99 Zinc Finger Nuclease Mediated Genomic Editing of CCR5 in Human Hematopoietic Stem Cells for Clinical Application in HIV Gene Therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright ©[.]
Trang 1Molecular Therapy Volume 19, Supplement 1, May 2011
STEM CELL THERAPIES I
97 LYL1, a Basic Helix-Loop-Helix
Transcription Factor, Promotes Hematopoietic
Differentiation of Common Marmoset Embryonic
Stem Cells
Hirotaka Kawano,1 Tomotoshi Marumoto,1,2 Michiyo Okada,1
Tomoko Inoue,1 Takenobu Nii,1 Jiyuan Liao,1 Saori Yamaguchi,1
Yoko Nagai,1 Hiroyuki Inoue,1,2 Atsushi Takahashi,1,2 Erika Sasaki,3
Yoshie Miura,1 Kenzaburo Tani.1,2
1 Department of Molecular Genetics, Medical Institute of
Bioregulation, Kyushu University, Fukuoka, Japan; 2 Department
of Advanced Molecular and Cell Therapy, Kyushu University
Hospital, Fukuoka, Japan; 3 Central Institute for Experimental
Animals, KEIO University, Kawasaki, Japan.
Since the successful establishment of human embryonic stem
cell (ESC) lines by James Thomson in 1998, transplantation of
differentiated ES cells to the specifi c impaired organ has been
expected to cure its defective function For the establishment of the
regenerative medicine, the preclinical studies using animal model
systems including non-human primates are essential We have
recently demonstrated that non-human primate of common marmoset
(CM) is suitable for the preclinical studies of hematopoietic stem
cells (HSCs) therapy [Hibino H et al., Blood 93: 2839-48, 2009]
Since then we have continuously investigated the in vitro and in
vivo differentiation of CM ESCs to hematopoietic cells by the
exogenous hematopoietic gene transfer In earlier study, we showed
that the induction of CD34+ cells having a blood colony forming
capacity from CM ESCs is promoted by the ectopic expression of
TAL1/SCL However those CD34+ cells did not have a bone marrow
reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull)
mice, suggesting that transduction of TAL1/SCL gene is not enough
to induce functional HSCs which have self-renewal capability and
multipotency Thus we tried to fi nd other hematopoietic genes being
able to promote hematopoietic differentiation more effi ciently than
Tal1/SCL when they are expressed in CM ESCs We found that
lentiviral transduction of LYL1, a basic helix-loop-helix transcription
factor, in embryoid bodies(EBs) derived from Cj11, one of CM ESC
lines, dramatically increased the number of cells positive for CD34, a
marker for hematopoietic stem/progenitors We also found that
LYL1-overexpressing EBs showed the formation of multi-lineage blood cells
consisting of erythroid cells, granulocytes and macrophages by CFU
(colony-forming unit) assay These results indicate that the ectopic
expression of LYL1 can promote the differentiation of CM ESCs to
HSCs in vitro Furthermore we found that the combined activation
of TAL1/SCL and LYL1 can enhance the emergence of CD34+ cells
from CM ESCs than the activation of TAL1/SCL or LYL1 alone
This technology to induce hematopoietic differentiation of ESCs
by the transduction of specifi c transcription factors are novel, and
might be applicable for human regenerative medicine to cure human
blood diseases
98 Genome-Wide Defi nition of
Transcriptionally Active Regulatory Elements in
Human Stem Cells by Retroviral Scanning
Valentina Poletti,1 Alessia Cavazza,2 Annarita Miccio,2 Danilo
Pellin,3 Ermanno Rizzi,4 Giorgio Corti,4 Clelia Di Serio,3 Gianluca
De Bellis,4 Fulvio Mavilio.1,2
1 Gene Expression Unit, H.San Raffaele Scientifi c Institute,
Milano, Italy; 2 Center for Regenerative Medicine, University of
Modena and Reggio Emilia, Modena, Italy; 3 CUSSB, Vita-Salute
San Raffaele University, Milano, Italy; 4 Institute of Biomedical
Technologies, CNR, Milano, Italy.
The rapidly expanding information on the structural and functional
characteristics of the human genome allows the development of
genome-wide approaches to the understanding of the molecular
circuitry wiring the genetic and epigenetic programs of somatic stem cells High-throughput approaches are essential to study transcriptomes, chromatin dynamics and the use of regulatory elements in the genome Very few studies exist for somatic stem cells, due to the absence of unambiguous markers for their prospective isolation We have recently developed a tool to map functional regions
of chromatin in stem and progenitor cells, based on the integration properties of the Moloney murine leukemia virus (MLV) By using
a deep sequencing technology, we mapped >32,000 MLV integration sites in the genome of human CD34+ hematopoietic stem/progenitor cells (HPCs), and designed a detailed integration map throughout the genome MLV integrations cluster in regions that co-map with transcriptionally active, cell-specific promoters and regulatory regions bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin confi gurations (H2A.Z) Single locus analysis showed association of MLV clusters with known enhancers and promoters, and allow the identifi cations of new regions with putative regulatory function (Cattoglio et al., Blood 2010) Functional genome mapping through retroviral “scanning” may be used as a tool to identify regulatory regions involved in the execution of stem cell genetic programs, providing a substantial contribution to a comprehensive description of the molecular circuitry associated with stem cell functions We are exploiting this technique to identify regulatory elements associated with stemness, commitment and differentiation
of prospectively isolated or retrospectively defi ned human stem cells and their progeny, such as HPCs and erythroid and myeloid committed precursors, embryonic stem (ES) cells and ES-derived neural precursors, epidermal stem/progenitor cells (EpSCs) cells and EpSC-derived keratinocytes “Integromes” of at least 30,000 independent integrations are correlated to gene expression profi les and epigenetic genome annotations, and compared by rigorous statistical analysis to discover shared or lineage-specifi c regulatory regions
99 Zinc Finger Nuclease Mediated Genomic Editing of CCR5 in Human Hematopoietic Stem Cells for Clinical Application in HIV Gene Therapy
Lijing Li,1 Anitha Rao,1 Lan-Feng Cao,1 Kenneth Kim,3 Ursula Hofer,2 Paula Cannon,2 Philip D Gregory,3 Michael C Holmes,3
John Zaia,1 Qing Liu,1 John Rossi,1 David DiGiusto.1
1 Virology, City of Hope National Medical Center, Duarte, CA;
2 University of Southern California, Los Angeles, CA; 3 Sangamo BioScience, Richmond, CA.
The HIV co-receptor CCR5 is a validated target for HIV/AIDS
therapy We are currently developing methodologies for ex vivo
treatment of hematopoietic stem cells (HSC) with Zinc Finger Nucleases (ZFN) designed to disrupt CCR5 expression with the intent
of using these cells for autologous transplantation into patients with AIDS Adenoviral vectors have been developed to deliver and express Green Fluorescence Protein (Ad5/F35-GFP) or a CCR5-specifi c ZFN (Ad5/F35-SB728) for these studies We compared several compounds (interferon α, pleiotrophin and phorbol 12-myristate 13-acetate - PMA) known to have an effect on HSC activation, proliferation and differentiation for their ability to enhance adenoviral transduction, expression and (ultimately) ZFN activity at the CCR5 locus in HSCs CD34+ HSC were isolated from G-CSF mobilized peripheral blood and prestimulated with SCF, Flt-3L and Tpo and then transduced with various amounts of adenovirus or prestimulated (as above) plus either IFNα, PTN, or PMA prior to viral transduction Cells were assayed
for viability, GFP expression, ZFN activity and in vitro hematopoietic
potential over a 4 week period Our results demonstrate that while
as much as 50% of the cells are transduced and express GFP (from Ad5/F35-GFP) when exposed to vector alone, only PMA treatment resulted in both increased frequency of transduction (>98%), protein expression and ZFN activity (using Ad5/F35 vector expressing the
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S40
CARDIOVASCULAR GENE & CELL THERAPY
CCR5 ZFNs) with up to 45% of the genomic CCR5 target sites
modifi ed We have established doses of PMA and virus that support
high levels of ZFN activity with minimal impact on hematopoietic
potential as measured in in vitro assays We also tested endosomal
disruption agents (chloroquine and oakadeic acid) for their ability to
enhance ZFN expression in this system Both compounds enhanced
ZFN activity alone and Chloraquine also enhanced PMA-mediated
ZFN activity These results demonstrate that ZFN activity is enhanced
in CD34+ cells following PMA treatment at least in part due to
induction of very high levels of protein expression and that endosomal
disruption further enhances ZFN activity in this system We are
currently comparing these conditions in vivo in an immunodefi cient
mouse model (NSG) to determine the effect of low dose PMA on
engraftment and lymphoid lineage potential
100 Human Neural Stem Cells of
Parthenogenetic Origin
Ruslan Semechkin,1 Dmitry Isaev,1 Tatiana Abramihina,1 Nikolay
Turovets,1 Richard A West,2 Tatjana Zogovic-Kapsalis,1 Andrey
Semechkin.1
1 International Stem Cell Corporation, Oceanside, CA; 2 Western
Michigan University, Kalamazoo, MI.
The use of multipotent neural stem cells (NSC) in regenerative
medicine may provide a potential treatment for Alzheimer’s and
Parkinson’s diseases, amyotrophic lateral sclerosis and other
neurodegenerative diseases, with human pluripotent stem cells
providing a potentially unlimited source of NSC In this study, we
demonstrated for the fi rst time that human multipotent NSC stable
to long-term passaging can be derived from pluripotent human
parthenogenetic stem cells, followed by differentiation into neurons
and glial cells Human parthenogenetic stem cells (hpSC) are derived
from the inner cell mass of blastocysts obtained from unfertilized
oocytes that have been activated using parthenogenesis These cells
demonstrate characteristics typical for human embryonic stem cells
(hESC), including extensive self-renewal and in vivo as well as in
vitro differentiation into cells of all three germ layers Differentiated
derivatives of HLA homozygous hpSC with common haplotypes
may reduce the risk of immune rejection after transplantation; thus
offering signifi cant advantages for application in cell-based therapies
due to their enhanced ability to be immune-matched over hESC lines
that carry HLA genes in heterozygous form Moreover, derivation
of hpSC overcomes ethical hurdles associated with the derivation of
hESC from fertilized human embryos For NSC derivation we use an
approach based on the adherent model with modifi cations Rosettes of
neuroepithelial cells were formed in the colonies of hpSC grown in
the absence of feeder cells The generated rosettes expressed specifi c
neuroepithelial markers such as PAX6, SOX1, NES (Nestin), MSI1
(Musashi-1), and did not express pluripotency marker OCT4 The
rosettes were isolated, disaggregated into single cell suspension and
then propagated as an adhesion culture The human parthenogenetic
NSC (hpNSC) were passaged every 4-5 days enzymatically with a
1:2 split rate for more than 15 passages Reverse transcriptase
real-time quantitative PCR (qRT-PCR) revealed the expression of specifi c
neural markers NES (Nestin), SOX2 and MSI1 (Musashi-1) in the
hpNSC which was also confi rmed by immunocytochemical staining
of corresponding proteins At the same time, the expression of OCT4
was not detected at RNA and protein levels The cells keep uniform
morphology up to at least 15 passages and do not show constantly
increasing levels of ectomesenchymal markers SNAI1 and FOXD3 as
detected by qRT-PCR To demonstrate the ability to produce neurons
and glia, we allowed spontaneous differentiation for three weeks
After this time most of the differentiated cells have acquired specifi c
neuron morphology and were positive for anti-Tuj1 (Tubulin beta
III) labeled antibodies The expression of neuronal markers TUBB3
(Tubulin beta III) and MAP2 as well as glial markers FOXO4 and
GFAP was detected in these differentiated cells by qRT-PCR The ethical and immunomatching advantages make hpSC a very promising source of multipotent NSC for cell-based transplantation therapy On this basis, we derived for a fi rst time hpNSC that can be maintained
in culture and expanded for clinical usage
Cardiovascular Gene & Cell Therapy
101 Initial Results from a First-in-Man Study Delivering Non-Viral Gene Therapy JVS-100 To Treat Ischemic Heart Failure
Marc S Penn,1 Joseph Pastore,2 Rahul Aras,2 Didier Rouy,3 Gary Schaer,4 MaryJane Farr,5 Warren Sherman,5 Farrell Mendelsohn,6
Douglas Losordo.7
1 Cleveland Clinic Foundation, Cleveland, OH; 2 Juventas Therapeutics, Cleveland, OH; 3 BioCardia, San Carlos, CA; 4 Rush University Medical Center, Chicago, IL; 5 Columbia University, New York, NY; 6 Cardiology PC, Birmingham, AL; 7 Northwestern University, Chicago, IL.
Stromal cell-derived factor-1 (SDF-1) has been shown to have signifi cant effects in wound healing in multiple organ systems in a variety of animal models, including heart failure (HF) SDF-1 has been shown to recruit bone marrow derived stem cells, cardiac stem cells and to reduce cardiac myocyte cell death JVS-100 is a non-viral plasmid encoding human SDF-1 under control of the CMV promoter
We previously demonstrated that JVS-100 was safe at doses up to 100
mg and improved cardiac function and angiogenesis at doses up to
30 mg in a GLP study delivering JVS-100 via the BioCardia Helical Infusion Catheter (HIC) to pigs with ischemic HF We propose to present 4 month results for all cohorts of a Phase 1 open-label study
to test safety and bioactivity of JVS-100 in patients (pts) with class III
HF due to prior myocardial infarction (MI) Methods and Results:
17 NYHA Class III pts with prior MI (mean time from MI > 5 years) and ejection fraction ≤ 40% were enrolled 3 doses of JVS-100: 5 mg (n=4 pts), 15 mg (n=6 pts), and 30 mg (n=7 pts), were delivered to the peri-MI region via 15 endomyocardial injections with the BioCardia HIC Pts are being followed at 1, 4, and 12 months post-injection Safety is being evaluated by tracking adverse events with the primary safety endpoint being the number of major adverse cardiac events at
30 days Bioactivity is assessed by measuring changes from baseline in: echocardiographic parameters including left ventricular volume and ejection fraction, cardiac perfusion via SPECT imaging at baseline and 4 months after treatment and clinical parameters including NYHA class, 6 minute walk distance and quality of life All pts received 15 injections of JVS-100 To date, pts have been followed for a total
of 64 months Follow-up at 1 and 4 months in the 5 mg cohort and
1 month in the 15 mg cohort has been completed There have been
no adverse events likely related to the drug at 1 month in the 15 pts who have reached their 1 month follow-up visit At 4 months post-injection, all 4 pts in the 5 mg group improved resting perfusion compared to baseline, demonstrating bioactivity of JVS-100 Using
a composite endpoint of clinically signifi cant changes in combining echocardiographic and clinical endpoints, 3 of 4 pts improved in the 5 mg cohort at 4 months and 4 of 6 pts improved at 1 month in the 15 mg cohort 4 month safety and effi cacy data will be available
for all pts in April 2011 Conclusions: The initial fi rst-in-man data
suggests delivery of a non-viral SDF-1 gene therapy, JVS-100, via endomyocardial injection to post-MI heart failure patients is safe JVS-100 demonstrates bioactivity at the 5 mg dose level by improving resting perfusion Follow-up at all dose levels is ongoing and will provide further insight into JVS-100 safety and bioactivity at higher dose levels