Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis.

BACKGROUND: Mesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime. METHODS: In this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking. RESULTS: Firstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively. CONCLUSIONS: Taken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment.

BACKGROUND: Mesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene. METHODS: Modified MSCs were preserved in a commercially available, GMP-grade cryopreservative-CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC). RESULTS: We found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers. CONCLUSION: This study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment.

A highly efficient non-viral process for programming mesenchymal stem cells for gene directed enzyme prodrug cancer therapy.

Mesenchymal stem cells (MSCs) driven gene-directed enzyme prodrug therapy has emerged as a potential strategy for cancer treatment. The tumour-nesting properties of MSCs enable these vehicles to target tumours and metastases with effective therapies. A crucial step in engineering MSCs is the delivery of genetic material with low toxicity and high efficiency. Due to the low efficiency of current transfection methods, viral vectors are used widely to modify MSCs in preclinical and clinical studies. We show, for the first time, the high transfection efficiency (> 80%) of human adipose tissue derived-MSCs (AT-MSCs) using a cost-effective and off-the-shelf Polyethylenimine, in the presence of histone deacetylase 6 inhibitor and fusogenic lipids. Notably, the phenotypes of MSCs remained unchanged post-modification. AT-MSCs engineered with a fused transgene, yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) displayed potent cytotoxic effects against breast, glioma, gastric cancer cells in vitro. The efficiency of eliminating gastric cell lines were effective even when using 7-day post-transfected AT-MSCs, indicative of the sustained expression and function of the therapeutic gene. In addition, significant inhibition of temozolomide resistant glioma tumour growth in vivo was observed with a single dose of therapeutic MSC. This study demonstrated an efficient non-viral modification process for MSC-based prodrug therapy.

A facile and scalable in production non-viral gene engineered mesenchymal stem cells for effective suppression of temozolomide-resistant (TMZR) glioblastoma growth.

BACKGROUND: Mesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP). METHODS: MSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and ß tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice. RESULTS: At > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated. CONCLUSIONS: Collectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.

Enhanced transfection of a macromolecular lignin-based DNA complex with low cellular toxicity.

Cationic polymers remain attractive tools for non-viral gene transfer. The effectiveness of these vectors rely on the ability to deliver plasmid DNA (pDNA) into the nucleus of cells. While we have previously demonstrated the potential of Lignin-PGEA-PEGMA as a non-viral gene delivery vector, alterations of cellular phenotype and cytotoxicity were observed post transfection. The present study aims to explore transfection conditions for high efficiency and low toxicity of the Lignin-PGEA-PEGMA based gene delivery system. Cellular toxicity was significantly reduced by using the centrifugation protocol, which enables rapid deposition of DNA complexes. Replacement of media post centrifugation resulted in minimal exposure of cells to excess polymers, which were toxic to cells. At an optimized DNA amount (500-750 ng) and molar ratios of nitrogen (N) in polymer to phosphate (P) in pDNA (N/P = 30-40), with the use of a novel transfection enhancer that facilitates endosomal escape and nuclear trafficking, the efficiency of gene delivery was increased significantly 24 h post transfection. The present study demonstrated an appropriately optimized protocol that enabled the utility of a novel cationic polymer blend with a mixture of fusogenic lipids and a histone deacetylate inhibitor in non-viral transfection, thereby providing an attractive alternative to costly commercial gene carriers.

Enhanced non-viral gene delivery by coordinated endosomal release and inhibition of ß-tubulin deactylase.

Efficient non-viral gene delivery is highly desirable but often unattainable with some cell-types. We report here that non-viral DNA polyplexes can efficiently transfect differentiated neuronal and stem cells. Polyplex transfection centrifugation protocols was enhanced by including a simultaneous treatment with a DOPE/CHEMS lipid suspension and a microtubule inhibitor, Tubastatin A. Lipoplex transfection protocols were not improved by this treatment. This mechanism of action was unravelled by systematically identifying and rationally mitigating barriers limiting high transfection efficiency, allowing unexpected improvements in the transfection of mesenchymal stem cells (MSC), primary neuron and several hard-to-transfect cell types beyond what are currently achievable using cationic polymers. The optimized formulation and method achieved high transfection efficiency with no adverse effects on cell viability, cell proliferation or differentiation. High efficiency modification of MSC for cytokine overexpression, efficient generation of dopaminergic neuron using neural stem cells and enhanced genome editing with CRISPR-Cas9 were demonstrated. In summary, this study described a cost-effective method for efficient, rapid and scalable workflow for ex vivo gene delivery using a myriad of nucleic acids including plasmid DNA, mRNA, siRNA and shRNA.

MicroRNA as potential modulators in chemoresistant high-grade gliomas.

Gliomas account for 70% of human malignant primary brain tumours. The most common form is glioblastoma multiforme, World Health Organization grade IV. Despite the implementation of post-operative adjuvant radiotherapy with concurrent temozolomide (TMZ), the disease's overall prognosis remains dismal. TMZ is currently the only mono-chemotherapeutic agent for newly-diagnosed high-grade glioma patients and acquired resistance inevitably occurs in the majority of such patients, further limiting treatment options. Therefore, there is an urgent need to better understand the underlying mechanisms involved in TMZ resistance, a critical step to developing effective, targeted treatments. An emerging body of evidence suggests the intimate involvement of a novel class of nucleic acid, microRNA (miRNA), in tumorigenesis and disease progression for a number of human malignancies, including primary brain tumours. miRNA are short, single-stranded, non-coding RNA (∼22 nucleotides) that function as post-transcriptional regulators of gene expression. This review provides an overview of the key treatment obstacles faced in patients with high-grade gliomas, especially in the context of recurrent, chemoresistant tumours and the potential roles of miRNA in chemoresistance and management of this disease.

Direct quantification of mRNA and miRNA from cell lysates using reverse transcription real time PCR: a multidimensional analysis of the performance of reagents and workflows.

Substantial efforts have been devoted to in vitro testing of candidate chemotherapeutics by profiling transcriptional changes across the collection of NCI-60 cell-lines. A work-flow with reagents that enable the direct quantification of RNA of different molecular sizes simultaneously in the same sample without laborious total RNA isolation will invariably increase the throughput and accuracy of the study. MicroRNAs (miRNAs) are known to regulate most cellular functions, acting post-transcriptionally by repressing numerous eukaryotic mRNAs. Recent findings on the remarkable stability of miRNA prompted us to investigate the feasibility of quantifying the expression levels of both mRNA and miRNA directly from cell lysates (cell-to-Ct). Multidimensional analyses of the expressions of mRNA and miRNA across seven NCI-60 cell lines and multiple reagents were conducted to assess the performances of these reagents and workflows for cell-to-Ct measurements using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Quantification of RNA species using lysates prepared from an in-house and one of the commercial reagents demonstrated comparable performance to those prepared by the more laborious and conventional method of using guanidinium-phenol-chloroform. Additionally, miRNA was found to be highly stable in the cell lysates when incubated at room temperature for prolonged period of time and subjected to multiple freeze-thaw cycles. In summary, this study demonstrated significant differences in pre-analytical performance of a variety of commercially available reagents and described a cost-effective reagent useful for rapid, scalable, and high-throughput workflow for the detection of mRNA and miRNA from the same biological sample.