Genetic and clinical characteristics including occurrence of testicular adrenal rest tumors in Slovak and Slovenian patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Objective: To analyze the mutational spectrum, clinical characteristics, genotype-phenotype correlations, testicular adrenal rests tumor prevalence, and role of neonatal screening in congenital adrenal hyperplasia (CAH) patients from Slovakia and Slovenia. Design and methods: Data were obtained from 104 patients with CAH registered in Slovak and Slovenian databases. Low-resolution genotyping was performed to detect the most common point mutations. To detect deletions, conversions, point mutations, or other sequence changes in the CYP21A2 gene, high-resolution genotyping was performed. Genotypes were classified according to residual 21-hydroxylase activity (null, A, B, C). Results: 64% of the individuals had the salt-wasting form (SW-CAH), 15% the simple virilizing form (SV-CAH), and 21% the non-classic (NC-CAH). CYP21A2 gene deletion/conversion and c.293-13A/C>G pathogenic variant accounted together for 55.5% of the affected alleles. In SV-CAH p.Ile172Asn was the most common pathogenic variant (28.13%), while in NC-CAH p.Val282Leu (33.33%), CYP21A2 gene deletion/conversion (21.43%), c.293-13A/C>G (14.29%), Pro30Leu (11.90%). The frequency of alleles with multiple pathogenic variants was higher in Slovenian patients (15.83% of all alleles). Severe genotypes (0 and A) correlated well with the expected phenotype (SW in 94.74% and 97.3%), while less severe genotypes (B and C) correlated weaklier (SV in 50% and NC in 70.8%). The median age of SW-CAH patients at the time of diagnosis was 6 days in Slovakia vs. 28.5 days in Slovenia (p=0.01). Most of the Slovak patients in the cohort were detected by NBS. (24 out of 29). TARTs were identified in 7 out of 24 male patients, of whom all (100%) had SW-CAH and all had poor hormonal control. The median age at the diagnosis of TARTs was 13 years. Conclusion: The study confirmed the importance of neonatal screening, especially in the speed of diagnosis of severe forms of CAH. The prediction of the 21-OH deficiency phenotype was reasonably good in the case of severe pathogenic variants, but less reliable in the case of milder pathogenic variants, which is consistent compared to data from other populations. Screening for TARTs should be realized in all male patients with CAH, since there is possible remission when identified early.

Intrinsic properties of tumour cells have a key impact on the bystander effect mediated by genetically engineered mesenchymal stromal cells.

BACKGROUND: Engineered mesenchymal stromal cells (MSC) have been used in many preclinical studies of gene directed enzyme/prodrug therapy. We aimed to compare the efficacy of two most frequently used systems, as well as evaluate the extent of a bystander effect mediated by therapeutic MSC towards cell lines derived from different tumours. METHODS: Two approaches were compared: (i) herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) and (ii) yeast cytosine deaminase fused with uracil phosphoribosyltransferase (CD::UPRT)/5-fluorocytosine (5-FC). The cytotoxic effect mediated by therapeutic MSC was evaluated in direct co-culture by a fluorimetric assay. The expression profile of tumour cells was analyzed by a quantitative polymerase chain reaction, and the ability of gap-junctional intercellular communication (GJIC) was evaluated by a dye transfer assay. RESULTS: Both systems were effective only on glioblastoma cells (8-MG-BA). The CD::UPRT-MSC/5-FC system showed efficiency on melanoma A375 cells. We decreased the sensitivity of 8-MG-BA cells and A375 cells to the CD::UPRT-MSC/5-FC system by pharmacological inhibition of thymidylate synthase, and we achieved a similar result in A375 cells by inhibition of thymidine phosphorylase. Although we demonstrated functional GJIC in A375 cells, TK-MSC were ineffective in mediating the bystander effect similarly to HeLa cells, which were also relatively resistant to CD::UPRT-MSC/5-FC treatment. TK-MSC/GCV treatment had a strong cytotoxic effect on MDA-MB-231 cells (breast carcinoma), whereas CD::UPRT-MSC/5-FC treatment failed as a result of overexpression of the gene for ABCC11. Transfection of the MDA-MB-231 cell line with small interference RNA specific to ABCC11 led to a significantly increased sensitivity to the CD::UPRT-MSC/5-FC approach. CONCLUSIONS: GJIC, expression of enzymes involved in drug metabolism and ABC transporters correlate with the response of tumour cells to treatment by MSC-expressing prodrug-converting genes.

Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo.

MSC-driven, gene-directed enzyme prodrug therapy (GDEPT) mediated by extracellular vesicles (EV) represents a new paradigm-cell-free GDEPT tumor therapy. In this study, we tested the efficacy of yeast cytosine deaminase::uracilphosphoribosyl transferase (yCD::UPRT-MSC)-exosomes, in the form of conditioned medium (CM) to inhibit the growth of C6 glioblastoma cells both in vitro and in vivo. MSCs isolated from human adipose tissue, umbilical cord, or dental pulp engineered to express the yCD::UPRT gene secreted yCD::UPRT-MSC-exosomes that in the presence of the prodrug 5-fluorocytosine (5-FC), inhibited the growth of rat C6 glioblastoma cells and human primary glioblastoma cells in vitro in a dose-dependent manner. CM from these cells injected repeatedly either intraperitoneally (i.p.) or subcutaneously (s.c.), applied intranasally (i.n.), or infused continuously by an ALZET osmotic pump, inhibited the growth of cerebral C6 glioblastomas in rats. A significant number of rats were cured when CM containing yCD::UPRT-MSC-exosomes conjugated with 5-FC was repeatedly injected i.p. or applied i.n. Cured rats were subsequently resistant to challenges with higher doses of C6 cells. Our data have shown that cell-free GDEPT tumor therapy mediated by the yCD::UPRT-MSC suicide gene EVs for high-grade glioblastomas represents a safer and more practical approach that is worthy of further investigation.

Suicide Gene Therapy Mediated with Exosomes Produced by Mesenchymal Stem/Stromal Cells Stably Transduced with HSV Thymidine Kinase.

Mesenchymal stem/stromal cells (MSCs) prepared from various human tissues were stably transduced with the suicide gene herpes simplex virus thymidine kinase (HSVTK) by means of retrovirus infection. HSVTK-transduced MSCs express the suicide gene and in prodrug ganciclovir (GCV) presence induced cell death by intracellular conversion of GCV to GCV-triphosphate. The homogenous population of HSVTK-MSCs were found to release exosomes having mRNA of the suicide gene in their cargo. The exosomes were easily internalized by the tumor cells and the presence of ganciclovir caused their death in a dose-dependent manner. Efficient tumor cell killing of glioma cell lines and primary human glioblastoma cells mediated by HSVTK-MSC exosomes is reported. Exosomes produced by suicide gene transduced MSCs represent a new class of highly selective tumor cell targeted drug acting intracellular with curative potential.

Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice.

BACKGROUND: Previously, we validated capability of human adipose tissue-derived mesenchymal stem cells (AT-MSC) to serve as cellular vehicles for gene-directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT-MSC (CD y-AT-MSC) combined with systemic 5-fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo. METHODS: CD y-AT-MSC/5FC-mediated proliferation inhibition against a panel of human tumour cells lines was evaluated in direct and indirect cocultures in vitro. Antitumour effect was tested on immunodeficient mouse model in vivo. RESULTS: Although culture expansion of CD y-AT-MSC sensitized these cells to 5FC mediated suicide effect, expanded CD y-AT-MSC/5FC still exhibited strong bystander cytotoxic effect towards human melanoma, glioblastoma, colon, breast and bladder carcinoma in vitro. Most efficient inhibition (91%) was observed in melanoma A375 cell line when directly cocultured with 2% of therapeutic cells CD y-AT-MSC/5FC. The therapeutic paradigm of the CD y -AT-MSC/5FC system was further evaluated on melanoma A375 xenografts on nude mice in vivo. Complete regression in 89% of tumours was achieved when 20% CD y-AT-MSC/5FC were co-injected along with tumour cells. More importantly, systemic CD y-AT-MSC administration resulted in therapeutic cell homing into subcutaneous melanoma and mediated tumour growth inhibition. CONCLUSIONS: CD y-AT-MSC capability of targeting subcutaneous melanoma offers a possibility to selectively produce cytotoxic agent in situ. Our data further demonstrate beneficial biological properties of AT-MSC as a cellular vehicle for enzyme/prodrug therapy approach to molecular chemotherapy.

HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells.

Previously we have reported adipose-tissue derived human mesenchymal stem cells (AT-MSC) as cellular delivery vehicles for tumor-targeted cancer gene therapy. In this report we aimed to determine whether Herpes simplex virus - thymidine kinase (HSV-tk) expressing AT-MSC (TK-MSC) could exert cytotoxic effect on tumor cells upon treatment with prodrug ganciclovir (GCV). Direct co-cultures of human glioblastoma cells 8-MG-BA, 42-MG-BA and U-118 MG with TK-MSC/GCV resulted in substantial viability decrease in vitro. This therapeutic paradigm was most efficient against 8-MG-BA glioblastoma cells exhibiting cytotoxicity (>50%) in the presence of TK-MSC and 0.1microM GCV. Rapid apoptosis induction in three glioblastoma cell lines and TK-MSC demonstrated both bystander cytotoxic effect on tumor cells and GCV conversion-mediated suicide effect on TK-MSC. Furthermore, we were able to demonstrate formation of gap junctions between AT-MSC and human glioblastoma cells as a mechanism contributing to bystander cytotoxicity. Inability of human HeLa and MCF7 to form gap junctions with AT-MSC rendered these cell refractory to the TK-MSC/GCV mediated cytotoxicity. Gap junction intercellular communication (GJIC) capability of AT-MSC with tumor cells further supports the exploitation of mesenchymal stem cells for approaches relying on the bystander effect. Biological consequences of these capabilities remain to be further explored.