693 effect of IL 3 on transduction efficiency in CD34+ cell derived erythroid cultures with lentiviral vectors expressing the human beta flobin gene

693 Effect of IL 3 on Transduction Efficiency in CD34+ Cell Derived Erythroid Cultures with Lentiviral Vectors Expressing the Human beta Flobin Gene Molecular Therapy Volume 17, Supplement 1, May 2009[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

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STEM CELL THERAPIES II

690 Antioxidant Treatment of Muscle Stem

Cells for Transplantation Increases Cardiac Repair

Lauren Drowley,1 Masaho Okada,1 Bradley Keller,2 Kimimasa

Tobita,2 Johnny Huard.1

1 University of Pittsburgh, Pittsburgh, PA; 2 Pediatrics, Children’s

Hospital of Pittsburgh, Pittsburgh, PA.

One major issue with cellular transplantation for cardiac repair has

been the poor survival of the transplanted cells, which is related to

the limited functional recovery seen clinically This is in part due to

the local environment at the site of transplantation, with oxidative

stress, infl ammation, and ischemia all playing key roles The ability

of cells to survive in this harsh environment could be critical in the

repair process, and cells that have increased survival could have a

greater impact on functional repair We have previously shown that

muscle-derived stem cells (MDSCs) signifi cantly ameliorate the

remodeling process in the ischemic heart compared to myoblasts

after myocardial infarction MDSCs also have lower levels of

apoptosis than myoblasts when oxidative stress is induced with

hydrogen peroxide Based on these results, we hypothesized that

one major characteristic of stem cells was an enhanced ability to

survive in stressful environments and that by up or down-regulating

antioxidants, we could infl uence repair To examine this, we reduced

levels of glutathione, a prevalent antioxidant, in MDSCs with diethyl

maleate (DEM), and also treated cells with N-acetylcysteine (NAC),

a molecule that has direct antioxidant effects as well as increasing

glutathione synthesis We examined in vitro characteristics including

cell survival after stress, proliferation, differentiation, and VEGF

secretion and found that antioxidant levels played a role in each In

a mouse model of myocardial infarction, we found that NAC-treated

MDSCs signifi cantly increased cardiac function, have decreased

formation of scar tissue, and increased levels of angiogenesis

compared to untreated and DEM-treated MDSCs These results show

that antioxidant treatment prior to cell transplantation can have a

signifi cant benefi cial effect

691 Sleeping Beauty-Mediated Gene Transfer

into Hematopoietic Stem Cells

Kendra A Hyland,1 Erik R Olson,1 Ron T McElmurry,2 Bruce R

Blazar,2 R Scott McIvor,1 Jakub Tolar.2

1 Discovery Genomics, Inc., Minneapolis, MN; 2 Pediatrics, Medical

School, University of Minnesota, Minneapolis, MN.

The Sleeping Beauty (SB) transposon system can transpose

sequences into mammalian chromosomes, supporting long term

expression of both reporter and therapeutic genes Advantages of the

SB system include ease of manufacture, the lack of genotoxic effects

after transposition and the absence of immunological complications

due to repeated administration Hematopoietic stem cells (HSC) are an

ideal target cell as they are used in therapy for a variety of hematologic

and other conditions Optimization of electroporation conditions,

assessed with a green fl uorescent protein (GFP) reporter plasmid and

a CytoPulse electroporator, included varying voltage, pulse width,

and pulse number to improve the loading of purifi ed cell populations

containing HSC, such as mouse lineage negative cells and human

umbilical cord blood CD34+ cells Cell viability was reduced by

10-30% compared to untreated cells, 1 to 2 days after electroporation

alone We demonstrate successful in vitro electroporation of

transposon and reporter plasmid DNA into mouse HSC, such that

5-10% of lineage negative cKit+ Sca-1+ cells (SKL) expressed GFP

on day 1 and 25-30% of human CD34+ cord blood cells expressed

GFP on day 2 post electroporation SB-mediated transposition of

HSC with a transposon containing the L22Y mutated dihydrofolate

reductase (DHFR) gene conferred methotrexate resistance after

electroporation as assessed in hematopoietic progenitor cells by in

vitro colony forming cell assays Studies are in progress to confi rm

transposition-mediated integration of individual transgenes by

sequence analysis of transposon-chromosome junctions recovered

by linear amplifi cation-mediated PCR Our data provide a platform

to establish a system for SB-mediated transposition of HSC and subsequent application to the treatment of human patients

692 Mechanical Loading of Stem Cells for Improvement of Cellular Cardiomyoplasty

Lauren Drowley,1 Theresa Cassino,1 Masaho Okada,1 Bradley Keller,2 Kimimasa Tobita,2 Philip LeDuc,3 Johnny Huard.1

1 University of Pittsburgh, Pittsburgh; 2 Children’s Hospital of Pittsburgh, Pittsburgh; 3 Carnegie Mellon University, Pittsburgh.

Although stem cell therapy for tissue repair is a rapidly developing

fi eld, the factors which dictate the physiological responsiveness of stem cells remain under investigation In this study we tested the hypothesis that controlling the loading history of muscle derived stem cells (MDSCs) with cyclic mechanical stimulation prior to transplantation signifi cantly improves MDSC survival and angiogenic effects on injured recipient myocardium Murine MDSCs were fi rst isolated using a preplating technique and then underwent 24 hours

of cyclic mechanical stretch To test their effi cacy for this loading history based approach, an acute myocardial infarction was created by permanently ligating the left coronary artery, and both unstimulated and mechanically stretched MDSCs were transplanted into the injured left ventricular myocardium Hearts implanted with mechanically preconditioned cells attenuated post-infarcted dilatation and sustained cardiac contractility for 12 weeks after myocardial infarction, which was signifi cantly better than hearts transplanted with non-stretched MDSCs Myocardium transplanted with stretched MDSCs also displayed signifi cantly higher angiogenesis in comparison to the non-stretched MDSC transplanted myocardium, which was further supported by a signifi cant increase in vascular endothelial growth factor secretion after mechanical preconditioning Results suggest that transplantation of mechanically stretched MDSCs into acute infarcted myocardium ameliorates post-infarcted remodeling better than non-stretched MDSC transplantation by promoting higher levels

of angiogenesis through paracrine factor secretion These fi ndings highlight the importance of loading history for increasing the effi cacy

of stem cell transplantation

693 Effect of IL-3 on Transduction Effi ciency

in CD34+ Cell-Derived-Erythroid Cultures with Lentiviral Vectors Expressing the Human beta-Flobin Gene

Leda Ferro,1 Clare Taylor,1 Daniel Hollyman,1 Michel Sadelain,1,2,3,4 Isabelle Riviere.1,2,3,4

1 Gene Transfer and Somatic Cell Engineering Facility, Memorial Sloan-Kettering Cancer Center, New York; 2 Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York;

3 Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York; 4 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York.

Gene transfer approaches for β-thalassemia using recombinant LVs requires effi cient gene transfer into HSCs and high level, stable expression of the b-globin gene in the erythroid lineage Although LVs are able to transduce non-proliferating cells, HSCs display low permissiveness to LVs and require cytokine prestimulation and high vector doses for high transduction effi ciency Standard CD34+ prestimulation protocols use a combination of three early-acting cytokines, Stem Cell Factor (SCF), Thrombopoeitin (TPO) and Flt3 ligand (Flt3-L) IL-3 alone or in combination with other hematopoietic cytokines has been shown to support multilineage colony formation, improve lentiviral and retroviral transduction and expansion of different lineages in liquid culture However its effect

on the engraftment potential of cultured CD34+ cells is controversial

Molecular Therapy Volume 17, Supplement 1, May 2009

STEM CELL THERAPIES II

We have developed a method to investigate the effects of different

prestimulation conditions on transduction effi ciency in vitro in short

term culture of GCS-F mobilized human peripheral blood (PB)

CD34+ cells Selected CD34+ cells are prestimulated, transduced

and differentiated into either myeloid (Dendritic cell) or erythroid

(Red blood cell) lineages in order to quantify vector copy number

(VCN) and transgene expression in the different lineages Using this

system we sought to investigate the effects of IL-3 prestimulation

using TNS9.3, a LV carrying the human β-globin gene and associated

regulatory sequences imparting erythroid-restricted transgene

expression, as well as the enhanced green fl uorescent protein (eGFP)

reporter gene driven by the human PGK promoter We demonstrate

that addition of IL-3 to the regular prestimulation cocktail (SCF, TPO

and Flt3-L) increases the percentage of eGFP positive cells from

10% to 35% and the average VCN from 0.5 to 1 After normalization

of the VCN to the percentage of GFP+ cells, the VCN per cell was

approximately 2.5 in the groups in which IL-3 was added and 5 with

the conventional cytokines prestimulation alone The VCN of single

erythroid and myeloid methylcellulose colonies was 1-3 (average 1.6)

for the groups in which the IL-3 was added and in the range of 1-5

(average 2.3) in the absence of IL-3 These data suggest that addition

of IL-3 increases the number of CD34+ progenitor cells permissive

to LV transduction Where as the absolute number of transduced cells

increases upon treatment with IL-3, we observed an accelerated loss

of the CD34 marker and an earlier onset of glycophorin A receptor

expression and a skewing of colony formation manifested as a

reduction in large immature colonies (BFU-E, CFU-GM) and an

increase of BFU-E, CFU-M and CFU-G We are currently performing

LTC-IC assays to further investigate the effect of IL-3 prestimulation

on self-renewal and multilineage potential of CD34+ cells

694 Optimization of Vector Design for Lentiviral

Gene Transfer into Human Embryonic Stem Cells

Emi Aizawa,1 Kaoru Mitsui,1 Keiichiro Suzuki,1 Hirofumi

Suemori,2 Norio Nakatsuji,3,4 Ko Mitani.1

1 Gene Therapy Division, Research Center for Genomic Medicine,

Saitama Medical University, Saitama, Japan; 2 Laboratory of

Embryonic Stem Cell Research, Stem Cell Research Center, Kyoto

University, Kyoto, Japan; 3 Department of Development and

Differentiation, Institute for Frontier Medical Sciences, Kyoto

University, Kyoto, Japan; 4 Institute for Integrated Cell-Material

Sciences, Kyoto University, Kyoto, Japan.

Lentiviral vectors (LVs) have become a valuable tool for gene

delivery because they can stably transduce a variety of cells, including

primate embryonic stem (ES) cells, in high effi ciency An important

characteristic of LVs is that they form a number of pseudotypes with

different tropisms by incorporating envelopes derived from other

viruses into the vector particles We compared ten pseudotypes

to optimize stable gene delivery into 293 cells, mouse ES (mES)

cells, cynomolgus monkey ES (cES) cells, and human ES (hES)

cells The envelope genes used for this experiment were those from

vesicular stomatitis virus (VSV-G), amphortopic retrovirus 4070A,

amphortopic retrovirus 10A1, gibbon ape leukemia virus (GALV),

RD114 feline endogenous virus, baculovirus (gp64), rabies virus,

Mokola virus, lymphocytic choriomeningitis virus (LCMV)-WE,

and LCMV-Arm536 Titers on each cell line were determined by

counting the number of G418-resistant colonies after infection with

a LV encoding the PGK-neo marker gene The VSV-G was the

most effective for stable gene transfer into 293 cells, mES cells, and

cES cells For hES cells, the gp64, the 4070A and the 10A1 have

been effective, as well as the VSV-G However, at multiplicities of

infection (MOIs) of higher than 100 vector particles/cell, the transfer

effi ciency with the gp64 was the highest Next, we have compared

the relative strengths as well as the extent of suppression of the

fi ve ubiquitous promoters as an internal promoter for LV in hES

cells They are the cytomegalovirus (CMV) enhancer/promoter, the human phosphoglycerate kinase (PGK) promoter, the mutant murine leukemia virus LTR (MNDU3c) enhancer/promoter, the human translation elongation factor 1 alpha (EF-1α) promoter, and the CMV enhancer/chicken β-actin (CAG) promoter They were tested

to drive the downstream enhanced green fl uorescent protein (EGFP) gene expression and the mean fl uorescence intensity was compared Because the titer of LV with CAG promoter was consistently lower than that with CMV by ∼10-fold, LV with an internal CAG was not investigated further While CMV was the strongest in 293 cells, EF-1α promoter showed the activity, which was higher than other three by approximately 3-fold, in hES cells Interestingly, for all four promoters, only 2-4% of the integrated vector expressed the marker gene, suggesting that high degree of suppression observed in human ES cells might be caused not by an internal promoter but by vector backbone sequences By using the EF-1α promoter in a gp64-pseudotyped LV, as high as 75% and 45% of the infected KhES-1cells and KhES-3 cells became GFP(+) at an MOI of 300, respectively Our results indicate that LVs are highly effi cient and versatile methods for gene transfer into hES cells and potentially other types of human stem cells, such as induced pluripotent stem cells

695 Application of a “Double-Feature”

Promoter To Identify Cardiomyocytes Differentiated from human Embryonic Stem Cells

Tamas I Orban,1 Agota Apati,1 Andrea Nemeth,1 Nora Varga,1 Virag Krizsik,1 Anita Schamberger,1 Gyorgy Varady,1 Katalin Nemet,1 Zsuzsanna Izsvak,2 Balazs Sarkadi.1

1 Membrane Research Group of the HAS, Semmelweis University and National Blood Center, Budapest, Hungary; 2 Mobile DNA Group, Max-Delbruck Center for Molecular Medicine, Berlin, Germany.

Human embryonic stem (HuES) cells represent a great potential for cell- and gene-therapy applications Apart from technical and experimental diffi culties, however, serious ethical concerns must

be resolved before they are actually used in clinical studies Major obstacles of HuES-based applications include the development of effi cient and stable gene delivery systems, as well as the effi cient control of directed tissue differentiations Nowadays, viral and non-viral applications are both used for gene transfer experiments and they are combined with various methods to obtain the tissue(s) of interest, including antibiotic selection with tissue-specific promoters or applying artifi cial drug cocktails Here we describe a novel approach

to identify tissues differentiated from HuES cells using a “double-feature” behavior of one version of the CAG promoter We generated GFP-expressing HuES clones under the expression of different promoters using the Sleeping Beauty non-viral transposon system

or the SEW lentiviral system Neither of the gene delivery methods modifi ed the subsequent differentiation of these HuES clones, and all kinds of tissue types could be identifi ed after a spontaneous differentiation via the embryoid body formation method By using the CAG promoter, in contrast to several other constitutive promoter

GFP expression was observed in differentiated cardiomyocytes This phenomenon was independent of the transgene sequence, methods

of gene delivery, copy number, and the integration sites Such

“double-feature” promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifi cally labeling differentiated cardiomyocytes, was assessed

by transcriptional profi ling To provide evidence that this promoter behavior manifests itself in tissue-dependent transcriptional profi les,

we collected and studied cell populations with different GFP signal intensities by fl uorescence activated cell sorting after differentiating

real-time quantitative PCR, we could confi rm that GFP mRNA levels