497 human erythropoietin gene delivery using an arginine grafted bioreducible polymer system

497 Human Erythropoietin Gene Delivery Using an Arginine Grafted Bioreducible Polymer System Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy[.]

Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy

S192

HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II

495 Validation of Cellular Function and Imaging

Capability of CD34+ Stem Cells Labeled with a 19F

MRI Contrast Agent

Brooke Helfer,1 Anthony Balducci,1 Zhina Sadeghi,2 Chris Flask,3

Amy Wesa.1

1 Research and Development, Celsense, Inc., Pittsburgh, PA;

2 Department of Urology, Case Western Reserve University,

Cleveland, OH; 3 Depts of Radiology and Biological Engineering,

Center for Imaging Research, Case Western Reserve University,

Cleveland, OH.

Non-invasive detection and tracking of stem cell post-delivery

lends insightful information on the mechanism of action of an

intended therapy and/or possible reasons for therapeutic failure

However, rigorous examination of the effects of the contrast agent on

the cells in question must be performed to ensure minimal disruption

to the therapy In this work, we examine the effects of a novel

self-delivering 19F MRI contrast agent on hematopoietic stem cells (HSC)

Comparison of 19F-labeled HSC and unlabeled control cohorts in in

vitro colony forming assays resulted in equal numbers of total colony

forming units (CFU), as well as individual CFU types, indicating

that label presence did not effect multipotency (see Figure 1) In vivo

reconstitution studies in mice, using labeled and unlabeled murine

bone marrow HSC, resulted in equivalent development of CFU in

the spleen, as well as reconstitution of the lymphoid and myeloid

compartments The fact that these highly complex processes remain

largely unaltered in the presence of contrast agent provides strong

evidence of the inertness of the reagent and that the therapeutic

potential of the cells is likely maintained Further, proof of principle

MR imaging of injected and implanted HSC in a rat thigh was

performed, indicating that HSC can be detected with MR methods

with the use of this contrast agent The data suggests that imaging

data on the location and persistence of delivered CD34+ HSC can

be obtained without infl uencing the function and/or differentiation

potential of the therapeutic cells

Figure 1: Average of two independent counts of colony formation

and types (BFU= Blast forming unit, E=erythroid, GM=granulocyte,

macrophage, GEMM=granulocyte, erythroid, macrophage, megakaryocyte) after 14 days of culture in differentiation media

Figure 2: MRI imaging of cells and persistence after impantation

in a rat hindlimb A Proton image of rat hindlimb anatomy B 19F image of implanted labeled cells (acquisition time ∼1hr) C 19F/1H overlay, with 19F rendered in pseudocolor D 19F/1H overlay using

a post-sacrifi ce, 11-hour 19F scan

496 Optimised Factor VIII Vectors for Gene Therapy of Haemophilia A

Natalie J Ward,1 Suzanne M K Buckley,1 Ahad A Rahim,1 John

H McVey,2 Simon N Waddington.1

1 Gene Transfer Technology Group, Institute for Women’s Health, UCL, London, United Kingdom; 2 Thrombosis Research Institute, London, United Kingdom.

Haemophilia A is a compelling candidate for treatment with gene therapy as therapeutic benefi t only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and FVIII expression can be mediated by many cell types in vivo B-domain deleted (BDD) FVIII protein retains full procoagulant function and is expressed at higher levels than wild type FVIII However a partial deletion of the B-domain leaving

an N-terminal 226 amino acid stretch (N6) has been previously reported to increase in vitro secretion of FVIII tenfold compared

to BDD-FVIII Previously, our group has shown that expression of FVIII protein from a codon optimised cDNA sequence increases expression in comparison to the wild type sequence over 30-fold

in vivo yielding over 200% normal human FVIII levels in neonatal mice after injection of a SIN lentiviral vector A longstanding goal for treatment of haemophilia A using gene therapy is to maintain sustained production of FVIII in the absence of an immune response in adult mice In recent studies we have used an optimised vector system including our codon-optimised FVIII N6 cDNA sequence under control of the liver specifi c promoter LP1 to maximise expression in hepatocytes Further to this, we have also included target sequences for the hematopoietic specifi c microRNA, miR-142-3p, to eliminate off target expression in antigen presenting cells With this approach

we hope to achieve stable long term expression of FVIII in adult FVIII knock-out mice

497 Human Erythropoietin Gene Delivery Using

an Arginine-Grafted Bioreducible Polymer System

Youngsook Lee,1 Hye Yeong Nam,1 Jaesung Kim,1 Minhyung Lee,1,2 James W Yockman,1 Sug Kyun Shin,3 Sung Wan Kim.1,2

1 Center for Controlled Chemial Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT; 2 Department of Bioengineering, College

of Engineering, Hanyang University, Seoul, Republic of Korea;

3 Division of Nephrology, Department of Internal Medicine, NHIC, Ilsan Hospital, Gyeonggi-do, Republic of Korea.

Anemia is a major complication of chronic kidney disease (CKD) and is primarily due to impaired erythropoietin (EPO) production

by the failing kidney, leading to EPO defi ciency Erythropoiesis stimulating agents are widely used to treat anemia for CKD and cancer, however, several clinical limitations impede their effectiveness To overcome many of these clinical hurdles, gene therapy providing continuous release has been suggested as an attractive alternative to current intermittently administered erythropoiesis-stimulating agents

Molecular Therapy Volume 20, Supplement 1, May 2012

Copyright © The American Society of Gene & Cell Therapy S193

ENGINEERING OF GENE REGULATION

Recently, we developed an arginine-grafted bioreducible

poly(CBA-DAH, disulfi de amine) [ABP] polymer for non-viral polymer-based

gene delivery Here, we extended our previous in vitro studies by

evaluating the erythropoietic effect of a single systemic ABP

polymer-based human erythropoietin plasmid DNA (phEPO) delivery system

on hematocrit level, reticulocyte count, plasma hEPO protein levels,

and organ distribution of hEPO mRNA in rats Systemic non-viral

gene therapy has been impeded by the low levels of transfection

effi ciency and lack of sustained gene expression In the present study,

we found that a single systemic injection of ABP-complexed phEPO,

sustained higher hematocrit levels, increased reticulocytosis, raised

expression levels of plasma hEPO In addition, we observed that the

distinct temporal and spatial distribution of phEPO/ABP polyplexes

contributed to increased erythropoietic effects compared to those of

traditional EPO therapies Overall, Our studies demonstrate that the

ABP polymer may be used as an advanced carrier for hEPO gene

delivery, and provide a potent and attractive clinical approach to

enhance erythropoiesis in vivo.

498 Development of In Vivo Gene Therapy of

Hemophilia A Mice by Intra-Bone Marrow Injection

of Lentiviral Vectors Containing a B-Domain

Variant of Human Factor VIII

Xuefeng Wang,1 Andy Chiang,1 Peiqing Ye,1 Carol H Miao.1,2

1 Seattle Children’s Hospital Research Institute, Seattle, WA;

2 Department of Pediatrics, University of Washington, Seattle, WA.

Our previous studies confi rmed that hematopoietic stem cells

(HSCs) in bone marrow (BM) of hemophilia A (HemA) mice can

be effi ciently transduced to express the transgene one week after

direct intra-bone marrow (iBM) injection of lentiviral vectors (LVs)

containing a gene encoding GFP (GFP-LV) or B-domain variant

human factor VIII (hFVIII/N6) (E-FVIII-LV) driven by ubiquitous

promoters However, the transgene expression levels changed over

time GFP+HSC cell numbers in BM reached largest on day 9 after

injection of GFP-LV, and then fell quickly afterwards The plasma

FVIII levels (0.1-2 U/mL) in E-FVIII-LV-treated mice fl uctuated over

time and decreased to undetectable one month postinjection; in some

mice, anti-FVIII antibodies (Abs) were produced In order to achieve

persistent transgene expression, in this study, we combined iBM

injection of LVs with immunomodulation Firstly, we investigated

if transient depletion of cytotoxic CD8+ T cells by intraperitoneal

(I.P.) injection of antiCD8α Ab can prevent the elimination of the

LV-transduced cells We injected 0.08 mg antiCD8α Ab per mouse

on day -1 and 0.05 mg antiCD8α Ab per mouse on day 4, 11, 16 and

21, and GFP-LV (9.3 × 108 TU/mouse) on day 0, and tracked GFP

expression in HSCs in the BM Compared with LV-only treated mice,

there were more GFP+HSCs in the BM of LV + antiCD8α Ab treated

mice on day 9 (529±65 cells per 1 × 106 BM cells v.s 198±118 cells

per 1 × 106 BM cells) Although GFP+HSC number in these two types

of mice decreased over time, LV + antiCD8α Ab treated mice still had

relatively larger numbers of GFP+HSC on day 63 (38±33 cells per 1

× 106 BM cells v.s 5±4 cells per 1 × 106 BM cells) Flow cytometry

data further showed that during antiCD8α Ab treatment, over 80%

of CD3+CD8+ cells in BM were depleted on day 3, however, these

cells gradually recovered on day 9 and reached to the normal level

on day 15 Thus, transient depletion of CD3+CD8+ cells in BM by I.P

injection of antiCD8α Ab could partly rescue the transduced BM cells

Secondly, we employed 10 HemA/Foxp3-Tg mice in which all T cells

over-expressed Foxp3 as an immunosuppressive mouse model and

treated them with low (3.5 × 105 TU/mouse) or high (1.7 × 106 TU/

mouse) dosage of E-FVIII-LVs, respectively None of the 10 treated

HemA/Foxp3-Tg produced anti-FVIII Abs; however plasma FVIII

levels in all 10 mice fell to low levels one month after treatment

Thus, increase of CD4+Foxp3+Tregs did not inhibit the elimination

of transduced cells by cytotoxic T cells despite that it prevented

anti-FVIII Ab production Based on the above results, in order to achieve persistent transgene expression of hFVIII in the HemA mice treated with iBM injection of E-FVIII-LVs, we will continue to develop more effi cient protocols to inhibit cytotoxic-T-cell-mediated elimination of the transduced cells and to expand FVIII-specifi c Treg cell populations

to block anti-FVIII Ab formation simultaneously

Engineering of Gene Regulation

499 Upregulation of the Pluripotency-Associated miRNA 302-367 Cluster Using Engineered Transcription Activator-Like Effector (TALE) Activators

Morgan L Maeder,1,2 James F Angstman,1 Sam J Linder,1 Deepak Reyon,1 J Keith Joung.1,2

1 Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA; 2 Biological and Biomedical Sciences Program, Harvard University, Boston, MA.

microRNAs (miRNAs) play critical roles in gene expression, development and pluripotency Certain families of miRNAs are highly expressed in embryonic stem cells (ESCs) and have been shown to play a role in regulating pluripotency For example, recent work has shown that the miRNA 302-367 cluster is a direct target of Oct4, Sox2 and Nanog and plays a critical role in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) Transcription activator-like effectors (TALEs) are DNA-binding proteins from the Xanthomonas genus of bacteria that can be engineered to bind novel DNA sequences Engineered TALEs can be fused to activation domains and act as artificial transcription factors to increase expression of endogenous genes in human cells However,

to our knowledge, TALE activators have only been used to stimulate expression of protein coding genes and not of miRNAs Here, we describe the generation of engineered TALE-activators targeted to the miRNA 302-367 promoter region Using single TALE-activators,

we observed modest increases in expression of the miRNA

302-367 cluster in primary human fi broblasts By expressing multiple TALE activators targeted to neighboring sequences simultaneously,

we were able to induce larger increases in miRNA 302-367 cluster expression, presumably due to synergistic transcriptional activation

We tested the use of both plasmid DNA and in vitro transcribed mRNA to express TALE-activators in human cells and investigated the duration of elevated miRNA expression over time Our results demonstrate the feasibility of using engineered TALE activators

to upregulate expression of an endogenous miRNA cluster The capability to purposefully alter miRNAs levels should provide a useful tool for exploring their critical roles in gene expression, development and pluripotency and for affecting the expression of downstream gene targets Such tools may also enable manipulation of complex phenotypes, such as reprogramming, which require changes in the expression level of hundreds of genes regulated by miRNAs

500 Light-Inducible Spatiotemporal Gene Regulation Using Engineered Transcription Factors

Lauren R Polstein,1 Charles A Gersbach.1

1 Biomedical Engineering, Duke University, Durham, NC.

Systems that facilitate the precise control of gene expression have numerous applications in the fi eld of gene therapy Current methods control gene expression via constitutive, tissue-specifi c,

or molecularly-induced promoters or by the expression of a gene-specifi c transcriptional activator However, these systems have not been designed to facilitate simultaneous spatial and temporal control

of gene expression, which is desirable for many gene therapy and