mRNA biotherapeutics landscape for rare genetic disorders.

The medical emergency of COVID-19 brought to the forefront mRNA vaccine technology where the mRNA vaccine candidates mRNA-1273 and BNT162b2 displayed superlative and more than 90% efficacy in protecting against SARS-CoV2 infections. Rare genetic disorders are rare individually, but collectively they are common and represent a medical emergency. In mRNA biotherapeutic technology, administration of a therapeutic protein-encoding mRNA-nanoparticle formulation allows for in vivo production of therapeutic proteins to functionally complement the protein functions lacking in rare disease patients. The platform nature of mRNA biotherapeutic technology propels rare disease drug discovery and, owing to the scalable and synthetic nature of mRNA manufacturing, empowers parallel product development using a universal production pipeline. This review focuses on the advantages of mRNA biotherapeutic technology over current therapies for rare diseases and provides summaries for the proof-of-concept preclinical studies performed to demonstrate the potential of mRNA biotherapeutic technology. Apart from preclinical studies, this review also spotlights the clinical trials currently being conducted for mRNA biotherapeutic candidates. Currently, seven mRNA biotherapeutic candidates have entered clinical trials for rare diseases, and of them, 3 candidates entered in the year 2023 alone. The rapid pace of clinical development promises a future where, as with mRNA vaccines for COVID-19, mRNA biotherapeutic technology would combat an emergency of rare genetic disorders.

Spinal muscular atrophy: Molecular mechanism of pathogenesis, diagnosis, therapeutics, and clinical trials in the Indian context.

Spinal muscular atrophy (SMA) is a neuromuscular, rare genetic disorder caused due to loss-of-function mutations in the survival motor neuron-1 (SMN1) gene, leading to deficiency of the SMN protein. The severity of the disease phenotype is inversely proportional to the copy number of another gene, SMN2, that differs from SMN1 by a few nucleotides. The current diagnostic methods for SMA include symptom-based diagnosis, biochemical methods like detection of serum creatine kinase, and molecular detection of disease-causing mutations using polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and exome or next-generation sequencing (NGS). Along with detection of the disease-causing mutation in the SMN1 gene, it is crucial to identify the copy number of the SMN2 gene, which is a disease modifier. Therapeutic options like gene therapy, antisense therapy, and small molecules are available for SMA, but, the costs are prohibitively high. This review discusses the prevalence, diagnosis, available therapeutic options for SMA, and their clinical trials in the Indian context, and highlights the need for measures to make indigenous diagnostic and therapeutic interventions.

Regulation of transferrin receptor trafficking by optineurin and its disease-associated mutants.

Transferrin receptor (TFRC) is a transmembrane protein that plays a crucial role in mediating homeostasis of iron in the cell. The binding of transferrin (that is bound to iron) to TFRC at the cell membrane generally starts endocytosis of TFRC-transferrin complex, which leads to formation of vesicles that are positive for TFRC. These vesicles travel to the early endosomes and later to the endocytic recycling compartment. Release of iron occurs in the early endosomes because of acidic pH. Major fraction of the transferrin and TFRC is transported back to the cell membrane; however, a minor fraction of it is transported to lysosomes through the process of autophagy. Optineurin (OPTN) is a multi-functional adaptor protein that plays a pivotal role in the control of TFRC trafficking, recycling and autophagy dependent degradation. Optineurin also plays a role in cargo-selective and non-selective autophagy. Here, we review our understanding of the function of OPTN in regulating TFRC trafficking, recycling and autophagy dependent degradation. We also discuss the mechanisms by which certain disease-associated mutations of OPTN alter these processes.

Detection of SARS-CoV-2 in the air in Indian hospitals and houses of COVID-19 patients.

To understand the transmission characteristics of severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) through air, samples from different locations occupied by coronavirus disease (COVID-19) patients were analyzed. Three sampling strategies were used to understand the presence of virus in the air in different environmental conditions. In the first strategy, which involved hospital settings, air samples were collected from several areas of hospitals like COVID-intensive-care units (ICUs), nurse-stations, COVID-wards, corridors, non-COVID-wards, personal protective equipment (PPE) doffing areas, COVID rooms, out-patient (OP) corridors, mortuary, COVID casualty areas, non-COVID ICUs and doctors' rooms. Out of the 80 air samples collected from 6 hospitals from two Indian cities- Hyderabad and Mohali, 30 samples showed the presence of SARS-CoV-2 nucleic acids. In the second sampling strategy, that involved indoor settings, one or more COVID-19 patients were asked to spend a short duration of time in a closed room. Out of 17 samples, 5 samples, including 4 samples collected after the departure of three symptomatic patients from the room, showed the presence of SARS-CoV-2 nucleic acids. In the third strategy, involving indoor settings, air samples were collected from rooms of houses of home-quarantined COVID-19 patients and it was observed that SARS-CoV-2 RNA could be detected in the air in the rooms occupied by COVID-19 patients but not in the other rooms of the houses. Taken together, we observed that the air around COVID-19 patients frequently showed the presence of SARS-CoV-2 RNA in both hospital and indoor residential settings and the positivity rate was higher when 2 or more COVID-19 patients occupied the room. In hospitals, SARS-CoV-2 RNA could be detected in ICUs as well as in non-ICUs, suggesting that the viral shedding happened irrespective of the severity of the infection. This study provides evidence for the viability of SARS-CoV-2 and its long-range transport through the air. Thus, airborne transmission could be a major mode of transmission for SARS-CoV-2 and appropriate precautions need to be followed to prevent the spread of infection through the air.

Autophagy-independent cytoprotection by optineurin from toxicity of aggregates formed by mutant huntingtin and mutant ataxin-3.

An important feature of several neurodegenerative diseases is the formation of pathological structures containing aggregated proteins. The autophagy receptor optineurin/OPTN is frequently observed in these structures. The role played by optineurin in these aggregates is not clear. In this study, we explored whether optineurin has a cytoprotective role in the cells having mutant protein aggregates. We overexpressed mutant huntingtin having 97 glutamine repeats (mHtt) and mutant ataxin-3 having 130 glutamine repeats (mAtax-3) in wild-type and optineurin-deficient neuronal (N2A) and non-neuronal cells (Optn-/- mouse embryonic fibroblasts) and determined the percentage of dead cells with mutant protein aggregates. Optineurin-deficient cells having mHtt or mAtax-3 aggregates showed higher cell death as compared to wild-type cells having mutant protein aggregates. Confocal microscopy revealed that optineurin formed a shell around mHtt and mAtax-3 aggregates through its C-terminal domain. The C-terminal domain of optineurin, which lacks LC3-interacting region required for autophagy, was necessary and sufficient to reduce cytotoxicity of mHtt and mAtax-3 aggregates. Our results show that in the absence of optineurin, mutant protein aggregates are highly toxic, revealing an autophagy-independent cytoprotective function of optineurin, which is mediated by its C-terminal domain.

Optineurin modulates ER stress-induced signaling pathways and cell death.

We have investigated the physiological role of the autophagy receptor Optineurin/Optn in endoplasmic reticulum (ER) stress response using cellular and animal models. In comparison to their normal counterparts, Optn-deficient mouse embryonic fibroblasts showed significantly higher cell death and caspase-3 activation upon treatment with tunicamycin and thapsigargin, inducers of ER stress. The transcript levels of some of the genes regulated by the IRE1-XBP1 and PERK-ATF4 pathways were upregulated in Optn-deficient cells, in comparison with normal cells, upon treatment with tunicamycin, and also in the brain cortex and liver of tunicamycin treated Optn-deficient mice. Also, the basal levels of IRE1α and PERK were higher in Optn-deficient cells. These results suggest that Optn modulates ER stress-induced signaling pathways and provides protection from ER stress-induced cell death.

Identification of a splice variant of optineurin which is defective in autophagy and phosphorylation.

Optineurin (Optn) is an autophagy receptor that performs various functions in cargo-selective and non-selective autophagy. Here, we have identified and characterized a splice variant of mouse optineurin mRNA, which produces a truncated protein lacking N-terminal 157 amino acids (d157mOptn). This mRNA and protein are expressed in several tissues and cells. d157mOptn has an intact LC3-interacting region and a serine (S187) in it. However, unlike normal optineurin, the d157mOptn was not phosphorylated at this site when expressed in mammalian cells, and showed reduced interaction with TBK1 (tank binding kinase) that mediates phosphorylation at S187 (S177 in human OPTN). This phosphorylation of Optn required intact N-terminal sequence as well as functional C-terminal ubiquitin-binding domain. Unlike normal optineurin, d157mOptn was unable to promote autophagosome and autolysosome formation upon expression in Optn-deficient cells. d157mOptn was recruited to mutant huntingtin aggregates, but unlike wild type optineurin, it was unable to clear these aggregates by autophagy in neuronal NSC-34 cells. Phospho-TBK1 was seen around mutant Huntingtin aggregates in Optn overexpressing cells but it was reduced in cells overexpressing d157mOptn. Thus, we have identified an isoform of mouse optineurin which is defective in cargo-selective and non-selective autophagy possibly due to loss of phosphorylation and impaired interaction with TBK1. This isoform, which inhibits autophagosome formation in neuronal cells, might be involved in selectively modulating some of the functions of Optn, such as autophagy. Our results provide an insight into the role of N-terminal domain of Optn in various autophagic functions.

Autophagy receptor optineurin promotes autophagosome formation by potentiating LC3-II production and phagophore maturation.

Autophagy is an essential physiological process that maintains cellular homeostasis by eliminating harmful protein aggregates, damaged organelles and certain pathogens through lysosomal degradation. During autophagy specialized structures, known as autophagosomes are formed that recruit the cargo through autophagy receptors, and deliver it to lysosomes. Optineurin (Optn) is an autophagy receptor that mediates cargo selective autophagy. Recently, we have identified a novel function of Optn that promotes autophagosome formation during non-selective autophagy. Optn-deficient cells show reduced formation of autophagosomal protein LC3-II and lower number of autophagosomes as well as autolysosomes. Interestingly, formation of phagophores is increased in Optn-deficient cells. This suggests that Optn promotes autophagosome formation by potentiating LC3-II production and phagophore maturation. Phosphorylation of Optn at Ser-177 is required for promoting autophagosome formation. Here, we discuss various aspects of the role of Optn in the formation of autophagosomes and Atg16L1-positive vesicles. We also discuss the potential role of Rab1a-Optn interaction.

Optineurin promotes autophagosome formation by recruiting the autophagy-related Atg12-5-16L1 complex to phagophores containing the Wipi2 protein.

Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.