12 research outputs found
Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies
Two Scaffolds from Two Flips: (α,β)/(β,γ) CH<sub>2</sub>/NH “Met-Im” Analogues of dTTP
Novel
α,β-CH<sub>2</sub> and β,γ-NH (<b>1a</b>) or α,β-NH and β,γ-CH<sub>2</sub> (<b>1b</b>) “Met-Im” dTTPs were synthesized
via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate
synthons (<b>4a</b>, <b>4b</b>), formed via a base-induced
[1,3]-rearrangement of precursors (<b>3a</b>, <b>3b</b>) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization
during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu
conjugation with dT was avoided by microwave conditions. <b>1a</b> was 9-fold more potent in inhibiting DNA polymerase β, attributed
to an NH-group interaction with R183 in the active site
Small Molecule Inhibition of SAMHD1 dNTPase by Tetramer Destabilization
SAMHD1
is a GTP-activated nonspecific dNTP triphosphohydrolase
that depletes dNTP pools in resting CD4+ T cells and macrophages and
effectively restricts infection by HIV-1. We have designed a nonsubstrate
dUTP analogue with a methylene bridge connecting the α phosphate
and 5′ carbon that potently inhibits SAMHD1. Although pppCH<sub>2</sub>dU shows apparent competitive inhibition, it acts by a surprising
allosteric mechanism that destabilizes active enzyme tetramer
Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine
A bone
imaging toolkit of 21 fluorescent probes with variable spectroscopic
properties, bone mineral binding affinities, and antiprenylation activities
has been created, including a novel linking strategy. The linking
chemistry allows attachment of a diverse selection of dyes fluorescent
in the visible to near-infrared range to any of the three clinically
important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate,
and minodronate or their analogues). The resultant suite of conjugates
offers multiple options to “mix and match” parent drug
structure, fluorescence emission wavelength, relative bone affinity,
and presence or absence of antiprenylation activity, for bone-related
imaging applications
In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels
Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases
In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels
Most known pathogenic point mutations in humans are C center dot G to T center dot A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.Base editors are effective and safe for cholesterol reduction in non-human primates.ISSN:1546-1696ISSN:1087-015
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Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies