Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing

Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using H2K mdx mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2′-O-methyl and 2′-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5–20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.

Khorana and colleagues laid the foundation for synthetic nucleic acid technology by demonstrating the first successful chemical synthesis of sequence-defined oligonucleotides in 1958 (1, 2). However, the process was inefficient due to its low yield and labor demands. A significant breakthrough was achieved in 1981 by Caruthers and coworkers who developed phosphoramidite chemistry for the synthesis of oligonucleotides, an approach that has become the most widely used method of modern oligonucleotide synthesis (3, 4). In the last 2 decades, improvements in synthesis, purification, and characterization techniques have allowed oligonucleotides, prepared by the phosphoramidite methodology, to be synthesized in large scale, with adequate yield and purity, for use as therapeutics. These advances have driven the remarkable progress of nucleic acid technologies in therapeutic and diagnostic applications—particularly antisense oligonucleotide (ASO) drugs. Following the approval of Vitravene by the US Food and Drug Administration in 1998 for the treatment of cytomegalovirus retinitis in immunocompromised patients (5), eight ASO drugs have been approved for clinical use, including Kynamro (2013) (6), Exondys 51 (2016) (7), Spinraza (2017) (8), Tegsedi (2018) (9), Waylivra (2019) (10), Vyondys 53 (2020) (11), Viltepso (2020) (12), and Amondys 45 (2021) (13). These approvals demonstrate the tremendous potential of ASOs for the treatment of various diseases.Synthetic oligonucleotides, based upon using natural DNA and RNA, are not clinically relevant because of their vulnerability to nucleases and poor target binding affinities. In contrast, chemically modified nucleic acid analogs offer superior therapeutic properties, such as increased target binding affinity, improved resistance to nuclease degradation, and diminished immunogenicity. A comprehensive review of these analogs can be found elsewhere (14). Currently, there are three main chemically modified chemistries that have been utilized in the synthesis of approved oligonucleotide drugs: 2′-O-methyl (2′-OMe) and 2′-O-methoxyethyl (2′-MOE) nucleosides having a phosphorothioate (PS) backbone and also phosphorodiamidate morpholino oligomers (PMOs) (5–15) carrying a N,N-dimethylamino phosphorodiamidate backbone. However, there are significant limitations associated with these chemistries. 2′-OMe and 2′-MOE oligonucleotides can exhibit toxicity (16, 17), whereas PMOs, despite their excellent safety profile, are challenging to synthesize on a scale large enough for use as therapeutic drugs. Additionally, PMOs are rapidly excreted after administration in vivo and thereby require high dosages that subsequently contribute to the cost of treatment. Another limitation of PMOs is their inability to form complexes with commercial transfection reagents. This limitation challenges the rapid evaluation of any PMO drug in an in vitro system. Previously, we have demonstrated a novel chemistry leading to the synthesis of thiomorpholino oligonucleotides (TMOs) (18,19). In the present study, we utilize a well-known Duchenne muscular dystrophy (DMD) cellular model (H2K^b-tsA58 mdx [H2K mdx] mouse myotubes) (20, 21) to systematically evaluate these TMOs. Herein, we report the design, synthesis, and evaluation of TMOs for their exon 23 skipping activity using H2K mdx myotubes as an in vitro model system.     

The next generation: continuous glucose monitors

These "real-time" monitors promise to change the way we chart ups and downs, by delivering readings every few minutes around the clock. But don't toss your old monitors just yet!     

Cardiac cell therapies: the next generation

Although significant advances have been made in terms of pharmacological, catheter-based, and surgical palliation, heart failure remains a fatal disease. As a curative concept, regenerative medicine aims at the restoration of the physiologic cellular composition of diseased organs. So far, clinical cardiac regeneration attempts have only been moderately successful, but a better understanding of myocardial cell homeostasis and somatic as well as embryonic stem cell biology has opened the door for the development of more potent therapeutic cardiac regeneration strategies. Accumulating evidence indicates that the postnatal mammalian heart retains a pool of tissue-specific progenitor cells and is also repopulated by cells from extracardiac sources. However, this intrinsic myocardial regeneration potential clearly needs to be augmented by either manipulation of the cell cycle of differentiated cells, activation of resident cardiac progenitor cells, and/or the transplantation of exogenous cells. This review summarizes the recent developments in cardiac regenerative medicine, many of which may find their way into the clinical setting in the foreseeable future.     

Next generation sequencing for clinical diagnostics and personalised medicine: implications for the next generation cardiologist

The fast moving field of genomic medicine is already impacting on clinical care and cardiologists are fortunate to be in a position to benefit early from the transformative advances in genomics. However, the challenges associated with genomics in the clinic in general, and with next generation sequencing technologies in particular, are significant and cardiologists need to be prepared if they wish to surf the wave of genomic opportunity. This paper presents an overview of the implications of next generation sequencing for clinical diagnostics and personalised medicine in the cardiology clinic.     

Choice of prosthetic heart valves:update for the next generation

Intervention for valvular heart disease poses unique clinical challenges in cardiology because the diseases are of relatively low prevalence, the interventions do not lend themselves to randomized comparative trials, and important clinical end points are assessed only after decades of follow-up. In addition, continuing advances in prosthetic heart valve technology make follow-up a moving target because long-term data by definition are available only for older prostheses. Newer tissue and mechanical prostheses afford superior hemodynamics compared with their older counterparts, and data suggest that durability and patient mortality are superior with newer compared with older bioprostheses. Arbitrary cutoffs dictating valve choice based predominantly on patient age may not give appropriate weight to individual patient perspectives. In educating and counseling patients regarding choices in heart valve prostheses, the clinician should help the patient weigh the relative merits for the individual patient of projected mortality, valve durability, and requirement for anticoagulation, with associated freedom from re-operation, hemorrhagic and thromboembolic risk, and impact on lifestyle.     

Porcine bioprosthetic heart valves: The next generation

There have been significant advances in organ xenotransplantation (cross-species transplantation), especially in the development of genetically engineered pigs, but clinical trials of solid organ transplants are still a time away. However, there is a form of pig-to-human xenotransplantation that has been taking place since the 1960s-bioprosthetic heart valve (BHV) replacement. Recently, there has been increasing evidence that, despite glutaraldehyde fixation of BHVs, there is a significant immune reaction to the valves, leading to calcification, rapid structural deterioration, and failure, particularly in young patients who have a more vigorous immune system and metabolism than the elderly. However, it is the young patients who would most benefit from such BHVs because these avoid the complications associated with the lifelong anticoagulation required with mechanical valves. In this review, we examine pathologic and immunohistochemical reports of failed BHVs that suggest that there is an immune response to these valves. Small animal studies that link the development of calcification and BHV failure to the immune response are reviewed. We draw parallels between the problems of glutaraldehyde-fixed tissue xenotransplantation and those currently being faced in live organ xenotransplantation. Finally, we discuss the advances being made in the production of genetically modified pigs and the evidence that these pigs may become a source of BHVs that can be used worldwide to treat valvular heart disease in children and young adults (for whom there is no ideal valve replacement in existence today). The design of a BHV that is resistant to the host's immune response would be a major step forward in cardiac surgery.     

Bacteria and fungi in acute cholecystitis. A prospective study comparing next generation sequencing to culture

ObjectivesGuidelines for antibiotic treatment of acute cholecystitis are based on studies using culture techniques for microbial identification. Microbial culture has well described limitations and more comprehensive data on the microbial spectrum may support adjustments of these recommendations. We used next generation sequencing to conduct a thorough microbiological characterization of bile-samples from patients with moderate and severe acute cholecystitis.MethodsWe prospectively included patients with moderate and severe acute cholecystitis, undergoing percutaneous or perioperative drainage of the gall bladder. Bile samples were analyzed using both culture and deep sequencing of bacterial 16S rRNA and rpoB genes and the fungal ITS2-segment. Clinical details were evaluated by medical record review.ResultsThirty-six patients with moderate and severe acute cholecystitis were included. Bile from 31 (86%) of these contained bacteria (29) and/or fungi (5) as determined by sequencing. Culture identified only 40 (38%) of the 106 microbes identified by sequencing. In none of the 15 polymicrobial samples did culture detect all present microbes. Frequently identified bacteria often missed by culture included oral streptococci, anaerobic bacteria, enterococci and Enterobacteriaceae other than Klebsiella spp. and Escherichia coli.ConclusionsCulture techniques display decreased sensitivity for the microbial diagnostics of acute cholecystitis leaving possible pathogens undetected.     

Digital health competencies for the next generation of physicians

As health care continues to change and evolve in a digital society, there is an escalating need for physicians who are skilled and enabled to deliver care using digital health technologies, while remaining able to successfully broker the triadic relationship among patients, computers and themselves. The focus needs to remain firmly on how technology can be leveraged and used to support good medical practice and quality health care, particularly around resolution of longstanding challenges in health care delivery, including equitable access in rural and remote areas, closing the gap on health outcomes and experiences for First Nations peoples and better support in aged care and those living with chronic disease and disability. We propose a set of requisite digital health competencies and recommend that the acquisition and evaluation of these competencies become embedded in physician training curricula and continuing professional development programmes.