ABSTRACT
Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.
Subject(s)
Codon, Nonsense , Cystic Fibrosis Transmembrane Conductance Regulator , RNA, Transfer , Animals , Mice , Amino Acids/genetics , Codon, Nonsense/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , RNA, Transfer/administration & dosage , RNA, Transfer/genetics , RNA, Transfer/therapeutic use , Base Pairing , Anticodon/genetics , Protein Biosynthesis , Nasal Mucosa/metabolism , Ribosome ProfilingABSTRACT
A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.
Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/administration & dosage , Alphavirus/genetics , Alphavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , COVID-19 Vaccines/biosynthesis , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Female , Gene Expression , Humans , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-4/genetics , Interleukin-4/immunology , Mice , Mice, Transgenic , Replicon/immunology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/virology , Transgenes , Treatment Outcome , Vaccination/methods , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , mRNA VaccinesABSTRACT
Few studies have investigated the possible role of higher-level cognitive mechanisms in color constancy. Following up on previous work with successive color constancy [J. Exper. Psychol. Learn. Mem. Cogn. 37, 1014 (2011)], the current study examined the relation between simultaneous color constancy and working memory-the ability to maintain a desired representation while suppressing irrelevant information. Higher working memory was associated with poorer simultaneous color constancy of a chromatically complex stimulus. Ways in which the executive attention mechanism of working memory may account for this are discussed. This finding supports a role for higher-level cognitive mechanisms in color constancy and is the first to demonstrate a relation between simultaneous color constancy and a complex cognitive ability.
Subject(s)
Color Perception/physiology , Memory, Short-Term/physiology , Female , Humans , Male , Retention, Psychology/physiology , Time FactorsABSTRACT
We explored the relation between individual differences in working memory (WM) and color constancy, the phenomenon of color perception that allows us to perceive the color of an object as relatively stable under changes in illumination. Successive color constancy (measured by first viewing a colored surface under a particular illumination and later recalling it under a new illumination) was better for higher WM individuals than for lower WM individuals. Moreover, the magnitude of this WM difference depended on how much contextual information was available in the scene, which typically improves color constancy. By contrast, simple color memory, measured by viewing and recalling a colored surface under the same illumination, showed no significant relation to WM. This study reveals a relation between WM and a low-level perceptual process not previously thought to operate within the confines of attentional control, and it provides a first account of the individual differences in color constancy known about for decades.
Subject(s)
Color Perception/physiology , Memory Disorders/physiopathology , Memory, Short-Term/physiology , Adolescent , Color Perception Tests , Female , Humans , Male , Photic Stimulation/methods , Psychophysics , Visual Fields/physiology , Young AdultABSTRACT
OBJECTIVES: Parents of premature infants often perceive their infants as medically vulnerable. High parental perception of child vulnerability (PPCV) is associated with disproportionately high health care utilization. The objectives of this study were to determine whether higher PPCV is correlated with worse developmental outcome in premature infants at 1-year adjusted age and to identify factors, present at neonatal discharge, that predict high PPCV. METHODS: This prospective cohort study assessed mothers of 116 premature infants who were
