52 Cell Therapy Improved the Lifespan of Murine Models of Accelerated Aging with Defective Proliferation and Myogenic Differentiation Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © Th[.]
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in the ES or iPS cells Furthermore, gene defects with unknown sites
of mutation and those involving large deletions cannot be restored by
homologous recombination Conventional gene transfer techniques
can also insert DNA randomly into the host genome, possibly even
resulting in cancer The use of human artifi cial chromosomes (HACs)
as a vector for gene therapy may solve these problems, because HACs
exhibit several important characteristics for an ideal gene therapy
vector, including stable episomal maintenance and the capacity to
carry large genomic loci with regulatory elements, thus allowing
the physiological regulation of the introduced gene similar to the
native chromosome Duchenne muscular dystrophy (DMD) is caused
by dysfunction in dystrophin gene Although several DMD gene
therapy vector have been developed, no episomal vector containing
the entire dystrophin genomic region has been reported, due to its
extremely large size (2.4Mb) As a proof of concept, we herein
report the correction of a genetic defi ciency in iPS cells derived
from DMD model (mdx) mice and human DMD patient, using a
HAC with a entire genomic dystrophin (Dys-HAC) which we have
recently developed The Dys-HAC was transferred to the mdx- or
DMD patient-specifi c iPS cells via microcell-mediated chromosome
transfer (MMCT) FISH and multiplex PCR analyses showed that
the Dys-HAC was present independently in the iPS cells and the
transferred Dys-HAC corrected the deletion of dystrophin in the iPS
cells, respectively Next, the iPS cells containing the Dys-HAC were
injected into nude mice to assess the capacity of the differentiation and
the expression in the teratoma The transplanted mdx-iPS (Dys-HAC)
cells gave rise to differentiate to three germ layers and the human
dystrophin expression was detected in the tissues Furthermore,
chimeric mice from the mdx-iPS (Dys-HAC) were produced to assess
the capacity of differentiation and human dystrophin expression in
vivo The Dys-HAC was detected in the all tissues examined and
the dystrophin was detected in the sarcolemmal membrene of the
chimeric muscle Therefore, the combination of patient-specifi c iPS
cells and HAC containing defective gene(s) provides a powerful tool
for gene and cell therapies
51 Contribution of PDGFRa-Positive Bone
Marrow Cells for Epithelial Regeneration in
Genetic Blistering Skin Disease, RDEB
Katsuto Tamai, Takehiko Yamazaki, Takenao Chino, Yasufumi
Kaneda
Gene Therapy Science, Osaka University Graduate School of
Medicine, Suita, Osaka, Japan.
Physiologic homeostasis of epithelial structures is maintained
by resident stem cells residing in the epithelia themselves In the
skin of patients with recessive dystrophic epidermolysis bullosa
(RDEB), the epidermal stem cell pool is continually depleted as a
result of detachment of full-thickness epidermis from dermis due to
genetic dysfunction of the dermo-epidermal basement membrane
zone Nevertheless, the epidermal regeneration mechanisms in
RDEB still adequately maintain functional epithelium in the skin,
suggesting epithelial stem/progenitor cell-supplementation from
extracutaneous sources In this study, we demonstrate that detached
RDEB epithelia release a 25kDa protein, designated hear as KOIKOI
(KOI2; come on come on in Japanese), to recruit platelet-derived
growth factor receptor alpha (PDGFRa)-positive bone marrow
cells via circulation Separated RDEB mouse epithelia immediately
releases KOI2in the blister fl uid within 10 to 20 seconds Flow
cytometry analysis indicated that intravenous inoculation of KOI2
induced mobilization of PDGFRa-positive cells in mouse circulation,
resulting in robust elevation of the PDGFRa-positive cell population
in the blood To elucidate the role of the mobilized PDGFRa-positive
bone marrow cells in the regeneration of RDEB skin in vivo, we
combined PDGFRa-positive/GFP-positive bone marrow cells
(P/G-BMCs) with wild-type bone marrow cells, and transplanted those
cells to the lethally irradiated mice to generate P/G-BMT mouse, followed by engraftment of RDEB mouse skin on the back of the P/G-BMT mouse In 2 weeks after the RDEB skin engraftment, signifi cant numbers of GFP-positive cells were observed in both mesenchymal and epithelial tissues of the engrafted RDEB skin The GFP-positive epithelial cells were then shown to express keratin 5
in the regenerating RDEB epithelia, clearly demonstrating that the PDGFRa-positive bone marrow cells contain particular population
to provide epithelial cells in the RDEB skin Collectively, our data suggest that KOI2, which is rapidly released from the separated RDEB epithelia of the blisters, contributes to mobilize PDGFRa-positive bone marrow cells in circulation, and accelerates regeneration of the skin lesions by recruiting those cells to raise marrow-derived mesenchymal and epithelial cells in the RDEB skin Finally we intravenously administered KOI2 to the mouse with full-thickness wound on the back of the skin KOI2 administration signifi cantly accelerated wound closure, and more extraordinary, inhibited scar formation by recruiting circulating mesenchymal cells in the dermis
of the regenerating skin Appropriate application of KOI2 protein or the expressing DNA vector may provide a novel therapeutic strategy to recruit PDGFRa-positive stem/progenitor cells of both mesenchymal and epithelial lineages from bone marrow to the damaged tissues for accelerating damaged tissue regeneration
52 Cell Therapy Improved the Lifespan of Murine Models of Accelerated Aging with Defective Proliferation and Myogenic Differentiation
Mitra Lavasani,1 Aiping Lu,1 Joseph Feduska,1 Andria R
Robinson,2 Laura J Niedernhofer,2 Johnny Huard.1
1 Department of Orthopaedic Surgery, Stem Cell Research Center, Pittsburgh, PA; 2 Department of Microbiology and Molecular Genetics, University of Pittsburgh Cancer Institute, Pittsburgh, PA.
Genetic depletion of ERCC1 (Excision repair
cross-complementation-1) or XPF (complementation group F) in humans
or mice causes accelerated aging of multiple organ systems including the musculoskeletal, nervous, hepatobiliary, renal and hematopoietic
ERCC1-XPF-defi cient (Ercc1 -/- and Ercc1 -/∆) mice express a unique
phenotype that mimics human progeria (or symptoms of accelerated
aging) XPF levels in Ercc1-/∆ mice are 10% that of wild type (WT)
mice, whereas Ercc1-/- mice have 0% of the normal level of XPF
Ercc1-/∆ mice have maximum lifespan of 7 months while Ercc1
-/-mice only live for 1 month Here we found that the number of slow
adhering cells (SAC) isolated from progeroid Ercc1 -/∆ mice using a modifi ed preplate technique was substantially lower than that yielded
from control littermates (Ercc1+/∆), indicating a loss of stem cells with
aging In vitro Ercc1 +/∆ cells were mitotically active and proliferate in
a similar fashion as the WT-SAC previously isolated and characterized
in our lab, while the Ercc1 -/∆ cells showed much reduced mitotic activity and proliferated poorly Cells derived from Ercc1+/∆ mice fused to form signifi cantly more and larger multi-nucleated myotubes
than their Ercc1 -/∆ counterparts (68.4% versus 21.9% respectively,
p<0.01) We also examined the potential of WT-SAC transplantation,
as a cell-mediated therapy to prevent or delay the onset of age-related
histopathologic changes and to extend the lifespan of Ercc1 -/- mice,
we transplanted The LacZ-expressing WT-SAC were detected in the
marrow of the long bones and a variety of abdominal organs including the liver, spleen, diaphragm, and pancreas Allowing the animals to live indefi nitely until “natural “death, we found that compared to the paired MDSC-PBS littermates, the mice injected with WT-SAC experienced pronounced weight gain (greater than 50% of initial weight) and have not only outlived their PBS littermates, but have also signifi cantly exceeded the normal life expectancy of the
PBS-treated Ercc -/- mice These results demonstrate that the number and function of SAC isolated from progeroid ERCC1-defi cient mice is impaired This suggests that either depletion of the adult stem cell
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compartment or their inability to proliferate and differentiate may
contribute to the dramatically accelerated aging of ERCC1-defcient
mice Our study also revealed that the delivery of WT-SAC, via
IP injection, was associated with an overall improvement in the
health of the mice including typical histopathologic symptoms of
accelerating aging and premature lifespan We believe this line of
research is highly signifi cant and it has the potential of developing
stem cell therapy approaches which could delay or ameliorate the
pathologies associated with premature aging while revealing the
underlying contributions that stem cells have on the basic biological
mechanisms of aging
53 A Vector System for Simultaneous Tracking
of Individual Reprogramming Factor Expression
during Human iPSC Induction and Differentiation
Eirini P Papapetrou,1,2 Mark J Tomishima,3,4 Stuart M Chambers,3
Yvonne Gruber,5 Lorenz Studer,1,3 Michel Sadelain.1,2
1 Center for Cell Engineering, Memorial Sloan-Kettering Cancer
Center, New York; 2 Molecular Pharmacology and Chemistry
Program, MSKCC, New York; 3 Developmental Biology Program,
MSKCC, New York; 4 SKI Stem Cell Research Facility, MSKCC,
New York; 5 Gerstner Graduate School, MSKCC, New York.
Human induced pluripotent stem cells (hiPSCs) are generated from
somatic cells by retroviral transduction of the four reprogramming
factors (RFs) Oct-4, Sox2, Klf4 and c-Myc Direct reprogramming
occurs with a low frequency which may, at least in part, be attributed
to the requirement for a stringent stoichiometry of RF expression A
problem faced by all efforts aimed at enhancing iPSC generation is
the lack of a consistent way to report effi ciency, since effects on RF
delivery cannot be separated from genuine effects on reprogramming
effi ciency Furthermore, silencing of the vector-encoded RFs in
iPSCs generated with lentiviral vectors and its signifi cance for
reprogramming and differentiation is unclear To better define
the stoichiometric requirements and dynamic expression patterns
required for successful hiPSC induction, we developed 4 bicistronic
vectors coexpressing each of the 4 RFs together with a discernable
fl uorescent protein, allowing real-time tracking of expression of
each individual RF during hiPSCs induction, maintenance and
directed differentiation Using this system, we accurately quantify
the reprogramming effi ciency to be 0.4-1% of quadruple transduced
human fetal fi broblasts We demonstrate for the fi rst time in a direct
prospective manner that stoichiometric deviations in RF expression
have a strong impact on the effi ciency of hiPSC induction We defi ne
the optimal stoichiometry of factor expression to be highly sensitive to
Oct-4 dosage and constrained as to the relative ratio of the three other
RFs and we quantify the effect of various stoichiometric aberrations
on reprogramming effi ciency These data will be critical for guiding
the implementation of optimized protocols for hiPSC induction and
the rational design of improved reprogramming vectors, such as
polycistronic, non-integrating vectors and protein delivery regimens
In addition, we show that profound lentiviral vector silencing is a
hallmark of successfully reprogrammed clones and demonstrate
that our system enables rapid screening of hiPSC clones that have
silenced the four vectors, as well as easy monitoring of potential RF
reactivation during maintenance or differentiation of established
hiPSC lines We show that residual low-level RF expression in
established hiPSC lines does not hinder early lineage specifi cation
towards all 3 germ layers Monitoring of the kinetics of expression
in single cells during the course of reprogramming reveals that
silencing follows acquisition of the pluripotent cell state and is not
selective for the RFs, indicating that silencing is an epiphenomenon
of the epigenetic remodeling of pluripotent cells rather than a factor
required for its acquisition The novel vector system presented here
presents a powerful tool for optimization of hiPSC generation and
mechanistic studies of the reprogramming process
54 Towards Safe Genetic Modifi cation of Human Epidermal Stem Cells by Zinc Finger Nucleases and Integrase Defective Lentiviral Vectors
Giulietta Maruggi,1 Angelo Lombardo,2 Pietro Genovese,2 Michael
C Holmes,3 Philip D Gregory,3 Fulvio Mavilio,1 Luigi Naldini.2
1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2 San Raffaele Telethon Institute for Gene Therapy and Vita Salute San Raffaele University, San Raffaele Scientifi c Institute, Milan, Italy; 3 Sangamo BioSciences, Inc., Richmond, CA.
Transplantation of autologous, genetically corrected Epidermal Stem Cells (ESC) has been successfully used to treat the skin genetic disorder Junctional Epidermolysis Bullosa (JEB) Whereas the therapeutic activity of this approach has been proven in the clinic, safety concerns were raised by the use of a gamma-retroviral vector
to express the laminin-5 beta 3 (LAMB3) Thus, the development
of new approaches aimed at safely correcting patient-derived ESC is urgently needed to further advance the treatment of JEB
We previously developed a gene targeting platform based on Zinc Finger Nucleases (ZFN) and Integrase-Defective Lentiviral Vectors (IDLV) to insert transgenes into pre-determined sites of the genome
by means of Homologous Recombination (HR) Here we assessed the feasibility of this approach in human keratinocytes by using two different sets of ZFN, one designed to target the CCR5 gene and the other to target the AAVS1 locus on chromosome 19 In ESC these two loci can be considered “safe harbors” for targeted transgene insertion since neither locus is involved in the self-renewal, growth and/or differentiation potential of these cells To identify which of the two platforms performs better in human keratinocytes, we fi rst compared their targeting effi ciency in the HaCat keratinocyte cell line using HR-donor molecules containing a GFP-expression cassette
By IDLV-mediated delivery of the ZFN pair and the cognate donor molecule for either site we reached up to 20% GFP-positive cells The mean GFP expression was higher in the cells treated with the AAVS1 targeting system We then assessed targeting specifi city by extensive molecular analyses of single-cell clones isolated from the ZFN/IDLV treated populations and found high rates of site-specifi c integration into the CCR5 gene As previously shown in other cell types, integration of the GFP expression cassette mostly occurred by an HR-mediated mechanism, in which either a single expression cassette
or its concatemers were inserted into the ZFN target site We then tested the CCR5 targeting platform in human primary keratinocytes derived from healthy donors and found up to 5% GFP-positive cells with evidence of site-specifi c transgene integration Overall, these experiments provide proof-of-principle of the feasibility of ZFN/ IDLV driven targeted integration in keratinocytes and establish a path toward genetically corrected skin implants derived from ESC carrying the therapeutic transgene in safe and pre-determined genomic location, and thus opening new possibilities for the treatment of genetic skin diseases
55 Effi cient Gene Transfer to Central Nervous System Progenitor Cells by Early Intraamniotic Delivery of Lentiviral Vector
David H Stitelman,1 Masayuki Endo,2 Philip Zoltick,3 Alan W Flake,4 Tim Brazelton.5
1 The Center for Fetal Research, The Children’s Hospital of Philadelphia, Philadelphia, PA.
Early gestational gene transfer may provide developmental opportunities for transduction of accessible stem cell populations that are not available later in development Early in gestation the neural ectoderm is exposed to the amniotic cavity via the open neural tube
At this developmental stage, nascent stem cells of the nervous system