709 phase 1 2 clinical trial in patients with decompensated liver cirrhosis treated with bone marrow derived endotelial progenitor cells: preliminary safety and efficacy analysis

709 Phase 1 2 Clinical Trial in Patients With Decompensated Liver Cirrhosis Treated With Bone Marrow Derived Endotelial Progenitor Cells Preliminary Safety and Efficacy Analysis Molecular Therapy Volu[.]

Molecular Therapy Volume 22, Supplement 1, May 2014

SOMATIC STEM CELLS

dmLT To determine the frequency of gene transfer; DC progenitor

cells, iDC or mDC were transduced with retroviral or lentiviral

vectors expressing GFP

The CD34+ cells expanded 10-20 fold during 2-3 week in vitro

culture When GM-CSF/IL-4 was added, proliferation ceased, but

cells acquired DC phenotypic and functional characteristics including

cell morphology, phenotype; phagocytosis, chemotaxis to lymph

node chemokines, and T cell stimulation properties The retroviral

vector LZRS-GFP transduced more than 40% CD34+ cells and iDC

Lentiviral vectors transduced iDC most effi ciently with >80% GFP+

Cultured DC were able to stimulate viral-specifi c IFN-γ ELIspot

forming units from autologous PBMC

In conclusion, a cytokine-based in vitro DC-generation method

was established, which started from rhesus CD34+ BM progenitor

cells and consisted of 3-steps: DC progenitor cell expansion, iDC

differentiation, and mDC activation Gene transfer into these

populations generated DC capable of stimulating antigen-specifi c

IFN-γ ELIspot Future studies are necessary to characterize the DC

function in vivo to maximize the clinical benefi t of the therapy

Env+ Cells

Stephen E Braun,1 Edith Walker,2 Gautam Sahu,2 Gail Skowron,2

Preston A Marx,1 Dorothee von Laer,3 Andrew MacLean,1 Richard

P Junghans.2

1 Tulane National Primate Research Center, Tulane University

School of Medicine, Covington, LA; 2 Infectious Diseases/Surgery,

Roger Williams Medical Center, Providence, RI; 3 Virology Section,

Innsbruck Medical University, Innsbruck, Austria.

Introduction: Infection with HIV-1 results in CD4+ T cell depletion

and the subsequent loss of immune function results in AIDS Although

HAART lowers plasma viremia, it requires life-long drug therapy

However, some patients control viral replication without HAART To

redirect CTL activity against HIV, the fi rst-generation CD4-chimeric

antigen receptors (CARs) used the CD4 extracellular domain to target

Envelope and the T-cell receptor zeta (TCRζ) intracellular signaling

domain to activate T cells These MLV-vectors targeted infected cells

in vitro, but failed to control viremia in clinical trials Previously, we

demonstrated that the membrane-associated C46 (maC46) fusion

inhibitor, when tethered to susceptible cells, binds HIV and blocks

viral replication – demonstrating protection and a strong selective

advantage in transduced cells To increase the immunosurveillance

of HIV; we genetically modifi ed autologous T cells to bolster and

redirect the “designer T cells” (dTc) towards an HIV-specifi c target

and measured CTL activity

Methods: To improve the CD4-CAR vectors, we added the

intracellular CD28 signaling domain to TCRz vector

CD4-TCRz and CD4-28z CAR were shuttled into PG13 packaging cells

(GaLV-pseudotyped) We also packaged MLV-vector expressing

the maC46 fusion inhibitor in Phoenix (amphotropic-pseudotyped)

clones We stimulated rhesus PBMCs with αCD3αCD28 and

co-transduced T cells with CD4-CAR and maC46 vectors To evaluate

the CTL activity of the dTc, we measured 1) HIV Env-specifi c

cytotoxicity using a novel real-time cytotoxicity assay as changes in

electrical impedance and 2) cytokine production using IFN-g ELIspot

Therefore, HEK 293T cells were transiently transfected with an HIV-1

Env expression plasmid (or control) and plated with CD4-CAR and

control-transduced populations at effector:target ratio of 1:1

Results: We observed gene transfer frequency up to 60-70%

of rhesus CD3+CD8+ T cells with the individual vectors and

co-transduction of 35% of the cells with both vectors In two experiments,

CD4-CAR populations specifically killed 293T-Env+ cells

Additionally, transduced populations showed increased frequency

of INF-γ spot-forming cells

Conclusions: In these studies, we showed that genetically modifi ed T cells were redirected to target HIV Env+ cells Additional intracellular signaling domains may increase CTL activity and/

or in vivo persistence Control of viremia without HAART would

revolutionize treatment for HIV patients

Somatic Stem Cells

Non-Invasive Imaging of Transplanted Cells in a Mouse Model of Hereditary Tyrosinemia Type 1

Raymond D Hickey,1 Shennen A Mao,1 Jaime Glorioso,1 Bruce Amiot,1 Lukkana Suksanpaisan,2 Kah Whye Peng,1 Scott L Nyberg,1 Stephen J Russell.1

1 Mayo Clinic, Rochester, MN; 2 Imanis Life Sciences, Rochester, MN.

Hepatocyte transplantation is a potential treatment for myriad metabolic liver disorders that are currently only curable by liver transplantation One major limiting factor is an inability to monitor cells longitudinally and non-invasively after transplantation to determine their biodistribution We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to monitor transplanted hepatocytes and set out to test this reporter system

in a rodent model of metabolic liver disease Wild-type C57Bl/6J mouse hepatocytes were transduced ex vivo using a lentiviral vector containing the mouse Slc5a5 (NIS) gene under the control

of a liver specifi c promoter Transduction effi ciencies of 75-80% were achieved and NIS-labeled cells could robustly concentrate radiolabeled iodine in vitro Next, NIS-labeled hepatocytes were transplanted into congenic fumarylacetoacetate hydrolase knockout (Fah-/-) mice, a small animal model of hereditary tyrosinemia type 1 NIS-labeled hepatocytes were readily imaged in vivo non-invasively

by single-photon emission computed tomography imaging with an increase in radiolabeled tracer detection correlating with an increase

in liver repopulation over time after intrasplenic injection of cells Additionally, NIS-imaging was able to unambiguously identify the extrahepatic biodistribution of transplanted hepatocytes in Fah-/- mice after intraperitoneal injection This work demonstrates the effi cacy

of NIS-labeling in the fi eld of cell transplantation and we anticipate that NIS-labeling of cells will allow further testing of hepatocyte and stem cell therapies for various liver disorders, not only in small animals, but in larger preclinical models also

709 Phase 1-2 Clinical Trial in Patients With Decompensated Liver Cirrhosis Treated With Bone-Marrow Derived Endotelial Progenitor Cells: Preliminary Safety and Effi cacy Analysis

Delia D’Avola,1 Veronica Fernandez-Ruiz,2 Francisco Carmona de

la Torre,5 Miriam Mendez,2 Felipe Prosper,3 Enrique Andreu,3 Jose Ignacio Herrero,1 Mercedes Iñarrairaegui,1 Carmen Fuertes,5 Jose Ignacio Bilbao,1 Bruno Sangro,1 Jesus Prieto,1 Jorge Quiroga.1

1 Liver Unit and Ciberehd, Clinica Universidad de Navarra, Pamplona, Spain; 2 Department of Hepatology and Gene Therapy, Centro Investigación Médica Aplicada (CIMA), Pamplona, Spain;

3 Cell Therapy, Clinica Universidad de Navarra, Pamplona, Spain;

4 Radiology, Clinica Universidad de Navarra, Pamplona, Spain;

5 Liver Unit, Clinica Universidad de Navarra, Pamplona, Spain.

Background and aims

Bone marrow-derived endothelial progenitor cells (EPC) promote liver regeneration and improve survival in animal models of acute and chronic liver disease

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

S274

SOMATIC STEM CELLS

This phase 1-2 clinical trial aimed to evaluate the safety, feasibility

and effi cacy of the treatment with autologous EPC in decompensated

cirrhosis

Matherials and methods

Child-Pugh>7 cirrhotic patients underwent aspiration of 50 ml of

bone marrow (BM) blood for ex vivo differentiation and selection of

EPC The fi nal product was resuspended in 50 ml and injected via

hepatic artery Patients were followed-up for 12 months

Results

Between May-11 and September-13, 14 patients (Child-Pugh 9,

range 8-11; MELD 17, range 8-27) were evaluated One patient died

before BM aspiration due to cause not related to liver disease and

another was a screening failure Among the 12 patients who underwent

BM aspiration, the feasibility of production of EPC was 84% and

91% with 1 and 2 punctions, respectively 11 patients were treated

Three patients died at day 25, 85 and 166 No treatment-related

severe adverse events after a median follow-up of 166 days (range

25-364) were observed MELD and Child-Pugh scores improved in

67% and 44% of patients at 30 days (n= 9) and in 66% and 44% at

90 days (n=9) Compared to baseline, hepatic vein pressure gradient

improved at 90 days in 55% of patients

Conclusion

Treatment with autologous BM-EPC is feasible and safe in

decompensated cirrhosis and may have benefi cial effects on liver

function

Airway Basal Stem/Progenitor Cells to Skew

Differentiation Towards the Secretory Cell Lineage

Matthew S Walters,1 Kazunori Gomi,1 Ann E Tilley,1 Ben-Gary

Harvey,1 Robert J Kaner,1 Ronald G Crystal.1

1 Weill Cornell Medical College, New York.

The human airway epithelium is a complex multi-cellular tissue

composed of basal, Clara, secretory and ciliated cells that line the

airway lumen Basal cells (BC) are the stem/progenitor cells of

the airway epithelium that differentiate into the other specialized

epithelial cell types during physiological turnover and repair In

response to environmental insult, including cigarette smoke, the

normal differentiation response of BC becomes dysregulated,

resulting in a disordered epithelium that characterizes chronic

obstructive pulmonary disease (COPD) Based on the concept that

gene transfer might be used to reestablish the normal differentiation of

the BC stem/progenitor cells, the objective of this study was to assess

whether it would be possible to shift the differentiation of primary

human airway BC to mucus-producing cells by genetic manipulation

To evaluate this concept, lentivirus gene transfer vectors were used

to infect human airway BC as they were differentiating on air-liquid

interface (ALI) culture Lentivirus reporter (GFP) constructs mediated

high transduction effi ciency (>90% cells GFP positive) and long-term

reporter expression (28 days on ALI culture) Quantifi cation of BC

differentiation at ALI Day 28 revealed no signifi cant deleterious

effects of lentivirus infection on differentiation of BC into a

mucociliated epithelium Based on the knowledge that the Notch

pathway is involved in a wide variety of cellular processes, including

turnover and repair of tissues and known to be dysregulated in the

airway epithelium of smokers and smokers with COPD relative to

healthy nonsmokers, as a demonstration of principal, we genetically

modifi ed BC activation of the Notch signaling pathway For sustained

Notch activation during BC differentiation, the constitutively active

intracellular form of the Notch 1, 2, 3 or 4 receptor (NICD1, 2, 3 or 4,

respectively) was expressed from lentivirus vectors and differentiation

assessed at ALI Day 28 Compared to control lentivirus-infected

cells, expression of NICD2 or 4 had no effect on differentiation of

BC into secretory and ciliated cells In marked contrast, expression of

NICD1 or 3 resulted in a signifi cant increase in the levels of secretory

cell genes and number of secretory cells, with a parallel decrease in expression of ciliated cell genes and number of ciliated cells These data suggest Notch 2 and 4 mediated signaling have no effect on human BC differentiation whereas activation of the Notch 1 and

3 pathways have a major effect on shifting differentiation towards the secretory lineage, i.e., genetic modifi cation of NICD 1 or 3 components of the Notch pathway of airway BC stem/progenitor cells can modulate the proportions of mucin-producing cells Overall, this study demonstrates the feasibility of genetic modifi cation of airway

BC to alter the proportion of mucin-producing cells, a strategy that may be used to suppress the increased numbers of mucin-producing cells that characterize the disordered airway epithelium in COPD

Expression on Hematopoietic Stem Cells -Improving the Chance of Finding an HLA-Matched Donor

Hiroki Torikai,1 Tiejuan Mi,1 Loren Gragert,2 Martin Maiers,2 Amer Najjar,1 Sonny Ang,1 Sourindra Maiti,1 Kirsten C Switzer,1 Helen Huls,1 Andreas Reik,3 Edward Rebar,3 Michael C Holmes,3 Philip

D Gregory,3 Richard E Champlin,1 Elizabeth J Shpall,1 Laurence

J N Cooper.1

1 MD Anderson Cancer Center, Houston; 2 National Marrow Donor Program, Minneapolis; 3 Sangamo BioSciences, Inc, Richmond.

Allogeneic hematopoietic stem cells (HSCs) are infused to restore

or replace absent or dysfunctional HSCs in the recipient and serve as

a source for generating specifi c hematopoietic cells The diversity of the human leukocyte antigen (HLA) system poses a hurdle to fi nd an HLA-compatible donor which is exacerbated by the impact of racial genetic polymorphism Furthermore, despite pre-banking umbilical cord blood (UCB) units and access to registered adult donors through the National Marrow Donor Program (NMDP), fi nding a suitable HLA-matched product remains challenging for many recipients, especially those from racial and ethnic minorities who are under-represented in the donor pool We hypothesized that the clinical application of donors already registered would be markedly increased

if allogeneic HSCs were genetically edited to eliminate expression

of the HLA-A locus Modeling from NMDP shows that the chance

of an African American recipient fi nding a HLA-matched donor increases from 18% to 73% when the need for a match at HLA-A is eliminated leaving HLA-B, C and DR We have previously shown that engineered zinc fi nger nucleases (ZFNs) can eliminate HLA-A expression in genetically edited T cells and cell lines To extend genetic editing to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus CD34+ lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB Electro-transfer of in vitro-transcribed mRNA species encoding the HLA-A-specifi c ZFNs generated 30% HLA-Aneg HSCs after one week ex vivo culture with defi ned cytokines (FLT3-L, SCF, TPO, and IL-6)

and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1) DNA sequence analysis revealed that HLA-Aneg HSCs display the

expected nucleotide changes at the ZFN target site In vitro assays

showed no signifi cant difference in lineage specifi c colony formation between HLA-Apos HSC and HLA-Aneg HSC Moreover, an in vivo engraftment assay, using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)

mice, demonstrated that engineered HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg multi-lineage hematopoietic cells Methods to improve access to the existing donor pool are anticipated to have clinical impact Indeed, the NMDP calculates that approximately 10,000 patients in the US could benefi t from unrelated allogeneic HSCT The elimination

of HLA-A expression in HSCs using ZFNs provides an appealing approach to increase the chance for fi nding HLA-matched donors This is anticipated to broaden the clinical application of allogeneic HSCT, especially for patients belonging to ethnic minority groups