[Correction of the pathogenic mutation in the G6PC3 gene by adenine base editing in mutant embryos].

Objective: To determine whether the adenine base editor (ABE7.10) can be used to fix harmful mutations in the human G6PC3 gene. Methods: To investigate the safety of base-edited embryos, off-target analysis by deep sequencing was used to examine the feasibility and editing efficiency of various sgRNA expression vectors. The human HEK293T mutation models and human embryos were also used to test the feasibility and editing efficiency of correction. Results: ①The G6PC3(C295T) mutant cell model was successfully created. ②In the G6PC3(C295T) mutant cell model, three distinct Re-sgRNAs were created and corrected, with base correction efficiency ranging from 8.79% to 19.56% . ③ ABE7.10 could successfully fix mutant bases in the human pathogenic embryo test; however, base editing events had also happened in other locations. ④ With the exception of one noncoding site, which had a high safety rate, deep sequencing analysis revealed that the detection of 32 probable off-target sites was <0.5% . Conclusion: This study proposes a new base correction strategy based on human pathogenic embryos; however, it also produces a certain nontarget site editing, which needs to be further analyzed on the PAM site or editor window.

Switch from protease inhibitor- to efavirenz-based antiretroviral therapy improves quality of life, treatment satisfaction and adherence with low rates of virological failure in virologically suppressed patients.

Regimen selection in antiretroviral therapy can impact treatment adherence, quality of life (QoL) and treatment satisfaction, and may influence clinical outcome. We evaluated the effect of regimen switching on virological, safety and patient-reported outcomes. In this 48-week, open-label, randomized, non-inferiority study, 262 HIV-1-infected adult patients with a viral load <50 copies/mL on protease inhibitor (PI)-based regimens were switched to either once-daily efavirenz, lamivudine and enteric-coated didanosine (efavirenz-A [QD]) or once-daily efavirenz plus continuation of current nucleoside reverse transcriptase inhibitors (efavirenz-B). In the primary outcome of patients who maintained virological suppression at week 48, efavirenz-A (QD) was non-inferior to efavirenz-B (81% versus 79%, respectively). Both regimens were associated with low virological failure rates and significant improvements in treatment satisfaction, adherence and QoL after switching from PI-based therapy, with no differences between regimens. Switching from a PI- to an efavirenz-based regimen was generally safe and well tolerated.

A novel N-acyl phosphatidylethanolamine-containing delivery vehicle for spermine-condensed plasmid DNA.

A unique method for formulation of plasmid DNA with phospholipids has been devised for the purpose of producing vehicles that can mediate gene delivery and transfection of living cells. The polycation, spermine, was used to condense plasmid DNA within a water-in-chloroform emulsion stabilized by phospholipids. After organic solvent removal, the particles formed could be extruded to a number average size of about 200 nm and retained DNA that was protected from nuclease digestion. This resulted in a relatively high protected DNA-to-lipid ratio of approximately 1 microg DNA/micromol lipid. The size distribution of the preparation was relatively homogeneous as judged by light microscopy and quasi-elastic light scattering. Electron microscopic studies showed structural heterogeneity, but suggested that at least some of the plasmid DNA in this preparation was in the form of the previously observed spermine-condensed bent rods and toroids and was encapsulated within liposomal membranes. Preparations with the fusogenic phospholipid composition, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoyl/ 1, 2-dioleoyl-sn-glycero-3-phosphocholine, showed transfection activity for several cells lines, particularly OVCAR-3 cells. The transfection activity sedimented with the lipid during centrifugation, confirming the association of active plasmid DNA with phospholipids. Transfection efficiency in culture was found to be of the same order of magnitude as cationic lipoplexes but much less toxic to the cells. Significant transfection of OVCAR-3 cells in tissue culture could also be observed, even in the presence of the intraperitoneal fluid from a mouse with an OVCAR-3 ascites tumor. These data indicate a new type of liposomal gene delivery system devoid of cationic lipids, phosphatidylethanolamine, cationic polymers and viral components.

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.

Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine.

N-acyl phosphatidylethanolamines (NAPEs) are natural lipid components of many organisms. N-acylation of unsaturated phosphatidylethanolamines with a saturated fatty acid converts them from non-lamellar organizing lipids into lamellar organizing, acidic lipids which can interact with cations and potentially return to non-lamellar structures. These special properties make NAPEs candidates for fusogens. We tested the fusogenicity of one of the NAPEs, N-dodecanoyl-di-oleoylphosphatidylethanolamine (N-C12-DOPE) mixed with dioleoylphosphatidylcholine (DOPC) in liposomes. Binding and fusion to erythrocyte ghosts in the presence of 3 mM Ca2+ required at least 60 mol% of N-C12-DOPE. Fusion was not observed when phosphatidylglycerol or phosphatidylserine was substituted for N-C12-DOPE, indicating specificity for properties of this lipid. Binding of N-C12-DOPE/DOPC (70:30) liposomes required 1 mM Ca2+ while 1.25 mM Ca2+ and Mg2+ were sufficient for lipid mixing and delivery of encapsulated dextrans to erythrocyte ghosts. These liposomes also bound and possibly mixed lipid with nucleated U-937 cells in a Ca2+ -and endocytosis-dependent manner. Low pH-dependent fusion with ghosts was observed in the absence of any divalent cation, indicating that fusion with U-937 cells could result after endocytosis into the acidic endosomes. The possible mechanisms for N-C12-DOPE mediated binding and fusion and the potential application of these liposomes as delivery vehicles for therapeutic agents are discussed.

Influenza-virus-liposome lipid mixing is leaky and largely insensitive to the material properties of the target membrane.

Monolayer intrinsic curvature, void stabilization, and membrane rupture tension have been suggested as important factors determining the rate of membrane fusion. Here, we have studied the kinetics of fusion between influenza virus and target liposomes as a function of various target membrane material properties. In order to examine the fusion process directly, a simple prebinding step is used and proven to be adequate to achieve fusion-rate-limiting kinetics. To test the hypothesis about membrane curvature and void stabilization, we studied the lipid mixing kinetics with dioleoylphosphatidylcholine (DOPC)/ganglioside GD1a (GD1a) liposomes containing lysooleoylphosphatidylcholine (LPC, positive curvature), dioleoyglycerol (DOG, negative curvature), arachidonic acid (AA, negative curvature), and hexadecane (HD, void stabilization). DOG, AA, and HD (at 4 mol%) showed no significant effect on the fusion kinetics, while LPC reversibly inhibited influenza HA mediated fusion only at very high concentrations. Using target liposomes with different membrane rupture tension values, no obvious correlation between membrane rupture tension and the rate of lipid mixing was observed. Moreover, a reported potential antiviral compound, tert-butylhydroquinone (t-b-HQ) (Bodian et al., 1993), showed no significant effect on the kinetics of influenza fusion. Finally leakage of liposome contents was detected during lipid mixing. For encapsulated molecules smaller than 450 MW, the kinetics of leakage is very similar to the kinetics of lipid mixing. In fact, leakage was also detected for encapsulated molecules up to 10 000 MW, suggesting that HA mediated lipid mixing is a very leaky process. Since "nonleaky fusion" has been the foundation of influenza fusion models, our work suggests the need for a major revision in the modeling of this process.