Clinical applications of aptamers and nucleic acid therapeutics in haematological malignancies

Haematological malignancies result from a heterogeneous mix of genetic mutations and chromosome aberrations and translocations. Targeted therapies, such as the anti-CD20 antibody rituximab, or the BCR-ABL1 inhibitor imatinib, have proven to be effective treatments in the management of some of these malignancies, though relapsing or refractory disease is still common. Nucleic acid-based therapies have also entered the clinical arena, providing an alternative, complementary approach. The forerunner of these therapies were the antisense oligonucleotides, but their scope has expanded to include short-interfering RNA (siRNA), microRNA, decoy oligonucleotides and aptamers. These can be used either as monotherapeutics, in conjunction with current chemotherapy regimens, or in combination with each other to improve therapeutic efficacy. Not only can these nucleic acid-based therapies silence target genes, they also have the potential of restoring gene function. While challenges remain in delivering effective doses of nucleic acid in vivo, these are steadily being met, suggesting an optimistic future in the treatment of haematological malignancies. This review summarizes the application of nucleic acid-based therapeutics, particularly aptamers, in the diagnosis and treatment of haematological malignancies.

Nucleic acid-based aptamers: applications, development and clinical trials

Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easier manufacturing protocol, freedom to introduce chemical modifications for further improvement, etc. Reiterative selection process of aptamers over 10-15 cycles starting from a large initial pool of random nucleotide sequences renders them with high binding affinity, thereby making them extremely specific for their targets. Aptamer-based detection systems are well investigated and likely to displace primitive detection systems. Aptamer chimeras (combination of aptamers with another aptamer or biomacromolecule or chemical moiety) have the potential activity of both the parent molecules, and thus hold the capability to perform diverse functions at the same time. Owing to their extremely high specificity and lack of immunogenicity or pathogenicity, a number of other aptamers have recently entered clinical trials and have garnered favorable attention from pharmaceutical companies. Promising results from the clinical trials provide new hope to change the conventional style of therapy. Aptamers have attained high therapeutic relevance in a short time as compared to synthetic drugs and/or other modes of therapy. This review follows the various trends in aptamer technology including production, selection, modifications and success in clinical fields. It focusses largely on the various applications of aptamers which mainly depend upon their selection procedures. The review also sheds light on various modifications and chimerizations that have been implemented in order to improve the stability and functioning of the aptamers, including introduction of locked nucleic acids (LNAs). The application of various aptamers in detection systems has been discussed elaborately in order to stress on their role as efficient diagnostic agents. The key aspect of this review is focused on success of aptamers on the basis of their performance in clinical trials for various diseases.

Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

DEVELOPMENT OF A NUCLEIC ACID-BASED SPECIFIC GROWTH MODEL FOR JUVENILE BLUE CRAB, CALLINECTES SAPIDUS

The evaluation and identification of habitats that function as nurseries for marine species has the potential to improve conservation and management. A key assessment of nursery habitat is estimating individual growth. However, the discrete growth of crustaceans presents a challenge for many traditional in situ techniques to accurately estimate growth over a short temporal scale. To evaluate the use of nucleic acid ratios (R:D) for juvenile blue crab (Callinectes sapidus), I developed and validated an R:D-based index of growth in the laboratory. R:D based growth estimates of crabs collected in the Patuxent River, MD indicated growth ranged from 0.8-25.9 (mg·g-1·d-1). Overall, there was no effect of size on growth, whereas there was a weak, but significant effect of date. These data provide insight into patterns of habitat-specific growth. These results highlight the complexity of the biological and physical factors which regulate growth of juvenile blue crabs in the field.

Clinical potential of oligonucleotide-based therapeutics in the respiratory system

The discovery of an ever-expanding plethora of coding and non-coding RNAs with nodal and causal roles in the regulation of lung physiology and disease is reinvigorating interest in the clinical utility of the oligonucleotide therapeutic class. This is strongly supported through recent advances in nucleic acids chemistry, synthetic oligonucleotide delivery and viral gene therapy that have succeeded in bringing to market at least three nucleic acid-based drugs. As a consequence, multiple new candidates such as RNA interference modulators, antisense, and splice switching compounds are now progressing through clinical evaluation. Here, manipulation of RNA for the treatment of lung disease is explored, with emphasis on robust pharmacological evidence aligned to the five pillars of drug development: exposure to the appropriate tissue, binding to the desired molecular target, evidence of the expected mode of action, activity in the relevant patient population and commercially viable value proposition.

Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles

Self-amplifying RNA or RNA replicon is a form of nucleic acid-based vaccine derived from either positive-strand or negative-strand RNA viruses. The gene sequences encoding structural proteins in these RNA viruses are replaced by mRNA encoding antigens of interest as well as by RNA polymerase for replication and transcription. This kind of vaccine has been successfully assayed with many different antigens as vaccines candidates, and has been shown to be potent in several animal species, including mice, nonhuman primates, and humans. A key challenge to realizing the broad potential of self-amplifying vaccines is the need for safe and effective delivery methods. Ideally, an RNA nanocarrier should provide protection from blood nucleases and extended blood circulation, which ultimately would increase the possibility of reaching the target tissue. The delivery system must then be internalized by the target cell and, upon receptor-mediated endocytosis, must be able to escape from the endosomal compartment into the cell cytoplasm, where the RNA machinery is located, while avoiding degradation by lysosomal enzymes. Further, delivery systems for systemic administration ought to be well tolerated upon administration. They should be safe, enabling the multiadministration treatment modalities required for improved clinical outcomes and, from a developmental point of view, production of large batches with reproducible specifications is also desirable. In this review, the concept of self-amplifying RNA vaccines and the most promising lipid-based delivery systems are discussed.

Nucleic Acid-Induced Aggregation and Pyrene Excimer Formation

Nucleic acid was found to induce the aggregation of the positively charged pyrene probe (compound 1); as a result, strong pyrene excimer emission was observed. The intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide length, sequence, and concentration. These results suggest a new strategy for label-free nucleic acid-based biosensing applications.

Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid analysis

Reagent pre-storage in a microfluidic chip can enhance operator convenience, simplify the system design, reduce the cost of storage and shipment, and avoid the risk of cross-contamination. Although dry reagents have long been used in lateral flow immunoassays, they have rarely been used for nucleic acid-based point-of-care (POC) assays due to the lack of reliable techniques to dehydrate and store fragile molecules involved in the reaction. In this study, we describe a simple and efficient method for prolonged on-chip storage of PCR reagents. The method is based on gelification of all reagents required for PCR as a ready-to-use product. The approach was successfully implemented in a lab-on-a-foil system, and the gelification process was automated for mass production. Integration of reagents on-chip by gelification greatly facilitated the development of easy-to-use lab-on-a-chip (LOC) devices for fast and cost-effective POC analysis.

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine

Conventional anticancer therapies, such as chemo- and/or radio-therapy are often unable to completely eradicate cancers due to abnormal tumor microenvironment, as well as increased drug/radiation resistance. More effective therapeutic strategies for overcoming these obstacles are urgently in demand. Aptamers, as chemical antibodies that bind to targets with high affinity and specificity, are a promising new and novel agent for both cancer diagnostic and therapeutic applications. Aptamer-based cancer cell targeting facilitates the development of active targeting in which aptamer-mediated drug delivery could provide promising anticancer outcomes. This review is to update the current progress of aptamer-based cancer diagnosis and aptamer-mediated active targeting for cancer therapy in vivo, exploring the potential of this novel form of targeted cancer therapy.

Label free electrochemiluminescence protocol for sensitive DNA detection with a tris(2,2'-bipyridyl)ruthenium(II) modified electrode based on nucleic acid oxidation

Label free electrochemiluminescence (ECL) DNA detection based on catalytic guanine and adenine bases oxidation using tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] modified glassy carbon (GC) electrode was demonstrated in this work. The modified GC electrode was prepared by casting carbon nanotubes (CNT)/Nafion/Ru(bpy)(3)(2+) composite film on the electrode surface. ECL signals of doublestranded DNA and their thermally denatured counterparts can be distinctly discriminated using cyclic voltammetry (CV) with a low concentration (3.04 x 10(-8) mol/L for Salmon Testes-DNA). Most importantly, sensitive single-base mismatch detection of p53 gene sequence segment was realized with 3.93 x 10(-10) mol/L employing CV stimulation (ECL signal of C/A mismatched DNA oligonucleotides was 1.5-fold higher than that of fully base-paired DNA oligonucleotides). Label free, high sensitivity and simplicity for single-base mismatch discrimination were the main advantages of the present ECL technique for DNA detection over the traditional DNA sensors.

RALA-mediated delivery of FKBPL nucleic acid therapeutics

Aims: RALA is a novel 30 mer bioinspired amphipathic peptide that is showing promise for gene delivery. Here, we used RALA to deliver the FK506-binding protein like – FKBPL gene (pFKBPL) – a novel member of the immunophilin protein family. FKBPL is a secreted protein, with overexpression shown to inhibit angiogenesis, tumor growth and stemness, through a variety of intra- and extracellular signaling mechanisms. We also elucidated proangiogenic activity and stemness after utilizing RALA to deliver siRNA (siFKBPL). Materials & methods: The RALA/pFKBPL and RALA/siFKBPL nanoparticles were characterized in terms of size, charge, stability and toxicity. Overexpression and knockdown of FKBPL was assessed in vitro and in vivo. Results: RALA delivered both pFKBPL and siFKBPL with less cytotoxicity than commercially available counterparts. In vivo, RALA/pFKBPL delivery retarded tumor growth, and prolonged survival with an associated decrease in angiogenesis, while RALA/siFKBPL had no effect on tumor growth rate or survival, but resulted in an increase in angiogenesis and stemness. Conclusion: RALA is an effective delivery system for both FKBPL DNA and RNAi and highlights an alternative therapeutic approach to harnessing FKBPL's antiangiogenic and antistemness activity.

Aptamer-based therapeutics of the past, present and future: from the perspective of eye-related diseases

 Aptamers have emerged as a novel and powerful class of biomolecules with an immense untapped potential. The ability to synthesise highly specific aptamers against any molecular target make them a vital cog in the design of effective therapeutics for the future. However, only a minutia of the enormous potential of this dynamic class of molecule has been exploited. Several aptamers have been studied for the treatment of eye-related disorders, and one such strategy has been successful in therapy. This review gives an account of several eye diseases and their regulatory biomolecules where other nucleic acid therapeutics have been attempted with limited success and how aptamers, with their exceptional flexibility to chemical modifications, can overcome those inherent shortcomings.

Locked nucleic acid aptamer based microfluidic devices for capturing tumor cells

Design and fabrication of novel microfluidic devices for sensitive and specific capture of circulating tumor cells using locked nucleic acid modified aptamers and antibodies targeting EpCAM/Nucleolin expression. These devices also allow re-usability, on-chip characterization of multiple markers and release of viable captured cells for further culture and in vitro characterization for cancer diagnosis, prognosis and therapeutic planning.

Microarray based real-time analysis of nucleic acid hybridization kinetics and thermodynamics

Ziel dieser Dissertation ist die experimentelle Charakterisierung und quantitative Beschreibung der Hybridisierung von komplementären Nukleinsäuresträngen mit oberflächengebundenen Fängermolekülen für die Entwicklung von integrierten Biosensoren. Im Gegensatz zu lösungsbasierten Verfahren ist mit Microarray Substraten die Untersuchung vieler Nukleinsäurekombinationen parallel möglich. Als biologisch relevantes Evaluierungssystem wurde das in Eukaryoten universell exprimierte Actin Gen aus unterschiedlichen Pflanzenspezies verwendet. Dieses Testsystem ermöglicht es, nahe verwandte Pflanzenarten auf Grund von geringen Unterschieden in der Gen-Sequenz (SNPs) zu charakterisieren. Aufbauend auf dieses gut studierte Modell eines House-Keeping Genes wurde ein umfassendes Microarray System, bestehend aus kurzen und langen Oligonukleotiden (mit eingebauten LNA-Molekülen), cDNAs sowie DNA und RNA Targets realisiert. Damit konnte ein für online Messung optimiertes Testsystem mit hohen Signalstärken entwickelt werden. Basierend auf den Ergebnissen wurde der gesamte Signalpfad von Nukleinsärekonzentration bis zum digitalen Wert modelliert. Die aus der Entwicklung und den Experimenten gewonnen Erkenntnisse über die Kinetik und Thermodynamik von Hybridisierung sind in drei Publikationen zusammengefasst die das Rückgrat dieser Dissertation bilden. Die erste Publikation beschreibt die Verbesserung der Reproduzierbarkeit und Spezifizität von Microarray Ergebnissen durch online Messung von Kinetik und Thermodynamik gegenüber endpunktbasierten Messungen mit Standard Microarrays. Für die Auswertung der riesigen Datenmengen wurden zwei Algorithmen entwickelt, eine reaktionskinetische Modellierung der Isothermen und ein auf der Fermi-Dirac Statistik beruhende Beschreibung des Schmelzüberganges. Diese Algorithmen werden in der zweiten Publikation beschrieben. Durch die Realisierung von gleichen Sequenzen in den chemisch unterschiedlichen Nukleinsäuren (DNA, RNA und LNA) ist es möglich, definierte Unterschiede in der Konformation des Riboserings und der C5-Methylgruppe der Pyrimidine zu untersuchen. Die kompetitive Wechselwirkung dieser unterschiedlichen Nukleinsäuren gleicher Sequenz und die Auswirkungen auf Kinetik und Thermodynamik ist das Thema der dritten Publikation. Neben der molekularbiologischen und technologischen Entwicklung im Bereich der Sensorik von Hybridisierungsreaktionen oberflächengebundener Nukleinsäuremolekülen, der automatisierten Auswertung und Modellierung der anfallenden Datenmengen und der damit verbundenen besseren quantitativen Beschreibung von Kinetik und Thermodynamik dieser Reaktionen tragen die Ergebnisse zum besseren Verständnis der physikalisch-chemischen Struktur des elementarsten biologischen Moleküls und seiner nach wie vor nicht vollständig verstandenen Spezifizität bei.