypersensitiv-
ity to DNA crosslinking agents and bone marrow failure, which cause aplastic anemia, and an increased incidence of ma-
lignancy. 13 complementation groups are currently discovered, and 13 distinct genes have been cloned (FANCA,
FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FNACI, FANCJ, FANCL, FANCM, FANCN).
Stem cells can theoretically divide to other cells without limit as long as a person is still alive. The stem cells that form
blood and immune cells are known as hematopoietic stem cells. Hematopoietic stem cells can be acquired from a Fanconi
anemia patient, whereas genomic DNA can be obtained easily from blood cells of a normal person. Normal genes also can
be synthesised by PCR method.
Normal genomic DNA will be delivered into a patient’s stem cells via microinjection or transfection after enzyme diges-
tion; the defective genes might be repaired by homologous genetic recombination. The gene-corrected stem cells can be
transplanted into the same patient finally. It is possible that human genomic DNA to be considered as materials for ho-
mologous genetic recombination to repair defective genes in vivo. This might be an efficient method for gene therapy,
which has no or less immunological rejection for Fanconi anemia and some genetic diseases. Several related observations
and experiments are discussed to support this possible means of stem cell gene therapy of Fanconi anemia.
Keywords: Gene therapy, Hematopoietic stem cell, Microinjection, Transfection, Fanconi anemia, Genomic DNA, Homolo-
gous genetic recombination.

INTRODUCTION FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG,

FANCI, FANCJ, FANCL, FANCM, FNACN [10, 11, 12].
Fanconi anemia (FA) was first described by Swiss pae-
Of the 13 genes, only FANCB is X chromosome-linked; the
diatrician Guido Fanconi in 1927 [1-3]. FA is an inherited other 12 genes are autosomal genes [13]. Stem cells were
chromosomal recessive syndrome characterized by bone
first described by two Canadian scientists in 1963 [14]. Stem
marrow failure, which cause aplastic anemia, and an in-
cells have the property of self-renewal and the potential to
creased incidence of malignancy. FA occurred in 1 to 5 per
differentiate into different cell types. Stem cells act as a
million people and a carrier frequency of about 1 in 200 to
repair system for the body, and they are always ready to dif-
300 in most populations [2]. In Spain, 122 patients with FA
ferentiates to the corresponding differentiated cells when
were registered among about 40 million inhabitants. Due to there is a need throughout the life span, and they cope with
founder effects and isolation, some populations have a higher
the physiological changes inside the body, so as to keep a
prevalence of FA, for example, the Afrikaner population of
stable balance-homeostasis. There are two types of stem
South Africa [4], the Ashkenazi Jewish population [5], and
cells-embryonic stem cells and adult stem cells. Early em-
the Spanish Gypsies [6], with a carrier frequency of 1 in 77,
bryonic stem cells can differentiate into all of the specialized
1 in 90, and 1 in 64 to 1 in 79 respectively. In a study of 754
embryonic tissues, whereas adult stem cells can only
patients, 80% experienced the onset of bone marrow failure( differentiate into cell types of the organ from which they
BMF), and 23% had neoplasms [7]. Most FA patients die of
originate, such as skin, hair, intestine, heart, bone or blood
haematological diseases including bone marrow failure, my-
stem cells. The stem cells that form blood and immune cells
elodysplastic syndromes, and acute myelogenous leukemia.
are known as hematopoietic stem cells (HSC) [15-18].
FA patients often die before 30 years of age [8], the median
survival time was 24 years [7]. Hematopoietic stem cell transplantation (HSCT) is the
only proven useful cure for FA so far; by this method, a pa-
Cells from Fanconi anemia patients are sensitive to DNA
tient’s defective stem cells are replaced by normal stem cells
cross-linking agents such as mitomycin C and diepoxybutane
from a HLA matched donor. Locatelli F et al reported the
due to chromosomal instability, and it is the basis for a diag-
outcome of 64 FA patients (age range, 2–20 years) who un-
nosis test [3, 9].
derwent HSCT in Italy, the 8-year estimate of overall sur-
13 complementation groups are currently discovered, and vival rate was 87%, 69%, and 40% when the donors were
13 distinct genes have been cloned (FANCA, FANCB, HLA-identical siblings, HLA-partially matched relatives, and
unrelated donors respectively (p<0.01) [19]. Unrelated donor
is not suitable for transplantation because of higher risk of
*Address correspondence to this author at the Department of Chemistry,
University of Waterloo, 200 University Avenue West, Waterloo, ON N2L both acute and chronic graft-verse-host disease (GVHD) and
3G1, Canada; Tel: 416 733 1573; Fax: 416 733 1573; transplant-related mortality (TRM) [20, 21].
E-mail: ltsong@uwaterloo.ca; ltsong@yahoo.com

1566-5232/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

A Possible Approach for Stem Cell Gene Therapy of Fanconi Anemia
Fludarabine is a chemotherapy drug that is used for
cancer therapy. It is most commonly used to treat chronic
lymphocytic leukemia. By the combined use of fludarabine
and T-cell depletion in recipients of unrelated donors, lower
rates of GVHD, higher rates of hematopoietic recovery, and
a significant improvement in both early and overall survival
were achieved [20, 22, 23].
Since it is not easy to find HLA-matched donors, gene
therapy methods using viral vectors to deliver normal genes
into patient’s stem cells have been tried several years. Some
genetic diseases such as Fanconi anemia probably could be
treated by transferring correct genes into stem cells [24].
Viral vectors, such as adeno-associated virus vector [25],
retroviral vectors [26-28], and lentiviral vectors [29-33] were
used for FA gene therapy in mice and/or human cells.
Retroviral vectors can only transfect and integrate in ac-
tively dividing cells, these limited retroviral vectors may be
used as ideal vectors, because bone marrow cells from FA
patients grow poorly in vitro [26, 34]. Lentiviral vectors are
better than retroviral vectors because of their ability of trans-
duction into early hematopoietic progenitors in vivo, and
relatively higher transduction efficiency [35].
Lentiviral vectors derived from human immunodeficie-
ncy virus type 1 (HIV-1) hold great hope for gene therapy.
However, one of the safety concerns with the construction of
retroviral and lentiviral packaging systems is the possibility
of generating replication-competent retrovirus (RCR) and
replication competent lentivirus (RCL) through genetic re-
combination between overlapping sequences respectively. In
order to avoid this kind of possible hazards, modified lentivi-
ral vectors have been developed [36, 37].
Retroviral vectors randomly integrate at various positions
in the cellular genome of stem cells, and there have been
several reports and concerns of the integration of therapeutic
vectors causing cancers [38-47]. This is a major hurdle and
barrier for retroviral vectors being used as ideal vectors for
Fig. (1). Hematopoietic Stem Cell Transplantation after Gene Correction
Current Gene Therapy, 2009, Vol. 9, No. 1 27
gene therapy, and there is a real need to develop other safer
vectors for gene therapy.
The FA primary BM cells in hypoplastic or aplastic mar-
row are limited and they are very fragile in vitro; the current
gene transfer technology has many problems, physical or
chemical methods are very toxic to stem cells [28, 45]. New
ways aimed to target mutations at specific sites in the ge-
nome via homologous recombination have been tried for a
few years. The frequency of homologous recombination in
human cells is very low.
One promising method is the use of designed zinc-finger
nucl-eases to target a specific defective gene and induce a
double-strand break, and then this defective gene is repaired
by stimulating homologous recombination between the
chromosome and an extrachromosomal DNA donor. This
strategy increased the frequency of homologous recombi-
nation repair remarkably [48-53].
Here I submit a possible new approach for stem cell gene
therapy as briefly described in Fig. (1).
This approach includes the following steps:
First, Genomic DNA is extracted from blood cells of a
normal person. There are several genomic DNA extraction
kits available now, such as PAXgene Blood DNA Kit from
QIAGEN (http://www1.qiagen.com/Products/GenomicDna
StabilizationPurification/PAXgeneBloodDNASystem/PAX
geneBloodDNAKit.aspx?ShowInfo=1), this kit is used for
purification of up to 500 μg genomic DNA from whole
blood; and DNAzol® BD Reagent from Invitrogen (http://
tools.invitrogen.com/content/sfs/manuals/3898.pdf), typical-
lly 20-40 μg of genomic DNA can be obtained from 1 ml of
whole blood by using this kit.
Second, the purified genomic DNA will be transferred
into hematopoietic stem cells of a patient by microinjection
[54-56] or transfection [32] in vitro. Transfection can be per-
formed according to the instructions and protocols provided
28 Current Gene Therapy, 2009, Vol. 9, No. 1
by Invitrogen [https://resources.invitrogen.com/content/sfs/
manuals/11514015.pdf] after restriction endonuclease diges-
tion [57]. Normal genes may also be synthesised by PCR
method also[58, 59]; the PCR product will be transfected
into stem cells to replace mutated genes.
After homologous genetic recombination [60-63], the
mutated genes of Fanconi anemia might be corrected. Fi-
nally, the gene-corrected hematopoietic stem cells are then
transplanted into the same patient. Hopefully, the Fanconi
anemia problems will be solved by this method.
DISCUSSION
Microinjection technique has been successfully used in
transgenic animals [64-66] and in transferring foreign DNA
clones into mammalian cells for several years [60, 67, 68].
There was a report that 115Kb genomic DNA was trans-
ferred into human cells by an episomal vector and a genetic
deficiency was corrected [69]. Herpes simplex virus type 1
(HSV-1) amplicons are plasmid-based viral vectors that lack
viral coding sequences, therefore, these vectors are fully
nontoxic for the infected cells. HSV-1 amplicons have been
used to deliver >100 Kb complete genomic loci to rescue
phenotypes in cellular models of genetic diseases [70-72].
Fanconi anemia complementation test showed that after
fusion of 2 different complementation groups of cells, mu-
tated genes of Fanconi anemia could be repaired, so the hy-
brid cells were able to resist DNA cross-linking agents [2,
73, 74]. Somatic mosaicism, the presence of genetically dis-
tinct populations of somatic cells in a given organism due to
in vivo reversion of a pathogenic allele to wild type has been
described in several autosomal recessive disorders [75, 76],
such as hemophilia B [77], Duchenne muscular dystrophy
[78], tyrosinemia type I [79], Bloom syndrome [80], atypical
X-linked severe combined immunodeficiency [81], adeno-
sine deaminase deficiency [82], epidermolysis bullosa [83],
Wiskott-Aldrich syndrome [84, 85], androgen insensi-tivity
syndrome [86], T-cell immunodeficiency [87], leukocyte
adhesion deficiency type 1 [88, 89], and FA [90, 91]. Ap-
proximately 25% of patients with Fanconi anemia have evi-
dence of somatic mosaicism, and two subpopulations of
lymphocytes presence, one of which is hypersensitive to
cross-linking agents while the other behaves normally in
response to these agents [92]. Milder symptoms were often
observed in FA patients with somatic mosaicism [91, 93].
Several mechanisms have been proposed to account for
somatic mosaicism, including back mutation, intragenic mi-
totic recombination, gene conversion, DNA polymerase slip-
page, and compensatory mutations in cis [3, 75, 76, 84, 90,
91, 93-95].
It is very interesting that monozygotic twins with non-
hematologic symptoms of FA due to somatic mosaics were
discovered. A compensatory missense mutation was detected
in the hematopoietic cells of both sisters, but not in their fi-
broblasts and their parents. Their skin fibroblasts were sensi-
tive to DNA cross-linking agents, but not lymphocytes or
committed hematopoietic progenitors. Both sisters have been
free of hematologic symptoms for more than 28 years. It
might be due to a single HSC in one twin that acquired a
compensatory FANCA mutation, after many generations of
Liting Song
cell replication, the corrected hematopoietic stem cells took
over the defective ones, and resulting in both sisters gained
normal blood cells, via the shared circulation exist in
monozygotic twins. This kind of revertant mosaicism origi-
nally occurred in a stem cell is a way of natural stem cell
gene therapy. It provided a successful model of stem cell
gene therapy of FA in vivo, and it indicated that stem cell
gene therapy is possible and the 1958 feasible [84, 91, 93].
Homologous recombination was demonstrated in bacteria
by Nobel Laureate Joshua Lederberg in 1958 [96, 97]. Hom-
ologous recombination can take place between any two DNA
molecules that contain sequence homology. Homologous
recombination can be divided into three key steps: strand
exchange, branch migration and resolution. In E. coli, the
key processes of homologous pairing and strand exchange
are carried out by the protein RecA. RecA, ATP and single-
stranded DNA (ssDNA) form a helical filament that binds to
double-stranded DNA (dsDNA), searches for homology, and
then catalyses the exchange of the complementary strand,
producing a new heteroduplex [98, 99]. In yeast, the Rad51
protein has considerable structural and functional similarity
to the bacterial RecA protein [98, 100]. In eukaryotes, ho-
mologus recombination commonly occurs during meiosis as
chromosomal crossover between nonsister chromatids of two
homologus pairs of chromosomes. This process leads to off-
spring having different combinations of genes from their
parents and can produce new chimeric alleles, and hence
create genetic diversity [98, 101-103, and "crossing over."
Genetics. The Gale Group, Inc, 2003. Answers.com 10 Sep.
2008. http://www.answers.com/topic/ chromosomal- cross-
over]. Crossing-over may occur during both meiosis and
mitosis in eukaryotes, but the frequency of meiotic crossing-
over is much higher["crossing over." McGraw-Hill Encyclo-
pedia of Science and Technology.
The McGraw-Hill Companies, Inc., 2005. Answers.com
06 Sep. 2008. http://www.answers.com/topic/chromosomal-
crossover]. Chromosomal crossovers also occur in asexual
organisms and in somatic cells, since they are important in
some forms of DNA repair [104].
Gene targeting (gene knockout) as a result of homolo-
gous recombination between DNA sequences residing in the
chromosome and newly introduced DNA sequences inserted
into gene targeting vectors has been used to inactive (knock-
out) or modify/correct specific genes in mammalian cells for
several years [61, 62, 105-111]. More than 11,000 genes in
mice have been knocked out by using gene knockout tech-
nique [112].
The frequency of gene targeting is unaffected by the
length of nonhomologus DNA Sequences transferred to a
target chromosomal locus [63], so it is possible that large
genomic DNA being used to correct defective genes.
Meganucleases are endonucleases, which recognize long
( >12 bp ) DNA target sites [113]. Meganucleases have been
used to create site-specific DNA double-strand breaks (DSBs)
and to induce efficient gene targeting [114-119].
Based on the above mentioned observations and experi-
ments, it is very possible that by homologous genetic recom-
bination as well as other unknown ways, injected normal
genomic DNA could repair damaged or mutated genes, since
A Possible Approach for Stem Cell Gene Therapy of Fanconi Anemia
the genomic DNA from a healthy donor has the correct
genes and is very similar to the genomic DNA of a FA pa-
tient, although copy number variations contributed substan-
tially to genomic differences among humans [120-122].
We might inject normal genomic DNA or genomic DNA
fragments into stem cells directly, instead of targeting genes
inserted into vectors, so as to have a possible higher ho-
mologus recombination rate and/or lower random integration
rate because of the highly compatible homologus sequences
between humans, and therefore to have better gene therapy
results. We may also combine the microinjection of genomic
DNA and transfection of enzyme digested genomic DNA
and/or PCR synthesized targeting genes together, to get more
efficient gene correction by homologous genetic recombina-
tion.
The major unsolved problem for bone marrow transplan-
tation and HSCT is that it is often very difficult to find suit-
able human leukocyte antigen (HLA)-matched donors for
most of the patients [123-125]. Graft failure, graft-versus-
host disease (GVHD) and opportunistic infections were often
occurred in FA patients after HSCT.
HSCT with donors other than HLA-identical siblings is
associated with high morbidity and poor survival [8, 21, 22,
126, 127]. Up to now, the only practical and proven cure for
Fanconi anemia-associated bone marrow failure is successful
allogeneic HSCT. Cells seem to tolerate large foreign DNA
without immunological rejection; therefore, it is possible that
normal genomic DNA is transferred into stem cells from
patients and to correct the defective genes via homologus
genetic recombination without any immunological rejection.
This could be the major benefit of the new strategy. It is a
relatively simple but meaningful method that human normal
genomic DNA is considered as materials for homologous
genetic recombination to repair defective genes in vivo.
When 30 purified mouse stem cells were transferred,
50% lethally irradiated mice survived for more than 30 days
[15]. A few corrected HSCs should be capable of repopulat-
ing the bone marrow and blood of an affected FA patient [32,
93]. Accordingly, we might only need hundreds of gene-
corrected stem cells significantly to improve a patient’s
physiological condition. If this scheme works at cellular and
animal experimental level, further experimental therapies
might be considered for patients; hopefully we might have a
cure for Fanconi anemia and many other genetic diseases
like cystic fibrosis [128], Huntington's disease [129, 130],
and muscular dystrophy [131-133] in the near future.
ABBREVIATIONS
BMF = Bone marrow failure
DSBs = DNA double-strand breaks
dsDNA = Double-stranded DNA
FA = Fanconi anemia
GVHD = Graft-versus-host disease
HIV-1 = Human immunodeficiency virus type 1
HLA = Human leukocyte antigen
HSC = Hematopoietic stem cells
Current Gene Therapy, 2009, Vol. 9, No. 1 29
HSCT = Hematopoietic stem cell transplantation
HSV-1 = Herpes simplex virus type 1
RCL = Replication competent lentivirus
RCR = Replication-competent retrovirus
ssDNA = Single-stranded DNA
TRM = Transplant-related mortality
REFERENCES
[1] Lobitz S, Velleuer E. Guido Fanconi (1892-1979): a jack of all
trades. Nat Rev Cancer 2006; 6: 893-8.
[2] Joenje H, Patel KJ. The emerging genetic and molecular basis of
Fanconi anaemia. Nat Rev Genet 2001; 2: 446-57.
[3] Tischkowitz MD, Hodgson SV. Fanconi anaemia. J Med Genet
2003; 40: 1-10.
[4] Tipping AJ, Pearson T, Morgan NV, et al. Molecular and genea-
logical evidence for a founder effect in Fanconi anemia families of
the Afrikaner population of South Africa. Proc Natl Acad Sci USA
2001; 98: 5734-39.
[5] Verlander P C, Kaporis A, Liu Q, Zhang Q, Seligsohn U, Auerbach
A D. Carrier frequency of the IVS4 + 4 A-->T mutation of the Fan-
coni anemia gene FAC in the Ashkenazi Jewish population. Blood
1995; 86: 4034–38.
[6] Callén E, Casado JA, Tischkowitz MD, et al. A common founder
mutation in FANCA underlies the world's highest prevalence of
Fanconi anemia in Gypsy families from Spain. Blood. 2005; 105:
1946-49.
[7] Kutler DI, Singh B, Satagopan J, et al. A 20-year perspective on
the International Fanconi Anemia Registry (IFAR). Blood 2003;
101: 1249-56.
[8] Gluckman E, Auerbach AD, Horowitz MM, et al. Bone marrow
transplantation for Fanconi anemia. Blood 1995; 86: 2856-62.
[9] Giampietro PF, Verlander PC, Davis JG, Auerbach AD. Diagnosis
of Fanconi anemia in patients without congenital malformations: an
international Fanconi Anemia Registry Study. Am J Med Genet
1997; 68 :58-61.
[10] Levitus M, Joenje H, de Winter JP. The Fanconi anemia pathway
of genomic maintenance. Cell Oncol 2006; 28: 3-29.
[11] Smogorzewska A, Matsuoka S, Vinciguerra P, et al. Identification
of the FANCI protein, a monoubiquitinated FANCD2 paralog re-
quired for DNA repair. Cell 2007; 129: 289-301.
[12] Reid S, Schindler D, Hanenberg H, et al. Biallelic mutations in
PALB2 cause Fanconi anemia subtype FA-N and predispose to
childhood cancer. Nat Genet 2007; 39: 162-4.
[13] Meetei AR, Levitus M, Xue Y, et al. X-linked inheritance of Fan-
coni anemia complementation group B. Nat Genet 2004; 36: 1219-
24.
[14] Becker AJ, McCulloch EA, Till JE. Cytological demonstration of
the clonal nature of spleen colonies derived from transplanted
mouse marrow cells. Nature 1963; 197: 452-4.
[15] Spangrude GJ, Heimfeld S, Weissman IL. Purification and charac-
terization of mouse hematopoietic stem cells. Science 1988; 241:
58-62.
[16] Smith C. Hematopoietic stem cells and hematopoiesis. Cancer
Control 2003; 10: 9-16.
[17] Martinez-Agosto JA, Mikkola HKA, Hartenstein V, Banerjee U.
The hematopoietic stem cell and its niche: a comparative view.
Genes Dev 2007; 21: 3044-60.
[18] Barclay WW, Axanova LS, Chen W, et al. Characterization of
adult prostatic progenitor/stem cells exhibiting self-renewal and
multilineage differentiation. Stem Cells 2008; 26: 600-10.
[19] Locatelli F, Zecca M, Pession A, et al. The outcome of children
with Fanconi anemia given hematopoietic stem cell transplantation
and the influence of fludarabine in the conditioning regimen: a re-
port from the Italian pediatric group. Haematologica 2007; 92:
1381-88.
[20] Chewning JH, Castro-Malaspina H, Jakubowski A, et al. Fluda-
rabine-based conditioning secures engraftment of second hema-
topoietic stem cell allografts (HSCT) in the treatment of initial graft
failure. Biol Blood Marrow Transplant 2007; 13: 1313-23.
30 Current Gene Therapy, 2009, Vol. 9, No. 1
[21] Guardiola P, Pasquini R, Dokal I, et al. Outcome of 69 allogeneic
stem cell transplantations for Fanconi anemia using HLA-matched
unrelated donors: a study on behalf of the European Group for
Blood and Marrow Transplantation. Blood 2000; 95: 422-9.
[22] Wagner JE, Eapen M, MacMillan ML, et al. Unrelated donor bone
marrow transplantation for the treatment of Fanconi anemia.
Blood 2007; 109: 2256-62.
[23] Chaudhury S, Auerbach AD, Kernan NA, et al. Fludarabine-based
cytoreductive regimen and T-cell-depleted grafts from alternative
donors for the treatment of high-risk patients with Fanconi anae-
mia. Br J Haematol 2008; 140: 644-55.
[24] Bordignon C. Stem-cell therapies for blood diseases. Nature 2006;
441: 1100-02.
[25] Walsh CE, Nienhuis AW, Samulski RJ, et al. Phenotypic correc-
tion of Fanconi anemia in human hematopoietic cells with a re-
combinant adeno-associated virus vector. J Clin Invest 1994; 94:
1440-48.
[26] Liu JM, Kim S, Read EJ, et al. Engraftment of hematopoietic pro-
genitor cells transduced with the Fanconi anemia group C gene
(FANCC). Hum Gene Ther 1999; 10: 2337-46.
[27] Gush KA, Fu KL, Grompe M, Walsh CE. Phenotypic correction of
Fanconi anemia group C knockout mice. Blood 2000; 95: 700-4.
[28] Cohen-Haguenauer O, Péault B, Bauche C, et al. In vivo repopula-
tion ability of genetically corrected bone marrow cells from Fan-
coni anemia patients. Proc Natl Acad Sci U S A 2006; 103: 2340–
45.
[29] Galimi F, Noll M, Kanazawa Y, et al. Gene therapy of Fanconi
anemia: preclinical efficacy using lentiviral vectors. Blood 2002;
100: 2732-36.
[30] Yamada K, Olsen JC, Patel M, Rao KW, Walsh CE. Functional
correction of fanconi anemia group C hematopoietic cells by the
use of a novel lentiviral vector. Mol Ther 2001; 3: 485-90.
[31] Yamada K, Ramezani A, Hawley RG, et al. Phenotype correction
of Fanconi anemia group A hematopoietic stem cells using lentivi-
ral vector. Mol Ther 2003; 8: 600-10.
[32] Kelly PF, Radtke S, von Kalle C, et al. Stem cell collection and
gene transfer in Fanconi anemia. Mol Ther 2007; 15: 211-9.
[33] Müller LU, Milsom MD, Kim MO, Schambach A, Schuesler T,
Williams DA. Rapid lentiviral transduction preserves the engraft-
ment potential of Fanca(-/-) hematopoietic stem cells. Mol Ther
2008; 16: 1154-60.
[34] Kohn DB. Gene therapy for genetic haematological disorders and
immunodeficiencies. J Intern Med 2001; 249: 379-90.
[35] Miyoshi H, Smith KA, Mosier DE, Verma IM, Torbett BE. Trans-
duction of human CD34+ cells that mediate long-term engraftment
of NOD/SCID mice by HIV vectors. Science 1999; 283: 682-6.
[36] Wu X, Wakefield JK, Liu H et al. Development of a novel trans-
lentiviral vector that affords predictable safety. Mol Ther 2000; 2:
47-55.
[37] Westerman KA, Ao Z, Cohen EA, Leboulch P. Design of a trans
protease lentiviral packaging system that produces high titer virus.
Retrovirology 2007; 4: 96.
[38] Li Z, Düllmann J, Schiedlmeier B, et al. Murine leukemia induced
by retroviral gene marking. Science 2002; 296: 497.
[39] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious
adverse event after successful gene therapy for X-linked severe
combined immunodeficiency. N Engl J Med 2003; 348: 255–6.
[40] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. LMO2-
associated clonal T cell proliferation in two patients after gene
therapy for SCID-X1. Science 2003; 302: 415–9.
[41] Modlich U, Kustikova OS, Schmidt M, et al. Leukemias following
retroviral transfer of multidrug resistance 1 (MDR1) are driven by
combinatorial insertional mutagenesis. Blood 2005; 105: 4235-46.
[42] Du Y, Spence SE, Jenkins NA, Copeland NG. Cooperating cancer-
gene identification through oncogenic-retrovirus-induced inser-
tional mutagenesis. Blood 2005; 106: 2498-505.
[43] Neff T, Beard BC, Kiem H-P. Survival of the fittest: in vivo selec-
tion and stem cell gene therapy. Blood 2006; 107: 1751-60.
[44] Seggewiss R, Pittaluga S, Adler RL, et al. Acute myeloid leukemia
is associated with retroviral gene transfer to hematopoietic progeni-
tor cells in a rhesus macaque. Blood 2006; 107: 3865-67.
[45] Dunbar CE. The Yin and Yang of stem cell gene therapy: insights
into hematopoiesis, leukemogenesis, and gene therapy safety. He-
matology Am Soc Hematol Educ Program 2007; 460-5.
[46] Cornetta K, Pollok KE, Miller AD. Retroviral vectors for gene
transfer. CSH Protocols 2008; doi: 10.1101/pdb.top29.
Liting Song
[47] Nienhuis AW. Development of gene therapy for blood disorders.
Blood 2008; 111: 4431-44.
[48] Bibikova M, Carroll D, Segal DJ, et al. Stimulation of homologous
recombination through targeted cleavage by chimeric nucleases.
Mol Cell Biol 2001; 21: 289-97.
[49] Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene
targeting with designed zinc finger nucleases. Science 2003; 300:
764.
[50] Porteus MH, Baltimore D. Chimeric nucleases stimulate gene tar-
geting in human cells. Science 2003; 300: 763.
[51] Urnov FD, Miller JC, Lee YL, et al. Highly efficient endogenous
human gene correction using designed zinc-finger nucleases. Na-
ture 2005; 435: 646–51.
[52] Pruett-Miller SM, Connelly JP, Maeder ML, Joung JK, Porteus
MH. Comparison of zinc finger nucleases for use in gene targeting
in mammalian cells. Mol Ther 2008; 16: 707-17.
[53] Porteus MH. Design and testing of zinc finger nucleases for use in
mammalian cells. Methods Mol Biol 2008; 435: 47-61.
[54] Tsulaia TV, Prokopishyn NL, Yao A, et al. Glass needle-mediated
microinjection of macromolecules and transgenes into primary hu-
man mesenchymal stem cells. J Biomed Sci 2003; 10: 328-36.
[55] Shen YM, Hirschhorn RR, Mercer WE, et al. Gene transfer: DNA
microinjection compared with DNA transfection with a very high
efficiency. Mol Cell Biol 1982; 2: 1145-54.
[56] Davis BR, Brown DB, Prokopishyn NL, Yannariello-Brown J.
Glass needle-mediated microinjection of macromolecules and
transgenes into primary human blood stem/progenitor cells. Blood
2000; 95: 437-44.
[57] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a labora-
tory manual. Second edition. Cold Spring Harbor Laboratory Press,
New York 1989; pp 5.3-32.
[58] Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of
beta-globin genomic sequences and restriction site analysis for
diagnosis of sickle cell anemia. Science 1985; 230: 1350–54.
[59] Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase.
Science 1988; 239: 487–91.
[60] Folger KR, Wong EA, Wahl G, Capecchi MR. Patterns of integra-
tion of DNA microinjected into cultured mammalian cells: evi-
dence for homologous recombination between injected plasmid
DNA molecules. Mol Cell Biol 1982; 2: 1372-87.
[61] Smithies O, Gregg RG, Boggs SS, et al. Insertion of DNA se-
quences into the human chromosomal beta-globin locus by ho-
mologous recombination. Nature 1985; 317: 230-4.
[62] Capecchi MR. Altering the genome by homologous recombination.
Science 1989; 244: 1288-92.
[63] Mansour SL, Thomas KR, Deng CX, Capecchi MR. Introduction
of a lacZ reporter gene into the mouse int-2 locus by homologous
recombination. Proc Natl Acad Sci U S A 1990; 87: 7688-92.
[64] Charreau B, Tesson L, Soulillou JP, Pourcel C, Anegon I. Trans-
genesis in rats: technical aspects and models. Transgenic Res 1996;
5: 223-34.
[65] Tesson L, Cozzi J, Menoret S, et al. Transgenic modifications of
the rat genome. Transgenic Res 2005; 14: 531-46.
[66] Filipiak WE, Saunders TL. Advances in transgenic rat production.
Transgenic Res 2006; 15: 673-86.
[67] Anderson WK, Killos L, Sanders-Haigh L, Kretschmer PJ,
Diacumakos EG. Replication and expression of thymidine kinase
and human globin gene microinjected into mouse fibroblasts. Proc
Natl Acad Sci U S A 1980; 77: 5399–403.
[68] Capecchi MR. High efficiency transformation by direct microinjec-
tion of DNA into cultured mammalian cells. Cell 1980; 22: 479–88.
[69] Wade-Martins R, White RE, Kimura H, Cook PR, James MR.
Stable correction of a genetic deficiency in human cells by an epi-
some carrying a 115 KB genomic transgene. Nat Biotechnol 2000;
18: 1311-14.
[70] Epstein AL. HSV-1-based amplicon vectors: design and applica-
tions. Gene Ther 2005; 12: S154-8.
[71] Hibbitt OC, Wade-Martins R. Delivery of large genomic DNA
inserts >100 KB using HSV-1 amplicons. Curr Gene Ther 2006; 6:
325-36.
[72] Cuchet D, Potel C, Thomas J, Epstein AL. HSV-1 amplicon vec-
tors: a promising and versatile tool for gene delivery. Expert Opin
Biol Ther 2007; 7: 975-95.
A Possible Approach for Stem Cell Gene Therapy of Fanconi Anemia
[73] Duckworth-Rysiecki G, Cornish K, Clarke CA, et al. Identification
of two complementation groups in Fanconi anemia. Somat Cell
Mol Genet 1985; 11: 35-41.
[74] Levitus M, Rooimans MA, Steltenpool J, et al. Heterogeneity in
Fanconi anemia: evidence for 2 new genetic subtypes. Blood 2004;
103: 2498-503.
[75] Hall JG. Review and hypotheses: somatic mosaicism: observations
related to clinical genetics. Am J Hum Genet 1988; 43: 355-63.
[76] Hirschhorn R. In vivo reversion to normal of inherited mutations in
humans. J Med Genet 2003; 40: 721-8.
[77] Taylor SA, Deugau KV, Lillicrap DP. Somatic mosaicism and
female-to-female transmission in a kindred with hemophilia B
(factor IX deficiency). Proc Natl Acad Sci U S A 1991; 88: 39-42.
[78] Klein CJ, Coovert DD, Bulman DE, Ray PN, Mendell JR, Burghes
AH. Somatic reversion/suppression in Duchenne muscular dystro-
phy (DMD): evidence supporting a frame-restoring mechanism in
rare dystrophin-positive fibers. Am J Hum Genet 1992; 50: 950–9.
[79] Kvittingen EA, Rootwelt H, Berger R, Brandtzaeg P. Self-induced
correction of the genetic defect in tyrosinemia type I. J Clin Invest
1994; 94: 1657-61.
[80] Ellis NA, Lennon DJ, Proytcheva M, Alhadeff B, Henderson EE,
German J. Somatic intragenic recombination within the mutated lo-
cus BLM can correct the high sister-chromatid exchange phenotype
of Bloom syndrome cells. Am J Hum Genet 1995; 57: 1019-27.
[81] Stephan V, Wahn V, Le Deist F, et al. Atypical X-linked severe
combined immunodeficiency due to possible spontaneous reversion
of the genetic defect in T cells. N Engl J Med 1996; 335: 1563-67.
[82] Hirschhorn R, Yang DR, Puck JM, Huie ML, Jiang CK,
Kurlandsky LE. Spontaneous in vivo reversion to normal of an in-
herited mutation in a patient with adenosine deaminase deficiency.
Nat Genet 1996; 13: 290-5.
[83] Jonkman MF, Scheffer H, Stulp R, et al. Revertant mosaicism in
epidermolysis bullosa caused by mitotic gene conversion. Cell
1997; 88: 543-51.
[84] Wada T, Schurman SH, Otsu M, et al. Somatic mosaicism in
Wiskott-Aldrich syndrome suggests in vivo reversion by a DNA
slippage mechanism. Proc Natl Acad Sci U S A 2001; 98: 8697-
702.
[85] Konno A, Wada T, Schurman SH, et al. Differential contribution of
Wiskott-Aldrich syndrome protein to selective advantage in T- and
B-cell lineages. Blood 2004; 103: 676-8.
[86] Kohler B, Lumbroso S, Leger J, et al. Androgen insensitivity
syndrome: somatic mosaicism of the androgen receptor in seven
families and consequences for sex assignment and genetic
counseling. J Clin Endocrinol Metab 2005; 90: 106-11.
[87] Rieux-Laucat F, Hivroz C, Lim A, et al. Inherited and somatic
CD3zeta mutations in a patient with T-cell deficiency. N Engl J
Med 2006; 354: 1913-21.
[88] Tone Y, Wada T, Shibata F, et al. Somatic revertant mosaicism in a
patient with leukocyte adhesion deficiency type 1. Blood 2007;
109: 1182-84.
[89] Uzel G, Tng E, Rosenzweig SD, et al. Reversion mutations in
patients with leukocyte adhesion deficiency type-1 (LAD-1).
Blood 2008; 111: 209-18.
[90] Waisfisz Q, Morgan NV, Savino M, et al. Spontaneous functional
correction of homozygous Fanconi anaemia alleles reveals novel
mechanistic basis for reverse mosaicism. Nat Genet 1999; 22: 379-
83.
[91] Gregory JJ Jr, John E. Wagner JE, Verlander PC, et al. Somatic
mosaicism in Fanconi anemia: evidence of genotypic reversion in
lymphohematopoietic stem cells. Proc Natl Acad Sci U S A 2001;
98: 2532-37.
[92] Lo Ten Foe JR, Kwee ML, Rooimans MA, et al. Somatic mo-
saicism in Fanconi anemia: molecular basis and clinical signifi-
cance. Eur J Hum Genet 1997; 5: 137-48.
[93] Mankad A, Taniguchi T, Cox B, et al. Natural gene therapy in
monozygotic twins with Fanconi anemia. Blood 2006; 107: 3084-
90.
[94] Youssoufian H, Pyeritz RE. Mechanisms and consequences of
somatic mosaicism in humans. Nat Rev Genet 2002; 3: 748-58.
[95] Gross M, Hanenberg H, Lobitz S, et al. Reverse mosaicism in
Fanconi anemia: natural gene therapy via molecular self-correction.
Cytogenet Genome Res 2002; 98: 126-35.
[96] Tatum EL, Lederberg J. Gene recombination in the bacterium
Escherichia coli. J Bacteriol 1947; 53: 673-84.
Current Gene Therapy, 2009, Vol. 9, No. 1 31
[97] Lederberg J. Gene recombination and linked segregations in Es-
cherichia Coli. Genetics 1947; 32: 505-25.
[98] Hiom K. Homologus recombination. Curr Biol 2000; 10: R359-61.
[99] Chen Z, Yang H, Pavletich NP. Mechanism of homologous recom-
bination from the RecA-ssDNA/dsDNA structures. Nature 2008;
453: 489-94.
[100] Ogawa T, Yu X, Shinohara A, Egelman EH. Similarity of the yeast
RAD51 filament to the bacterial RecA filament. Science 1993; 259:
1896-99.
[101] Nelson DL, Cox MM. DNA recombination. In: Nelson DL, Cox
MM Eds , Lehninger principles of biochemistry. New York, W.H.
Freeman and Company 2005; pp. 978-988.
[102] Whitby MC. Making crossovers during meiosis. Biochem Soc
Trans 2005; 33: 1451–55.
[103] Lorenz A, Whitby MC. Crossover promotion and prevention. Bio-
chem Soc Trans 2006; 34: 537-41.
[104] Li X, Heyer WD. Homologous recombination in DNA repair and
DNA damage tolerance. Cell Res 2008; 18: 99–113.
[105] Thomas KR, Folger KR, Capecchi MR. High frequency targeting
of genes to specific sites in the mammalian genome. Cell 1986; 44:
419-28.
[106] Thomas KR, Capecchi MR. Introduction of homologous DNA
sequences into mammalian cells induces mutations in the cognate
gene. Nature 1986; 324: 34-8.
[107] Thomas KR, Capecchi MR. Site-directed mutagenesis by gene
targeting in mouse embryo-derived stem cells. Cell 1987; 51: 503-
12.
[108] Doetchman T, Gregg RG, Maeda N, et al. Targetted correction of a
mutant HPRT gene in mouse embryonic stem cells. Nature 1987;
330: 576–8.
[109] Thompson S, Clarke AR, Pow AM, Hooper ML, Melton DW.
Germ line transmission and expression of a corrected HPRT gene
produced by gene targeting in embryonic stem cells. Cell 1989; 56:
313-21.
[110] Vasquez KM, Marburger K, Intody Z, Wilson JH. Manipulating the
mammalian genome by homologous recombination. Proc Natl
Acad Sci U S A 2001; 98: 8403-10.
[111] Misra RP, Duncan SA. Gene targeting in the mouse: advances in
introduction of transgenes into the genome by homologous recom-
bination. Endocrine 2002; 19: 229-38.
[112] Vogel G. A knockout award in medicine. Science 2007; 318: 178-
9.
[113] Thierry A, Dujon B. Nested chromosomal fragmentation in yeast
using the meganuclease I-Sce I: a new method for physical map-
ping of eukaryotic genomes. Nucleic Acids Res 1992; 20: 5625–31.
[114] Rouet P, Smih F, Jasin M. Introduction of double-strand breaks
into the genome of mouse cells by expression of a rare-cutting en-
donuclease. Mol Cell Biol 1994; 14: 8096-106.
[115] Choulika A, Perrin A, Dujon B, Nicolas JF. Induction of homolo-
gous recombination in mammalian chromosomes by using the I-
SceI system of Saccharomyces cerevisiae. Mol Cell Biol 1995; 15:
1968–73.
[116] Cohen-Tannoudji M, Robine S, Choulika A, et al. I-SceI-induced
gene replacement at a natural locus in embryonic stem cells. Mol
Cell Biol 1998; 18: 1444-48.
[117] Donoho G, Jasin M, Berg P. Analysis of gene targeting and in-
trachromosomal homologous recombination stimulated by genomic
double-strand breaks in mouse embryonic stem cells. Mol Cell Biol
1998; 18: 4070-78.
[118] Epinat J-C, Arnould S, Chames P, et al. A novel engineered me-
ganuclease induces homologous recombination in yeast and mam-
malian cells. Nucleic Acids Res 2003; 31: 2952-62.
[119] Pâques F, Duchateau P. Meganucleases and DNA double-strand
break-induced recombination: perspectives for gene therapy. Curr
Gene Ther 2007; 7: 49-66.
[120] Sebat J, Lakshmi B, Troge J et al. Large-scale copy number poly-
morphism in the human genome. Science 2004; 305: 525-8.
[121] Iafrate AJ, Feuk L, Rivera MN et al. Detection of large-scale varia-
tion in the human genome. Nat Genet 2004; 36: 949-51.
[122] Nguyen D-Q, Webber C, Ponting CP (2006) Bias of selection on
human copy-number variants. PLoS Genet 2006; 2(2): e20.
[123] Bonfim CM, de Medeiros CR, Bitencourt MA, et al. HLA-matched
related donor hematopoietic cell transplantation in 43 patients with
Fanconi anemia conditioned with 60 mg/kg of cyclophosphamide.
Biol Blood Marrow Transplant 2007; 13: 1455-60.
32 Current Gene Therapy, 2009, Vol. 9, No. 1
[124] Torjemane L, Ladeb S, Ben Othman T, Abdelkefi A, Lakhal A,
Ben Abdeladhim A. Bone marrow transplantation from matched re-
lated donors for patients with Fanconi anemia using low-dose
busulfan and cyclophosphamide as conditioning. Pediatr Blood
Cancer 2006; 46: 496-500.
[125] Kohli-Kumar M, Morris C, DeLaat C, et al. Bone marrow trans-
plantation in Fanconi anemia using matched sibling donors. Blood
1994; 84: 2050-54.
[126] Grewal SS, Kahn JP, MacMillan ML, Ramsay NKC, Wagner JE.
Successful hematopoietic stem cell transplantation for Fanconi
anemia from an unaffected HLA-genotype-identical sibling se-
lected using preimplantation genetic diagnosis. Blood 2004; 103:
1147-51.
[127] Guardiola P, Socie G, Li X, et al. Acute graft-versus-host disease
in patients with Fanconi anemia or acquired aplastic anemia under-
going bone marrow transplantation from HLA-identical sibling do-
nors: risk factors and influence on outcome. Blood 2004; 103: 73-7.
[128] Riordan JR, Rommens JM, Kerem B, et al. Identification of the
cystic fibrosis gene: cloning and characterization of complementary
Received: May 23, 2008 Revised: September 17, 2008 Accepted: December 19, 2008
Liting Song
DNA. Science 1989; 245: 1066–73. Erratum in: Science 1989; 245:
1437.
[129] Gusella JF, Wexler NS, Conneally PM, et al. A polymorphic DNA
marker genetically linked to Huntington's disease. Nature 1983;
306: 234–8.
[130] MacDonald ME, Ambrose CM, Duyao MP et al. A novel gene
containing a trinucleotide repeat that is expanded and unstable on
Huntington's disease chromosomes. Cell 1993; 72: 971–83.
[131] La Spada AR, Roling DB, Harding AE, et al. Meiotic stability and
genotype-phenotype correlation of the trinucleotide repeat in X-
linked spinal and bulbar muscular atrophy. Nat Genet 1992; 2:
301–4.
[132] Brook JD, McCurrach ME, Harley HG, et al. Molecular basis of
myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at
the 3' end of a transcript encoding a protein kinase family member.
Cell 1992; 68: 799-808. Erratum in: Cell 1992; 69: 385.
[133] Rodino-Klapac LR, Chicoine LG, Kaspar BK, Mendell JR. Gene
therapy for Duchenne muscular dystrophy: expectations and chal-
lenges. Arch Neurol 2007; 64: 1236-41.
Correction for Liting Song, A possible approach for stem cell gene therapy of Fanconi
anemia. Curr Gene Ther 2009; 9: 26-32.
A few new minor errors were made during the editing and printing process.
1. On page 27, right column, in line 12, ‘nucl-eases’ the hyphen should be deleted
2. On page 28, left column, in line 4, ‘also’ should be deleted.
3. On page 28, right column, in line 8, ‘the 1958’ should be moved down to line 10
before the words ‘Nobel Laureate Joshua Lederberg’, the first letter of
‘Laureate’ should be lower case and the words ‘in 1958’ in line 10 should be
deleted.
4. On page 29, left column, in line 16 from the bottom, the word ‘to’ should be
moved to in front of the word ‘significantly’ or the word ‘significantly’ should
be moved to the end of this sentence.