Bioengineering MSCs for untapped therapeutic potential

Mesenchymal stromal cells (MSCs) are multipotent homeostatic regulators of the body that have both multilineage differentiation potential and self-renewal capabilities [1]. These ’stem-like’ cells are readily available and exist in the stroma of the bone marrow, adipose tissue, the synovium of the joints, dental pulps, the umbilical cord and the liver among other tissues [1,2]. MSCs are immune-evasive allowing for off-the-shelf use. Given MSC availability, their low immunogenicity, significant immunomodulatory and regenerative capabilities, MSCs are considered an attractive option for conversion to cellular therapies for treatment of immune disease [1,2].

Despite the promising potential of MSCs for the treatment of immune disease ranging from graft-vs.-host diseases (GVHD) to Crohn’s and amyotrophic lateral sclerosis, there have been no resultant US FDA approvals to date. However, MSCs have been approved for the treatment of steroid refractory acute GVHD and implications of colitis in other countries including Japan, Canada, Europe and New Zealand [2,3]. These approved MSC therapy products span from autologous to allogeneic sources and are derived from the bone marrow, adipose and umbilical cord sources. Among the clinical products furthest along in the path to FDA acceptance are an allogeneic bone marrow derived MSC, called Mesoblast (remestemcel-L) for use in pediatric steroid refractory GVHD. Most recently, Mesoblast treatment led to a 74.1% overall survival rate at day 100 in a phase 3, prospective, single-arm, multicenter study (NCT02336230) treating 54 children with primary steroid refractory GVHD [4].

Nonetheless, full approval in the USA has mostly been thwarted by lack of consistent MSC therapeutic efficacy across clinical trials [3,5,6]. MSC failures are characterized by low survival, engraftment, homing to damaged tissues as well as insufficient immunosuppression at inflammatory sites following their ex vivo culture [2,3,5]. Additionally, MSCs have high heterogeneity from donor to donor, as well as from different tissue and isolation sources, which further complicates the consistency of their therapeutic efficacy [6]. These underwhelming findings and untapped MSC therapeutic potential call forth the need to increase efforts in MSC cellular engineering to enhance therapeutic outcomes in the clinic.

To directly improve the shortcomings associated with MSC therapeutic efficacy, various researchers have employed genetic and nongenetic engineering strategies to enhance MSC function. Cellular engineering has been used to help overexpress beneficial proteins for MSC therapeutic function including the promotion of MSC survival, pro-apoptotic proteins, enhanced migration to target sites and enhanced immunosuppressive function [3,7,8].

1. Nongenetic MSC engineering

Among the most prevalent nongenetic engineering strategy used to improve MSC function is MSC priming. Priming approaches have been employed with cytokines, growth factors, alternative culture conditions, hypoxia, herbal components and biomaterials such as pharmacologic drugs, small molecules, among other factors [9,10].

1.1. Cytokine priming

Preconditioning MSCs with IFNy, among other inflammatory cytokines, leads to an upregulation of immunosuppressive IDO1 gene, priming the MSC to secrete immunomodulatory molecules PGE2, HGF, TGF-β and CCL2 as a feedback mechanism [9,11].

1.2. Culture conditioning

MSCs have also shown improvements based on their culture conditions. It has been reported that 3D culturing techniques of MSCs more closely mimics the in vivo state of MSCs as compared with 2D culture. As such, the development of 3D MSC spheroids has led to improved immunosuppressive outcomes in murine peritonitis, colitis and rheumatoid arthritis [12].

1.3. Hypoxic priming

Another strategy to improve MSC proliferative capabilities is through culturing MSCs in hypoxic environments [9]. MSCs adapt to the reduced oxygen in hypoxia by increasing their proliferative capabilities, reducing their rates of apoptosis and increasing HIF1a-associated expression of chemokines involved in MSC trafficking [9,13].

Altogether, these strategies are used in attempt to mimic the inflammatory environment and other cell stressors that MSCs react to in vivo, but in an ex vivo cell culture environment [12,14].

2. Genetic engineering

Advances in genetic engineering tools have allowed various transient and stable protein delivery to improve MSC therapy:

2.1. MSC viability

One notable limitation observed in some instances of MSC therapy is low viability following MSC transplantation into hosts. Researchers have transiently overexpressed growth factors in MSCs to improve this viability. For example, FGF-2 gene has been overexpressed in MSCs ex vivo to improve cardiac tissue repair. This MSC genetic modification results in a threefold increase in cell viability and cardiac marker expression for greater therapeutic outcomes in myocardial infarction repair [7,15]. Integrin-linked kinase has also been overexpressed in MSCs to increase their survival for myocardial infarction repair leading to improved angiogenesis at week three post-MSC transplantation [7,16].

2.2. MSC homing

Another major therapeutic limitation addressed through MSC cellular engineering is the limited homing and migration of MSC to target regions following administration [3,5]. The induction of CXCR4 and CXCR7 has been applied for enhanced MSC migration. CXCR4 and CXCR7 chemokine receptors are known to play a pivotal role in canonical MSC migration and adhesion capacity. As such, these studies reported that the overexpression of CXCR4/CXCR7 in adipose derived MSCs improved their paracrine, proliferative and migratory abilities yielding a powerful therapeutic improvement to MSC treatment [8,17].

2.3. MSC immunosuppression

Cellular engineering has also been applied to directly improve MSC immunosuppressive function to better treat inflammatory disease. This has been done through the stable lentiviral overexpression of potent anti-inflammatory cytokine IL10 in combination with CXCR4 creating CXCR4-IL10-MSCs. These cells not only induced expression of functional CXCR4 receptor on the MSC cell surface, but also allowed for the increased secretion of IL10 [17,18]. In preclinical GVHD mouse models, CXCR4-IL10-MSCs displayed increased therapeutic capacity and marked reduction in the number of pro-inflammatory Th1 and Th17 CD3⁺ cells with polarization toward anti-inflammatory IL10⁺ CD3⁺ T-cells [18]. Engineered MSCs have also been created to overexpress both inhibitory marker PDL1 as well as pro survival kinase AKT (adMSC-PDL1-AKT) simultaneously [19,20]. This dual engineering not only allowed for enhanced proliferation rates and resistance to oxidative stress by the MSCs through AKT, but also led to a robust upregulation of immunosuppressive regulatory T-cell phenotypes through increased interaction with PDL1 on the MSCs to PD1 on T-cells [19]. These modifications further highlight the functional and therapeutic potential of genetically engineered MSCs.

3. Synthetic biology

Most recently, our group has applied synthetic biology to MSCs creating chimeric antigen receptor MSCs (CAR-MSCs). A CAR is a synthetic protein composed of an extracellular antigen binding domain fused to a functional intracellular signaling domain. CAR was first applied with resounding success to the field of hematological malignancies through the creation and eventual FDA approval of CART-cell therapy [21,22]. CART became the most successful cellular therapy to date by taking known functions of immunogenic T-cells and enhancing them to target B-cell malignancies. Specifically, the first FDA approved CART-cells contained CARs designed to recognize CD19 antigen expressed almost exclusively on malignant B-cells and was fused to a intracellular 4-1BB and CD3z signaling domain to mediate activation and killing of target cancer cells by T-cells [21,22].

By creating CAR-MSCs, we equipped the MSCs with a gain of more than one function including enhanced 1) homing to target tissues due to a tissue-specific antigen binding domain and 2) immunosuppression due to inclusion of an immunosuppressive intracellular signaling domain [23].

The bioengineering of CAR-MSCs was verified for safety and maintenance of stemness as well as high lentiviral transduction efficiency of CAR at >80% in human adipose-derived MSCs. As a proof-of-concept model, CAR-MSCs were engineered to target E-cadherin (Ecad) for the treatment of GVHD. CAR-MSCs displayed Ecad antigen-specific immunosuppression of T-cells in vitro and in vivo GVHD Xenograft Models. Compared with standard nontransduced MSC therapy, CAR-MSC treatment led to a significant prevention of mouse weight loss, decreased clinical symptom scores, increased human T-cell suppression, differentiation of T-cells toward a Treg-like phenotype and improved overall survival of mice [23]. A distinct upregulation of immunosuppressive gene signatures was found in Ecad stimulated CAR-MSCs, further supporting functionality of the CAR receptor. Finally, anti-Ecad CAR-MSCs displayed preferential homing to mouse Ecad⁺ colonic tissue and greater efficacy as compared to nonspecific control scFv CAR-MSCssupporting the importance of CAR scFv design [23].

The stability and increased functionality of MSCs engineered to overexpress genes and synthetic CAR receptors represents a promising step for cell therapy technologies used to treat immune disease. They directly address the shortcomings of previous MSC clinical trials and display promising superiority to the current standard of care. The possibilities here are endless where MSC functionality can be mediated by intracellular signaling domains or inclusion of immunosuppression cytokines [18], while MSC homing and migration can be mediated by increased expression of chemokine receptors or incorporation of a target tissue specific antigen-binding domain [8,17,23].

Engineered MSCs have only recently entered early phase clinical trials. Although terminated early due to insufficient trial participants, autologous MSCs retrovirally expressing tumor-specific herpes simplex virus thymidine kinase were tested in a phase I/II clinical trial (EuDRA CT Numberi: 2012-003741-15) for patients with gastrointestinal tumors and reported a median overall survival of 15.6 months [24,25]. A second completed phase I/II clinical trial for patients with relapsed solid tumors utilized autologous BM-MSCs engineered to carry oncolytic adenovirus called Celyvir. Celyvir showed minimal clinical efficacy with 83% of patients demonstrating progressive disease following treatment. However, the engineered Celyvir product demonstrated robust safety and tolerability in patients guiding future opportunities for selecting off-the-shelf MSCs from healthy donors with clinically favorable characteristics in future trials [25,26].

These foundations and early clinical findings in MSC cellular engineering open the door for the creation of novel therapeutic platforms that can be designed and expanded to treat a wide range of immune diseases. The successful transduction of CAR-MSCs with evidence of improved antigen-specific immunosuppression serves as a proof-of-concept study to be implemented in other disease-specific contexts. For example, cytokine secreting or differentiation-promoting intracellular signaling domains could be incorporated into CAR-MSCs to initiate an anti-inflammatory milieu and encourage tissue regeneration. Additionally, these functionalities could be directed to a wide range of tissues including the heart, lungs and liver based on CAR-MSC antigen-binding domain or chemokine design ultimately broadening the therapeutic scope of engineered MSCs.

Funding Statement

This study was supported by the NIHMS grant R37CA266344, R01AI179974.

Author contributions

O Sirpilla and SS Kenderian wrote, edited and approved the final version of the manuscript.

Financial disclosure

This study was supported by the NIHMS grant R37CA266344, R01AI179974. The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

SS Kenderian is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania and Novartis), Humanigen (through Mayo Clinic), Mettaforge (through Mayo Clinic). SS Kenderian receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, LifEngine Animal Health Laboratories Inc and Lentigen. SS Kenderian has participated in advisory meetings with Kite/Gilead, Humanigen, Juno/BMS, Capstan Bio and Novartis. SS Kenderian has served on the data safety and monitoring board with Humanigen and Carisma. SS Kenderian has severed a consultant for Torque, Calibr, Novartis, Capstan Bio, BMS, Kite/Gilead and Humanigen. O Sirpilla and SS Kenderian have intellectual property in the CAR-MSC technology.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.