Characterisation of blood-derived exosomal hTERT mRNA secretion in cancer patients: a potential pan-cancer marker.

BACKGROUND: Telomerase (human telomerase reverse transcriptase (hTERT)) is considered a hallmark of cancer. The aim of our study was to evaluate the feasibility of the detection of hTERT transcripts in serum as a 'pan-cancer' diagnostic method. METHODS: Human telomerase reverse transcriptase mRNA levels were determined in serum and serum-derived exosomes from 133 patients with different malignancies and 45 healthy controls. In four patients hTERT mRNA levels were measured in different clinical stages. RESULTS: Human telomerase reverse transcriptase transcript was absent in all controls and was variably detected in 67.5% of patients with all cancer types. A correlation between hTERT transcript levels and the clinical course was found in several cases. CONCLUSIONS: Human telomerase reverse transcriptase mRNA levels may reflect the tumour burden and the clinical status of the patient. In patients with detectable levels, this assay may potentially serve as a diagnostic and follow-up 'pan-cancer' marker. Owing to the large variety of patients and small sample size in each diagnosis, the statistical power is limited and will be explored further in larger groups.

[EXOSOMES AS MEDIATORS OF INTERCELLULAR COMMUNICATION: THE CANCER AND TELOMERASE CONNECTION].

INTRODUCTION: Exosomes, nano-vesicles secreted from all types of cells in the human body, function as inter-cell communicators. This role of exosomes is fulfilled by their specific content, dependent on the origin of donor cells from which they are secreted. Exosomes contain a plethora of nucleic acids (DNA, RNA and micro RNA), proteins and lipids. These molecules are packed in the donor cells into the exosomes which are subsequently secreted and transferred through various body fluids into the target cells which may be located far from the donor cells. Recently, the issue of exosome research has been widely expanded and data has been accumulated regarding the relevance and involvement of exosomes in cancer. Exosomes play a role in the process of malignant transformation, in avoiding the surveillance of the immune system, the expansion of the tumor and its establishment in the cancer microenvironment. In addition, exosomes promote the process of metastasis formation. Among other subjects, exosomal research focuses around the characterization of the content of cancer-derived exosomes in order to identify markers that can be used for diagnosis and prognosis of cancer patients. Telomerase, a unique reverse transcriptase, has been widely shown to be crucial for the process of malignant transformation and the perpetuation of the malignant clone. In the current review, we describe the importance of exosomes to the various themes of cancer and their potential use as diagnostic and prognostic markers as well as their therapeutic potential. In addition, the results and implications of our study with regards to the secretion of telomerase transcript into exosomes derived from cancer cells will be discussed.

Blood-Derived Exosomal hTERT mRNA in Patients with Lung Cancer: Characterization and Correlation with Response to Therapy.

Background: Telomerase (human telomerase reverse transcriptase (hTERT) is considered a hallmark of cancer, being active in cancer cells but repressed in human somatic cells. As such, it has the potential to serve as a valid cancer biomarker. Exosomal hTERT mRNA can be detected in the serum of patients with solid malignancies but not in healthy individuals. We sought to evaluate the feasibility of measuring serum exosomal hTERT transcripts levels in patients with lung cancer. Methods: A prospective analysis of exosomal hTERT mRNA levels was determined in serum-derived exosomes from 76 patients with stage III-IV lung cancer (11 SCLC and 65 NSCLC). An hTERT level above RQ = 1.2 was considered "detectable" according to a previous receiver operating characteristic curve (ROC) curve. Sequential measurements were obtained in 33 patients. Demographic and clinical data were collected retrospectively from patients' charts. Data on response to systemic therapy (chemotherapy, immunotherapy, and tyrosine kinase inhibitors) were collected by the treating physicians. Results: hTERT was detected in 53% (40/76) of patients with lung cancer (89% of SCLC and 46% of NSLCC). The mean hTERT levels were 3.7 in all 76 patients, 5.87 in SCLC patients, and 3.62 in NSCLC patients. In total, 25 of 43 patients with sequential measurements had detectable levels of hTERT. The sequential exosomal hTERT mRNA levels reflected the clinical course in 23 of them. Decreases in hTERT levels were detected in 17 and 5 patients with partial and complete response, respectively. Eleven patients with a progressive disease had an increase in the level of exosomal hTERT, and seven with stable disease presented increases in its exosomal levels. Another patient who progressed on the first line of treatment and had a partial response to the second line of treatment exhibited an increase in exosomal hTERT mRNA levels during the progression and a decrease during the response. Conclusions: Exosomal hTERT mRNA levels are elevated in over half of patients with lung cancer. The potential association between hTERT levels and response to therapy suggests its utility as a promising cancer biomarker for response to therapy. This issue should be further explored in future studies.

Therapeutic inhibitory RNA in head and neck cancer via functional targeted lipid nanoparticles.

Currently there are no specific therapies addressing the distinctive biology of human papillomavirus (HPV)-induced cancer approved for clinical use. Short interfering RNA (siRNA) has much potential for therapeutic manipulation of HPV E6/E7 oncoproteins. Lipid-based nanoparticles (LNPs) can be utilized for systemic transportation and delivery of siRNA at target site. We recently developed a recombinant protein linker that enables uniform conjugation of targeting antibodies to the LNPs. Herein, we demonstrate the therapeutic efficacy of anti-E6/E7 siRNA delivered via targeted LNPs (tLNPs) in a xenograft HPV-positive tumor model. We show that anti-epidermal growth factor receptor (EGFR) antibodies, anchored to the LNPs as targeting moieties, facilitate cargo delivery but also mediate anti-tumor activity. Treatment with siE6 via tLNPs resulted in 50% greater reduction of tumor volume compared to treatment with siControl encapsulated in isoLNPs (coated with isotype control antibodies). We demonstrate superior suppression of HPV oncogenes and higher induction of apoptosis by the tLNPs both in vitro and in vivo. Altogether, the coupling of inhibitory siE6 with anti-EGFR antibodies, that further elicited anti-tumor effects, successfully restricted tumor progression. This system that combines potent siRNA and therapeutically functional tLNPs can be modulated against various cancer models.

Progress and challenges towards CRISPR/Cas clinical translation.

CRISPR/Cas systems (clustered regularly interspaced short palindromic repeats) have emerged as powerful tools to manipulate the genome for both research and therapeutic purposes. However, the clinical use of this system is hindered by multiple challenges, such as the rate of off-target effects, editing efficiency, the efficacy of HDR, immunogenicity, as well as development of efficient and safe delivery vehicles that can carry these compounds. Tremendous efforts are being conducted to overcome these challenges, including the discovery and engineering of more precise and efficacious Cas nucleases. Moreover, in recent years multiple viral and non-viral delivery approaches have been explored for in vivo delivery of CRISPR components. Here, we summarize the available CRISPR/Cas toolbox for genome editing as well as the recently developed in vivo delivery vehicles for CRISPR/Cas system. Furthermore, we discuss the remaining challenges for successful clinical translation of this system and highlight the current clinical applications.

CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy.

Harnessing CRISPR-Cas9 technology for cancer therapeutics has been hampered by low editing efficiency in tumors and potential toxicity of existing delivery systems. Here, we describe a safe and efficient lipid nanoparticle (LNP) for the delivery of Cas9 mRNA and sgRNAs that use a novel amino-ionizable lipid. A single intracerebral injection of CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) into aggressive orthotopic glioblastoma enabled up to ~70% gene editing in vivo, which caused tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30%. To reach disseminated tumors, cLNPs were also engineered for antibody-targeted delivery. Intraperitoneal injections of EGFR-targeted sgPLK1-cLNPs caused their selective uptake into disseminated ovarian tumors, enabled up to ~80% gene editing in vivo, inhibited tumor growth, and increased survival by 80%. The ability to disrupt gene expression in vivo in tumors opens new avenues for cancer treatment and research and potential applications for targeted gene editing of noncancerous tissues.

Targeting central nervous system pathologies with nanomedicines.

One of the major challenges in drug development is the delivery of therapeutics to the central nervous system (CNS). The blood-brain barrier (BBB), which modulates the passage of molecules from the CNS, presents a formidable obstacle that limits brain uptake of therapeutics and, therefore, impedes the treatment of multiple neurological pathologies. Targeted nanocarriers present an excellent opportunity for drug delivery into the brain leveraging on endogenous receptors to transport therapeutics across the BBB endothelium. Receptor-mediated transport endows multiple benefits over other conventional delivery methods such as the transient permeabilization of the BBB or the direct depositioning of intracranial depots. Herein, different strategies for nanocarrier targeting to the CNS are discussed, highlighting the challenges and recent developments.

Harnessing nanomedicine for therapeutic intervention in glioblastoma.

INTRODUCTION: Glioblastoma is a type of brain cancer arises from glial cells. Glioblastoma multiforme (GBM), a subtype of glioblastoma, is the most common and most aggressive primary brain tumor. Currently, GBM therapy includes surgery and post-operative high-doses of radiation and chemotherapy. This therapeutic strategy has a limited contribution in extending the survival rate of GBM patients. Areas covered: Herein, we focus on harnessing nanoscale drug delivery strategies to treat brain malignancies. Specifically, we briefly discuss the challenges facing GBM therapy such as restricted passage across the blood-brain barrier (BBB) and low enhanced permeability and retention effect. Next, we describe different pathways to address these challenges. Finally, we discuss the field of nanomedicine, which emerged as a promising platform for drug delivery to brain malignancies. Expert opinion: Countless strategies have been applied in preclinical and clinical settings to treat GBM. Among them is the use of different types of nanoparticles (NPs) and viruses with different approaches to cross or bypass the BBB. We suggest here a paradigm shift in thinking about crossing the BBB and tumor penetration as fundamental issues that need to be address in order to improve the therapeutic outcome in GBM.

Tumor cells derived exosomes contain hTERT mRNA and transform nonmalignant fibroblasts into telomerase positive cells.

Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter-cell communicators and affecting biological processes in "recipient" cells upon their uptake. The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from cancer cells via exosomes into telomerase negative fibroblasts, where it is translated into a fully active enzyme and transforms these cells into telomerase positive, thus creating a novel type of cells; non malignant cells with telomerase activity. All tested telomerase positive cells, including cancer cells and non malignant cells with overexpressed telomerase secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. Similarly exosomes isolated from sera of patients with pancreatic and lung cancer contained hTERT mRNA as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including increased proliferation, extension of life span and postponement of senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by phleomycin and from apoptosis, indicating that also telomerase "extracurricular" activities are manifested in the recipient cells. The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the alterations of cancer microenvironment and its role in cancer. The described process has an obvious therapeutic potential which will be explored in further studies.