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Cancer Gene Therapy (2019) 26:11-16

ARTICLEInducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy

Filippo Rossignoli

1

Giulia Grisendi

1,2

Carlotta Spano

1,2

Giulia Golinelli

1

Alessandra Recchia

3

Giulia Rovesti

1

Giulia Orsi

1

Elena Veronesi

1,4

Edwin M. Horwitz

5

Massimo Dominici

1,2

Received: 22 March 2018 / Revised: 23 May 2018 / Accepted: 31 May 2018 / Published online: 29 June 2018

© The Author(s) 2018. This article is published with open access

Abstract

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and

oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face

this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once

infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have

been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a

variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell

death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in

MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested

for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor

approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we

generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data

show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the

tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-

based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.Introduction Mesenchymal stromal/stem cells (MSC) are a hetero-

geneous population offibroblast-like cells originallyisolated from bone marrow and other tissues including

adipose tissue, peripheral blood, umbilical cord blood, and Wharton's jelly, among others [1,2]. MSC retain ther- apeutic potential in several pathological conditions through different mechanisms such as differentiation into mature cells, secretion of cytokines, and release of microvesicles [3,4]. Moreover, growing evidences revealed that MSC retain unique immunological features that are relevant for the treatments of immune-related disorders [5] and for their possible use as cellular vehicles to deliver bioactive mole- cules inside tumor parenchyma and stroma [6]. In particular, this latter possibility gained a relevant interest in the last decade and we and others have been developing anti-tumor approaches based on MSC expressing the potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), variants demonstrating efficacy against several tumors [7-9,

Spano et al., Submitted].

Despite encouraging results, there is some level

of concern regarding the safety of inoculating both wild- type and gene-modified MSC in particular concerning their possible damage to normal organs, malignant*Filippo Rossignoli filippo.rossignoli@unimore.it *Massimo Dominici massimo.dominici@unimore.it 1 Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy 2

Rigenerand srl, Modena, Italy

3 Department of Life Sciences, University of Modena and Reggio

Emilia, Modena, Italy

4

Technopole of Mirandola TPM, Modena, Italy

5 Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta, Emory University,

Atlanta, GA, USA1234567890();,:

1234567890();,:

transformation, and promotion of cancer growth [10]. One possibility to face these possibilities is to incorporate a suicide gene in MSC; the most commonly used are the Herpes Simplex Virus Thymidine Kinase and the Cyto- sine Deaminase [11]. These transgenes confer the ability to convert a non-toxic prodrug into an active cytotoxic compound that kills the cell itself and the neighbors. This "bystander effect"has been exploited to specifically deliver cytotoxic compounds into tumor burdens with therapeutic benefit limiting possible off target toxicity [12]. However, these systems demonstrated a number of disadvantages starting from their possible immunogeni- city with consequent elimination by immune system [13] and the high cell-cycle dependency of the activated drugs that limits killing capacity to dividing cells [14]. Addi- tional drawbacks may also come from the limitation in concurrent administration of drugs (i.e., Ganciclovir) that, delivered for a possible CMV-viral infection, can lead to unintended elimination of gene-modified cells [15]; fur- ther evidences indicate also the possible onset of drug resistance on target cells [16]. In recent years, alternative strategies have been devel- oped to overcome these limitations. In particular, investi- gators described a novel suicide gene, namely, iCasp9, based on the sequence of human Caspase9 encoding for a modified protein having a high affinity to a synthetic, bioinert small molecule (B/B Homidimerizer, AP20187) resulting in iCasp9 dimerization and activation eventually leading to cell death [15]. This approach offers several advantages over the other suicide strategies, such as the human origin of the protein, as favorable condition to avoid immunogenicity. Moreover, the suicide trigger is indepen- dent from the cell cycle phase and does not interfere with any drug schedule [15]. Several studies showed the high efficacy and specificity of iCasp9 [10,17,18] and a safety study on healthy volunteers demonstrated the feasibility of the treatment with the dimerizer molecule in humans [19]. Based on this background, we challenge this approach on anti-cancer MSC delivering TRAIL to generate a proof of concept that would ultimately increase the safety of our cell therapy approach.

Materials and methods

Cell culture

The human embryonic kidney cell line 293T and the human Ewing sarcoma cell line A673, both from our laboratory, were maintained in culture at 37 °C in Dulbecco's Modified Eagle's Medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine

serum (PAA Laboratories Inc., Etobicoke, Canada), 1%penicillin/streptomycin (10,000 U/mL Penicillin, 10mg/mL

Streptomycin in 0.9% NaCl solution, PAA Laboratories Inc.). Adipose-derived MSC (AD-MSC) were obtained as previously described from lipoaspirate specimens of indi- viduals undergoing liposuction for esthetic purposes after approval by local Ethical Committee [7]. Cells from two different donors were used as biological replicates. After isolation, cells were grown inα-MEM (Gibco) containing

2.5% human platelet lysate (Modena Policlinic Blood

Bank, Modena, Italy), 1% L-Glutamine (200 mM in 0.85% NaCl solution, BioWhittaker, Lonza, Verviers, Belgium),

0.5% Ciprofloxacin (Fresenius Kabi Italia S.r.l., Verona,

Italy), 1IU/ml Heparin (Sigma-Aldrich, St. Louis, MO, USA). When confluent, the adherent AD-MSC cells were detached with trypsin/EDTA (Trypsin 0.05% EDTA 0.02% in PBS, EuroClone, Milan, Italy), counted and seeded at

6000 cells/cm

2 . Cells were incubated and maintained within a controlled atmosphere (5% CO 2 and temperature of

37 °C).

Vector generation and MSC transduction

The pMSCV-F-del Casp9.IRES.GFP plasmid was obtained from Addgene repository (Addgene plasmid #15567, http://www.addgene.org/). iCasp9 gene was subcloned into a third-generation lentiviral backbone (pCCL.PGK.WPRE) and the resulting construct was transiently transfected into

293T cells together with a mixture of helper plasmids

according to jetPEI protocol (Polyplus Transfection, Ill- kirch, France). After 48 h, conditioned medium containing lentiviral particles was collected and used to transduce AD-

MSC, thus obtaining AD-MSC iCasp9. AD-MSC expres-

sing TRAIL (AD-MSC TRAIL) from our laboratory [7] have been further engineered in the same way to express iCasp9 gene (AD-MSC TRAIL-iCasp9). AD-MSC trans- duced with the empty lentiviral backbone were used as control (AD-MSC EMPTY).

Dose-response apoptosis induction assay

Modified AD-MSC were tested for apoptosis induction after the addition of B/B Homodimerizer (AP20187, Clontech Laboratories, Inc., Mountain View, CA, USA) by MTS metabolic assay (CellTiter

96 AQueous One Solution Cell

Proliferation Assay, Promega Corporation, Madison, WI, USA). Briefly, for each tested sample and dimerizer con- centration, 5000 cells were seeded in a well of a 96-wells plate. The following day, medium was replaced with fresh one containing different concentrations of B/B Homo- dimerizer (0.01 nM, 0.1 nM, 1nM, 10 nM, 100 nM), and after 24 h the plate was analyzed with a Multiskan FC Microplate Photometer (Thermo Fisher Scientific Inc.), according to manufacturer's instructions.

12F. Rossignoli et al.

AD-MSC TRAIL-iCasp9 cytotoxicity assay

AD-MSC TRAIL-iCasp9 have been then tested for cyto- toxic activity against the A673 cell line through a 51
Cr release assay in coculture and compared with AD-MSC

TRAIL and AD-MSC EMPTY. Briefly, tumor cells were

labeled with 51

Cr and then cultured alone or together with

AD-MSC, AD-MSC TRAIL, or AD-MSC TRAIL-iCasp9

for 8 and 24h at several ratio of target:effector cells (1:1,

1:2, 1:5).

51

Cr released has been detected by 2450

microplate counter MicroBeta2 TM (PerkinElmer, Wal- tham, MA, USA). The experiment layout comprises a maximum 51

Cr release condition obtained by lysing

51
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