A 37-year-old man (Karnofsky Performance Score-KPS > 90) with multifocal glioblastoma multiform referred to the outpatient neurosurgery clinic of Shohadaye-Tajrish hospital on Oct 31, 2018. He had two gliomatous foci which were in the left frontal and left parietal lobes. Left parietal focus had been undergone resection at another centre 35 days ago. Another lesion at the frontal lobe was resected at our centre through frontal craniotomy on Nov 5, 2018. Pathology of both foci revealed that these tumors were grade IV astrocytoma (glioblastoma multiforme). According to the immunohistological and PCR tests, both foci had wild type of IDH1 and IDH2, and the promotor of MGMT was methylated under 5 percent. Standard radiation therapy was started on Nov 25, 2018 following Temozolomide (75mg/m2/day) was taken one hour before radiation therapy for the first week and then continued separately until 54 days until Jan 20, 2019. After the accomplishment of radio/chemotherapy, regarding the eligibility of the patient, he volunteered for participation in our stem cell-mediated gene therapy of glioblastoma program. This project was designed in accordance with the declaration of Helsinki and was approved by the Ethics committee in the Medical Research of the Shahid Beheshti University of Medical Sciences; license number: IR.SBMU.REC.1396.224 (Clinical trial registration code: IRCT20200502047277N2). All procedures were performed after written informed consent was obtained. The patient was fully aware of the experimental process of the treatment, unexpected outcomes, and possible adverse effects.
Collection of MSCs was performed in the operating room under sterile conditions. The patient was discharged after the procedure on the same day. Bone marrow blood (100 ml) was aspirated from the iliac bone for the collection of bone marrow stem cells according to the protocol that we used in our previous study and we assessed the MSCs characteristics through analysing the cell surface markers such as CD90, CD105, CD45, and CD73 (8).
Human embryonic kidney (HEK) 293 cell lines were obtained from Iranian Biological Resource Centre and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM, Gibco, USA) containing 10% Fetal Bovine Serum (FBS, Gibco, USA) and 1% nonessential amino acids (Invitrogen, USA), and L-glutamine (2 mM, Gibco, USA). Cells were cultured under a standard condition in 95% humidity and 5% CO2 at 37°C.
For the gain of function study, thymidine kinase was cloned into the pCDH-CMV-MCS-EF1-copGFP plasmid that was previously digested with BamHI and EcoRI endonuclease enzymes. For lentivirus production, HEK-293T cells were calcium phosphate co-transfected with pCDH- Thymidine kinase, pSPAX2 plasmid (packaging plasmid Contains Gag, Pol, Rev, and Tat) and pMD2 plasmid (containing vesicular stomatitis viruses (VSVs) glycoprotein (G)). The supernatants were harvested every 12 hours for 3 days after transfection and concentrated by ultracentrifuge at 47,000×g for 2 hours at 4°C. To induce thymidine kinase expression in MSCs; MSCs were cultured in DMEM/F12 and transduced with pCDH- thymidine kinase lentivirus. 3×105 cells were transferred to a T25 flask and transduced the following day with Multiplicity of infection (MOI) 40 (TU/cell). Also, transduced cells with pCDH-TK were selected by puromycin (2 µg/ml). After purification of transducing cells by puromycin, they were washed three times with normal saline afterwards; the cells were ready for injection.
Human MSCs highly expressed CD90 and CD73, and were positive for the CD105 marker. Furthermore, the expression of CD45 observed in low percentage of these cells.
After virus production and concentration, the virus titration was determined by the use of flow cytometry. Following the titration of recombinant viruses, the mesenchymal stem cells were transduced with a certain amount of the virus (MOI 40) (Fig. 1).
The patient was hospitalized and underwent general anaesthesia and a direct injection of MSCs transduced with lentivirus was given through the previous frontal craniotomy under the guidance of navigation in the bed of the frontal tumor. The patient received 5×105 MSCs transfected by lentivirus containing HSV-TK enzyme in 1 cc volume via the injection catheter which had been connected to the navigation system before using. The catheter was placed at the site of the injection for one minute to decrease leakage. Forty-eight hours after cell injection, the patient received intravenous ganciclovir with a dose of 5 mg/kg over 1 hour, every 12 h. The drug administration continued for a total of 28 doses over 14 days.
The patient was followed up after injections and during the prodrug therapy and assessed for any adverse effects based on common terminology criteria for adverse events (CTCAE, version 4.3). Blood and urine samples were analysed during hospitalization to control hematologic toxicity and renal impairment due to ganciclovir medication.
Radiological recurrence was assessed by MRI, obtained before injection, and then every 3 months and checked independently by two neurosurgeons. This assessment was done by MRI according to Response Evaluation Criteria in Solid Tumors (RECIST) where progression was defined as at least a 20% increase in the sum of the longest diameters of the target lesions.(9)
After the cell injection, our patient was in a good general state and he was conscious and comfortable. The patient had prior history of seizures, but due to the lack of regular medication, he experienced a seizures attack three months after gene therapy. Generally, the adverse events and systemic complications were not observed.
After the injection on Feb 19, 2019, the patient’s follow-up revealed recurrence of parietal focus on Dec 09, 2019; however, frontal focus had a slight and unremarkable enhancement. Until our last radiological follow-up (on Mar 18, 2020), frontal focus had no prominent recurrence and the size of the parietal focus increased and extended to the contralateral hemisphere through the corpus callosum (Fig. 2). Eventually patient has passed away on July 16, 2020 (PFS = 293 days, OS = 657 days).