Πρόσφατη μελέτη από ερευνητές του Πανεπιστημίου της Ναγκόγια στην Ιαπωνία αποκάλυψε πως τα microRNA στα ούρα θα μπορούσαν να αποτελέσουν μια πολλά υποσχόμενη «βιοένδειξη» (biomarker) για τη διάγνωση όγκων στον εγκέφαλο.
Τα ευρήματα, τα οποία δημοσιεύονται στο ACS Applied Materials & Interfaces, έχουν υποδείξει πως τα τακτικά τεστ ούρων θα μπορούσαν να βοηθήσουν στον έγκαιρο εντοπισμό και αντιμετώπιση όγκων στον εγκέφαλο, οδηγώντας σε αυξημένα επίπεδα επιβίωσης ασθενών.
Η έγκαιρη διάγνωση όγκων στον εγκέφαλο είναι συχνά δύσκολη, εν μέρει επειδή πολλοί κάνουν εξετάσεις (MRI κ.α.) μόνο μετά την εμφάνιση νευρολογικών προβλημάτων, για παράδειγμα προβλήματα κίνησης άκρων, δυσκολίες στην ομιλία κ.α. Όταν εντοπίζονται, σε πολλές περιπτώσεις είναι ήδη πολύ αργά για να αφαιρεθούν πλήρως.
Ως διαγνωστική βιοένδειξη καρκινικών όγκων, τα microRNA (μικροσκοπικά μόρια νουκλεϊκού οξέος) έχουν προσελκύσει έντονο ενδιαφέρον: Εκκρίνονται από διάφορα κύτταρα και υπάρχουν σε σταθερή κατάσταση, χωρίς βλάβες, εντός εξωκυτταρικών κυστιδίων σε βιολογικά υγρά όπως το αίμα και τα ούρα. Οι επιστήμονες εδώ εστίασαν στα microRNA στα ούρα.
Η νέα συσκευή που ανέπτυξαν διαθέτει 100 εκατομμύρια νανοκαλώδια οξειδίου του ψευδαργύρου, που μπορούν να αποστειρώνονται και να παράγονται μαζικά. Η συσκευή αυτή είναι σε θέση να εξάγει πολύ μεγαλύτερη ποικιλία και ποσότητα microRNA από μόλις ένα ml ούρων συγκριτικά με συμβατικές μεθόδους. Όπως έδειξε η ανάλυση, πολλά microRNA που προκύπτουν από όγκους στον εγκέφαλο συναντώνται στα ούρα, σε σταθερή κατάσταση.
Οι ερευνητές επίσης εξέτασαν εάν αυτά τα microRNA μπορούν να χρησιμοποιηθούν ως βιοενδείξεις όγκων. Τα αποτελέσματα έδειξαν ότι το μοντέλο που χρησιμοποίησαν μπορεί να διακρίνει τους καρκινοπαθείς από τους μη καρκινοπαθείς με εξαιρετικά υψηλά επίπεδα ευαισθησίας και ακριβείας, ανεξαρτήτως μεγέθους και κακοήθειας. ΝΑΥΤΕΜΠΟΡΙΚΗ
Abstract
mRNA vaccines have become a promising platform for cancer immunotherapy. During vaccination, naked or vehicle loaded mRNA vaccines efficiently express tumor antigens in antigen-presenting cells (APCs), facilitate APC activation and innate/adaptive immune stimulation. mRNA cancer vaccine precedes other conventional vaccine platforms due to high potency, safe administration, rapid development potentials, and cost-effective manufacturing. However, mRNA vaccine applications have been limited by instability, innate immunogenicity, and inefficient in vivo delivery. Appropriate mRNA structure modifications (i.e., codon optimizations, nucleotide modifications, self-amplifying mRNAs, etc.) and formulation methods (i.e., lipid nanoparticles (LNPs), polymers, peptides, etc.) have been investigated to overcome these issues. Tuning the administration routes and co-delivery of multiple mRNA vaccines with other immunotherapeutic agents (e.g., checkpoint inhibitors) have further boosted the host anti-tumor immunity and increased the likelihood of tumor cell eradication. With the recent U.S. Food and Drug Administration (FDA) approvals of LNP-loaded mRNA vaccines for the prevention of COVID-19 and the promising therapeutic outcomes of mRNA cancer vaccines achieved in several clinical trials against multiple aggressive solid tumors, we envision the rapid advancing of mRNA vaccines for cancer immunotherapy in the near future. This review provides a detailed overview of the recent progress and existing challenges of mRNA cancer vaccines and future considerations of applying mRNA vaccine for cancer immunotherapies.
Introduction
Cancer immunotherapies have gained tremendous attention since the U.S. Food and Drug Administration (FDA) approval of six checkpoint blockade modulators and two chimeric antigen receptor (CAR)-T cell immunotherapies [1, 2]. Cancer immunotherapies aim to activate the host anti-tumor immunity, modify the suppressive tumor microenvironment and ultimately result in tumor reduction and increased overall patients’ survival rate. Cancer vaccines are an attractive alternative immunotherapeutic option with both prophylactic and therapeutic potentials. The vaccines that target tumor-associated or tumor-specific antigens (TAAs or TSAs) can specifically attack and destroy malignant cells that overexpress the antigens and achieve chronic therapeutic response because of immunologic memory. Therefore, cancer vaccines offer specific, safe, and tolerable treatment compared to other immunotherapies. Despite considerable efforts to develop cancer vaccines, clinical translations of cancer vaccines into efficacious therapies have remained challenging for decades due to highly variate tumor antigens and relevantly low immune response. Nonetheless, U.S. FDA has recently approved two prophylactic vaccines, one for human papillomavirus (HPV) that accounts for 70% of cervical cancers, and another for hepatitis B virus that can cause liver cancer [3]. More encouragingly, PROVENGE (Sipuleucel-T), an immune cell-based vaccine has been approved by the U.S. FDA in 2010 as the first therapeutic cancer vaccine for treating hormone-refractory prostate cancer patients [4]. Besides these initial successful attempts in cancer vaccines, multiple personalized cancer vaccines combined with checkpoint blockage modulators or cytokine therapies are currently being evaluated in clinical trials, with some promising clinical responses in multiple solid or metastatic tumors [5, 6].
There are four types of cancer vaccines, including tumor or immune cell-based vaccines, peptide-based vaccines, viral vector-based vaccines, and nucleic acid-based vaccines [7]. Nucleic acid (DNA- or RNA-) based vaccine is a promising vaccine platform for multiple reasons. Firstly, nucleic acid vaccines allow simultaneous delivery of multiple antigens covering various TAAs or somatic tumor mutations, eliciting both humoral and cell-mediated immune response, increasing the likelihood of overcoming vaccine resistance. Secondly, unlike peptide vaccines, nucleic acid vaccines can encode full-length tumor antigens, allowing APCs to simultaneously present or cross-present multiple epitopes with both class I and II patient-specific human leukocyte antigen (HLA), thus are less restricted by the human HLA types and more likely to stimulate a broader T cell response [8]. Ultimately, nucleic acid vaccines are non-infectious, free of protein or virus-derived contaminations during production, and are thus considered well tolerated for both prophylactic and therapeutic applications [7]. Messenger RNA (mRNA) vaccine has recently emerged as an appealing alternative to DNA vaccine for infectious disease preventions and anti-cancer treatments. Advantages of mRNA over DNA as cancer vaccine strategy include: (1) mRNAs can be translated in both dividing and non-dividing cells, where RNA only needs to be internalized into the cytoplasm, followed by a one-step translation into the antigen(s) of interest. The rate and magnitude of protein expression of mRNA are typically higher than DNA vaccines. (2) Unlike DNA vaccines, mRNA vaccines cannot integrate into the genome sequence, thus free of insertional mutagenesis. The first report of the successful expression of in vitro transcription (IVT) mRNA in mouse skeletal muscle cells through direct injection into animals was published in 1990, underlining the feasibility of mRNA vaccine development [9]. However, this early attempt didn’t lead to substantial mRNA vaccine development investigations, largely stemmed from concerns regarding mRNA instability, insufficient in vivo delivery, and high intrinsic innate immunogenicity [10].
Over the past decades, major technological innovations have enabled mRNA as a more feasible vaccine candidate. Various modifications of mRNA backbone and untranslated regions make mRNA less RNase-sensitive, more stable, and highly translatable. Improved purification methods have allowed mRNA products free of double-stranded contaminations, thus reducing the non-specific activation of innate immunity. More efficient in vivo delivery of mRNA has been achieved by formulating mRNA into delivery vehicles, including but not limited to lipid nanoparticles (LNPs), polymers, and peptides. Lastly, IVT methods (free from isolation and purification of biological samples) have been widely applied to the production of mRNAs. With the maturation of scale-up manufacturing, mRNA vaccines have supreme advantages over other vaccine techniques due to the rapid, inexpensive production and large-scale deployment [11]. So far, non-replicating mRNAs are mostly investigated in clinical trials for cancer treatment. However, self-amplifying mRNAs (SAM) have gained extensive attention and are being evaluated in both cancer and infectious disease due to long-lasting efficacy and lower required dosages [12, 13].
Up to now, over twenty mRNA-based immunotherapies have entered clinical trials with some promising outcomes in solid tumor treatments. Besides anti-cancer immunotherapies, mRNA vaccines have a vast advantage to respond rapidly to the global explosion of the coronavirus disease 2019 (COVID-19). With the recent U.S. FDA’s approval of two mRNA-based vaccines from Pfizer-BioNTech and Moderna for emergency use in COVID-19 prevention, the mRNA vaccine field will encompass a dramatic rise in the market value and will attract widespread interest in both cancer and infectious disease applications [14, 15]. In this review, we discuss the improvements that have been made on mRNA structures to increase stabilities and translation efficiencies, highlight the advantages and limitations of various in vivo delivery vehicles for mRNA therapeutics, evaluate the applications of SAM in cancer vaccines, and summarize the current clinical applications of mRNA cancer vaccines. The data suggest mRNA vaccines have the potential to overcome several challenges for cancer immunotherapies.
Basic mRNA pharmacology, limitations and advantages
mRNA is a single-stranded macromolecule that corresponds to the genetic sequence of a DNA in the cell nuclei and is read by a ribosome and translated into proteins in the cytoplasm [16]. The rationale behind mRNA as an appealing cancer vaccination platform is to deliver the transcript of interest(s), encoding one or more TAAs or TSAs, into the host cell (typically APCs) cytoplasm, to be expressed into the targeted antigen(s). The expressed TAAs and TSAs can be presented to the surface of APCs by major histocompatibility complexes (MHCs) to activate anti-tumor immunity. mRNA vaccine could induce both antibody/B cell mediated humoral responses and CD4+ T/ CD8+ cytotoxic T cell responses, which are beneficial for efficient clearance of malignant cells. On the other side, mRNA is non-infectious and non-integrating, and therefore it’s quite tolerable and has posed no genetic risks. There are mainly three types of RNAs currently investigated as cancer vaccines: non-replicating unmodified mRNA, modified mRNA and virus derived SAM. IVT has been commonly used for synthesizing both non-replicating mRNA (modified and unmodified) and SAMs. The method utilizes a bacteriophage RNA polymerase, such as T3, T7 or SP6 RNA polymerase and a linearized DNA template containing the target antigen sequences. The IVT production precludes the usage of cells and their associated regulatory hurdles, and therefore the production of mRNA is undoubtedly simpler, quicker and cleaner than large-scale protein production and purification. The fundamental structure of conventional non-replicating IVT mRNA, which correspondent to “mature” eukaryotic mRNA, is composed of an open reading frame (ORF) region that encodes the target antigen sequences, flanked by five-prime (5′) and three-prime (3′) untranslated region (UTR), and further stabilized by 7-methylgaunosine (m7G) 5′ cap and 3′ poly (A) tails respectively. The 5′ cap and 3′ poly (A) can be added during the IVT or added enzymatically after initial IVT. In contrast, SAM comprises two ORFs, including one that encodes the targeted antigen sequences and another that encodes viral replication machinery which enables long-lasting RNA amplification intracellularly. Once mRNA or SAM is internalized and transited to the cytosol, it will be read by ribosomes, and translated into proteins that undergoes post-translational modifications, ultimately resulting in a properly folded functional protein. The remaining IVT mRNA template will be degraded by normal physiological process, decreasing the metabolite toxicity risk [11].
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