280 Generation of Integration Free iPSCs from an X CGD Patient''''s Blood Cells as Clinically Relevant Target for Gene Repair Using Designer ZFN or TALEN Molecular Therapy Volume 20, Supplement 1, May 20[.]
Trang 1Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
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accumulation is responsible for cutaneous skin photosensitivity whose
severity varies between diseases The only curative treatment for
severe cases of EP is allogenic bone marrow transplantation (ABMT)
that requires an HLA-matched donor Autologous hematopoietic
stem cell (HSC) gene therapy represents an alternative to ABMT
We have successfully used retroviral vectors for HSC gene therapy
in congenital erythropoietic porphyria (CEP) and erythropoietic
protoporphyria (EPP) mice However, due to multiple random
integrations of integrative vectors, insertional mutagenesis represents
a serious side effect that was observed in clinical trials Over the last
few years, researchers were able to generate induced pluripotent stem
cells (iPSCs) by reprogramming differentiated cells with retroviral
expression of stem cell factors (Oct3/4, Sox2, Klf4 and c-Myc) These
iPSCs cells shared many properties with embryonic stem cells
(self-renewal and totipotency) and give rise to many cell types, including
HSC We have induced and characterised iPSCs lines from EP mice
and evaluated their therapeutic potential after genetic correction and
hematopoietic differentiation We have reprogrammed adult skin
fi broblasts from EPP, CEP and wild type mice with a single integrative
lentiviral vector expressing Oct3/4, Klf4 and Sox2 proteins (0.05%
effi ciency) We obtained iPSCs from the three mice models which
presented ES-like morphology and expressed embryonic factors
using RT-PCR and immunocytochemistry analysis LoxP sequences
in the LTR of the provirus allowed us to remove the reprogramming
vector by transient adenoviral expression of the recombinase CRE
We showed that reprogramming vector-free iPSCs clones continue to
express pluripotent markers and are able to form embryoid bodies in
vitro and teratomas in immunodefi cient mice For genetic correction
of EP iPSCs cells, we used a lentiviral vector expressing either the
ferrochelatase (FECH) or the uroporphyrinogen-III-synthase (UROS)
cDNA from a chimeric erythroid-specifi c promoter We used the
LAM-PCR to select for safe therapeutic proviral integration, far
from known oncogenes The therapeutic effi ciency was analysed
after hematopoietic differentiation of corrected-iPSCs on OP9 cell
stroma We obtained up to 48% CD41+ hematopoietic progenitors
cells We are actually performing in vivo hematopoietic repopulation
assays in EP mice to evaluate the therapeutical potential of our
EP-iPSCs-derived hematopoietic progenitor cells Transplanted EP mice
will be monitored over time for metabolic and phenotypic correction
These experiments represent important steps in the development of
preclinical gene therapy protocols for erythropoietic porphyria
279 Derivation and Functional Analysis of
Patient Specifi c Induced Pluripotent Stem Cells
as an In Vitro Model of Chronic Granulomatous
Disease
Ulrich Siler,1 Yan Jiang,3 Sally A Cowley,2 Dario Melguzo,4
Katarzyna Tilgner,3 Cathy Browne,2 Angus deWilton,2 Stefan
Pryzborski,5 Gabriele Saretzki,6 William S James,2 Reinhard A
Seger,1 Janine Reichenbach,1 Majlinda Lako,1 Lyle Armstrong.1
1 Immunology, University Children’s Hospital Zürich, Switzerland;
2 James Martin Stem Cell Facility, Sir William Dunn School of
Pathology, University of Oxford, United Kingdom; 3 Institute
of Genetic Medicine, Newcastle University, Newcastle, United
Kingdom; 4 Centro de Investigacion Principe Felipe, Valencia,
Spain; 5 School of Biomedical Sciences, University of Durham,
United Kingdom; 6 Institute for Ageing and Health, Newcastle
University, Newcastle, United Kingdom.
Chronic granulomatous disease (CGD) is an inherited disorder
of phagocytes in which NADPH oxidase is defective in generating
reactive oxygen species In this study, we reprogrammed three normal
unrelated patient’s fi broblasts (p47 phox and gp91 phox) to pluripotency
by lentiviral transduction with defi ned pluripotency factors These
induced pluripotent stem cells (iPSC) share the morphological features
of human embryonic stem cells, express the key pluripotency factors
and posses high telomerase activity Furthermore, all the iPSC lines
formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo
They were isogenic with the original patient fi broblasts, exhibited
normal karyotype and retained the gp47 phox or gp91 phox mutations found in the patient fi broblasts We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profi le to blood-derived macrophages under resting conditions Most importantly, CGD-patient specifi c iPSC derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients Together these results suggest that CGD-patient-specifi c iPSC lines represent an important tool for modelling CGD disease phenotypes, screening candidate drugs and the development of gene therapy
280 Generation of Integration-Free iPSCs from an X-CGD Patient’s Blood Cells as Clinically Relevant Target for Gene-Repair Using Designer ZFN or TALEN
Jizhong Zou,1 Colin L Sweeney,2 Harry L Malech,2 Linzhao Cheng.1
1 Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; 2 Laboratory of Host Defenses, NIAID/NIH, Bethesda, MD.
Human induced pluripotent stem cells (iPSCs) provided an unlimited cell resource for gene and cell therapy As an alternative approach to current viral vector-based, random integration-mediated gene transfer method, patient-derived iPSCs bearing defi ned disease-causing mutations can be precisely corrected by homologous recombination mediated gene targeting, and then indefinitely expanded and differentiated into desired cell lineages for autologous transplantation Current genome engineering technologies offer two powerful DNA sequence-specifi c nucleases, zinc fi nger nuclease (ZFN) and transcriptional activator-like effector nuclease (TALEN)
to create double-strand break and thus signifi cantly stimulate the effi ciency of homologous recombination For clinical applications
of iPSCs it is advantageous to develop iPSC using reprogramming vectors and correction plasmids that either do not integrate or that can
be partially or completely excised following integration To develop clinically applicable iPSCs, we effi ciently generated integration-free patient-specifi c iPSCs from the CD34+ hematopoietic stem/ progenitor cells of an adult suffering X-chronic granulomatous disease (X-CGD), an inherited immunodefi ciency disease caused by
mutations in X-linked CYBB gene Using either a single polycistronic
or a combination of three EBNA1/OriP-based episomal vectors, we delivered the reprogramming factors effi ciently (>60%) to in vitro expanded CD34+ cells We obtained 3∼30 TRA-1-60+ colonies per million transfected CD34+ cells The derived
integration-free X-CGD iPSCs bearing a mutation in CYBB exon 5 have
normal ESC-like characteristics (such as AP+, SSEA-4+, OCT4+, NANOG+) and karyotype They form cystic embryoid body(EB) and teratoma containing three germ layer lineages during in vitro and in vivo differentiation, respectively Upon directed hematopoietic differentiation using a serum-free medium, 30∼50% CD34+CD45+ cells were detected 14 days after EB formation The episomal plasmids carrying reprogramming factors were undetectable after 10 passages following the iPSC derivation In order to enhance the gene targeting effi ciency, both ZFNs and TALENs targeting the disease-causing mutation were designed and synthesized Approximately 50%
of context-dependent assembly (CoDA)-based designs generate active ZFNs that can boost gene targeting effi ciency >100-fold TALEN offers more fl exibility, therefore more candidates can be tested around
Trang 2Molecular Therapy Volume 20, Supplement 1, May 2012
desired target sequence and offer more active DNA-cutting nucleases
We will report our effort on the TALENs targeting CYBB mutation in
X-CGD iPSCs and compare their effi cacy with ZFNs
Stem Cells (iPSCs) from the Urine of a Patient with
Duchenne Muscular Dystrophy
Xuan Guan,1 Yingai Shi,2 Chad D Markert,2 David L Mack,2 Tara
N Jones,2 Emily C Moorefi eld,2 Yuanyuan Zhang,2 Martin K
Childers.2
1 Department of Physiology and Pharmacology, School of
Medicine, Wake Forest University, Winston-Salem, NC; 2 Institute
for Regenerative Medicine, Wake Forest University,
Winston-Salem, NC.
Mature human somatic cells can be reprogrammed to a primitive
stage, resembling human Embryonic Stem cells (hES) isolated from
embryos These iPSCs acquire the same infi nite self-renewal ability
and pluripotent differentiation potential as ES cells Because of
these extraordinary properties, iPSCs can provide virtually infi nite
numbers of multiple types of somatic cells Thus, iPSCs hold great
potential for disease modeling, drug screening and regenerative
medicine New emerging technologies, such as microRNA delivery,
have vastly improved the effi ciency of the reprogramming process
However, iPSCs generation is still time-consuming (usually weeks)
with relatively low efficiency Moreover, most iPSCs derived
from skin fi broblasts or peripheral blood cells, require invasive
collection procedures Here we show, for the fi rst time, that iPSCs
can be generated from the urine of a patient with Duchenne muscular
dystrophy (DMD) Urine derived cells (UC) were expanded by
culturing DMD patient urine sediment in tissue-culture treated
plates Isolated UCs were fast-proliferating Moreover, they
intrinsically expressed high levels of c-myc and klf4, two factors in
the reprogramming cocktail For comparison, DMD patient UCs and
normal human fi broblasts were seeded on Matrigel-coated plates and
transduced with a polycistronic lentiviral vector expressing human
oct4, sox2, klf4 and c-myc Virus-infected cells were maintained in
original medium for 3 days before switching to hES medium mTeSR
ES-like colonies were observed around 7 days from UCs Those
colonies were alkaline phosphate positive, expressing the pluripotent
surface markers ssea4 and Tra-1-81 Exogenous transgenes, as
determined by red fl uorescence reporter, started to silence around
day 7 and complete transgene silenced colonies were noted around
day 10 to 14 Isolated large colonies could be manually picked and
passaged by day 12 In contrast, fi broblast derived iPSC colonies
generally require 3 weeks
RT-PCR array and immunostaining confi rmed the expression of pluripotent markers in several UC iPSC lines
An in vivo teratoma formation assay further confirmed the differentiation ability of iPSC to form three germ layers Together, these data demonstrate the feasibility of rapid iPSC generation from the urine of a DMD patient