THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE
J Gene Med 2006; 8: 646–653.
Published online 28 February 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.891
Phage φC31 integrase-mediated genomic
integration of the common cytokine receptor
gammachaininhumanT-celllines
Yoshinori Ishikawa
1
Nobuyuki Tanaka
1
*
Kazuhiro Murakami
2
Toru Uchiyama
3
Satoru Kumaki
3
Shigeru Tsuchiya
3
Hiroyuki Kugoh
2
Mitsuo Oshimura
2
Michele P. Calos
4
Kazuo Sugamura
1
1
Department of Microbiology and
Immunology, Tohoku University
Graduate School of Medicine, 2-1
Seiryo-machi, Aoba-ku, Sendai
980-8575, Japan
2
Department of Biomedical Science,
Graduate School of Medical Science,
Tottori University, 86 Nishimachi,
Yonago 683-8503, Japan
3
Department of Pediatric Oncology,
Institute of Development, Aging and
Cancer, Tohoku University, 4-1
Seiryo-machi, Aoba-ku, Sendai
980-8575, Japan
4
Department of Genetics, Stanford
University School of Medicine,
Stanford, CA 94305-5120, USA
*Correspondence to:
Nobuyuki Tanaka, Department of
Microbiology and Immunology,
Tohoku University Graduate School
of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan.
E-mail:
n-tanaka@mail.tains.tohoku.ac.jp
Received: 19 October 2005
Revised: 27 December 2005
Accepted: 28 December 2005
Abstract
Background X-linked severe combined immunodeficiency (SCID-X1, X-
SCID) is a life-threatening disease caused by a mutated common cytokine
receptor γ chain (γ c) gene. Although ex vivo gene therapy, i.e., transduction
of the γ c gene into autologous CD34
+
cells, has been successful for treating
SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated
integration, causing leukemias. Here, to explore an alternative gene transfer
methodology that may offer less risk of insertional mutagenesis, we employed
the φC31 integrase-based integration system using human T-cell lines,
including the γ c-deficient ED40515(-).
Methods A φC31 integrase and a neo
r
gene expression plasmid containing
the φC31 attB sequence were co-delivered by electroporation into Jurkat cells.
After G418 selection, integration site analyses were performed using linear
amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-)
cells were also transfected with a γ c expression plasmid containing attB,and
the integration sites were determined. IL-2 stimulation was used to assess the
functionality of the transduced γ c in an ED40515(-)-derived clone.
Results Following co-introduction of the φC31 integrase expression plasmid
and the plasmid carrying attB, the efficiency of integration into the unmodified
human genome was assessed. Several integration sites were characterized,
including new integration sites in intergenic regions on chromosomes 13 and
18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing
the integrated γ c gene exhibited stable expression of the γ c protein, with
normal IL-2 signaling, as assessed by STAT5 activation.
Conclusions This study supports the possible future use of this φC31
integrase-mediated genomic integration strategy as an alternative gene
therapy approach for treating SCID-X1. Copyright  2006 John Wiley &
Sons, Ltd.
Keywords site-specific integration; φC31 integrase; SCID-X1; hematopoietic
cells
Introduction
X-linked severe combined immunodeficiency (SCID-X1) is the most frequent
form of severe combined immunodeficiency (SCID) [1,2]. A number of stud-
ies have demonstrated that mutations within the common cytokine receptor
γ chain (γ c) gene cause this life-threatening disease. As a shared receptor
Copyright  2006 John Wiley & Sons, Ltd.

Phage φC31 Integrase-Mediated γcIntegration 647
component of multiple cytokine receptors, including the
interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21
receptors, the γ c protein is required for the normal
development of hematopoietic cell populations, including
T and NK cells, as well as the normal function of B cells,
in humans. Dysfunction of the γ c, therefore, causes the
SCID-X1 phenotypes. Because there is a single causative
gene for this lethal disease, patients can be cured by
transfusion with a population of their own CD34
+
bone
marrow cells transfected ex vivo with the normal γcgene
[3,4]. Indeed, a clinical trial using retroviral transfer of the
γ c cDNA into hematopoietic stem cells (HSC) successfully
reconstituted the hematopoietic cell populations of the
affected patients [5–7]. Unexpectedly, however, almost
3 years following the initial therapy, three of the ten
SCID patients developed severe adverse effects, such as
leukemia [8,9]. In at least two cases, the same proto-
oncogene, LMO-2, was transcriptionally activated, due
to integration of the retroviral vector near the LMO-2
promoter [10]. To reduce the risk of such insertional
activation of cellular proto-oncogenes, alternative safer
gene delivery/integration methods are desirable [11].
Bacteriophage integrases are enzymes that mediate
site-specific recombination between two DNA recognition
sequences [12]. Among such integrases, φC31 integrase,
which has a catalytic serine residue and is therefore
classified as a member of the serine integrase family,
mediates specific recombination between the phage
attachment site attP and the bacterial attachment site
attB. φC31 integrase mediates the integration of plasmids
bearing an attB site into a limited number of ‘pseudo-attP’
sites present in native mammalian genomes. Given that
φC31 integrase recognizes relatively short, yet still rather
specific, sequences in mammalian genomes, it is becoming
a promising genetic manipulation tool in eukaryotes
[13,14]. Indeed, by using this strategy, enzyme genes
have been stably integrated into the livers of mice
after high-pressure tail vein injection, resulting in the
prolonged expression of therapeutic genes [15,16]. The
φC31 integrase system has also been applied to the
ex vivo modification of human primary skin and muscle
progenitor cells for autotransplantation [17,18].
Because HSC can be manipulated ex vivo, SCID-X1
may be a feasible candidate for φC31 integrase-mediated
gene therapy. In this study, we demonstrate that the
φC31 integrase functions in human T-cell lines. We also
identify pseudo-attP sites into which the therapeutic gene
integrates and assess the feasibility of this strategy for
SCID-X1 gene therapy by demonstrating the correction of
a γ c-deficient human T-cell line.
Materials and methods
Plasmid construction
pCMVInt was used for φC31 integrase expression [12].
A carrier plasmid pCS was generated by removing the
integrase gene from pCMVInt using BamHI-SpeI followed
by blunt-end ligation. The attB sequence-containing
plasmids, pcDNA-attB and pCXB, were constructed as
follows: the EcoRI fragment containing the φC31 attB
sequence from pTA-attB [12] was blunt-ligated into the
BglII site of the backbone vector pcDNA3 and the HindIII
site of pCXN2 [19]. A 1.7-kb HindIII-XbaI fragment
containing the firefly luciferase gene was excised from
the pGL3-promoter (Promega, Madison, WI, USA), the
ends were blunted, and the fragment was subcloned
into pCXN2 or pCX-B at a blunted EcoRI site, generating
pCXL and pCXLB, respectively. The human γcgenewas
removed from pSRG1 [20] by digestion with XbaI and
inserted into EcoRI site of pCXB, generating pCXγB.
Plasmid constructs were verified by DNA sequencing of
their respective inserts.
Cell culture and transfection
An HTLV-transformed human T-cell line, ED40515(-),
lacks expression of the γ c [21]. Jurkat and ED40515(-)
cells were maintained in RPMI 1640 supplemented with
10% fetal calf serum and antibiotics. Jurkat cells (1× 10
7
)
were transformed by electroporation with 10 µgpcDNA-
attB and 10 µg pCMVInt, using pulses of 270 V, 960 µF
from a Gene Pulser II (Bio-Rad, Hercules, CA, USA). The
electroporation conditions for ED40515(-) were pulses of
350 kV at 250 µF. Cells were split into a 96-well plate
at the appropriate dilution. One day after transfection,
G418-containing medium was added to a final concentra-
tion of 600 µg/ml, and the cells were cultured for at least
3moreweeks.
Luciferase assay
Jurkat cells were seeded in 24-well plates at a density
of 1× 10
5
cells per ml, 36 h before they were trans-
fected with 1.5 µgofeitherpCXLorpCXLBand1.5 µg
of either carrier plasmid pCS or pCMVInt, using 4 µlof
DIMRIE-C (Invitrogen, Carlsbad, CA, USA). Twenty-four
hours after transfection, the cells were transferred into
flasks with 6 ml of medium. Seventy-two hours after the
transfection, 80% of the cells were harvested for use in the
assays. The remaining cells were further cultured with a
maintenance medium exchange every 3 days. Luciferase
assays were performed using a luciferase assay system
(Promega) according to the manufacturer’s instructions.
Linear amplification
mediated-polymerase chain reaction
(LAM-PCR)
LAM-PCR was performed using the previously reported
methodology with slight modifications [22]. Briefly,
genomic DNA from transfectant clones passaged at least
3 weeks was purified using the GenElute mammalian
genomic DNA Miniprep kit (Sigma, St.Louis, MO, USA),
and used as the template. Linear amplification was carried
out with the biotinylated primer B1 (5
′
-ctg acg ctg ccc cgc
Copyright  2006 John Wiley & Sons, Ltd. J Gene Med 2006; 8: 646–653.