Recent advances in lipid nanoparticles for delivery of nucleic acid,mRNA, and gene editing-based therapeutics

Lipid nanoparticles (LNPs) are becoming popular as a means of delivering therapeutics, including those based on nucleic acids and mRNA. The mRNA-based coronavirus disease 2019 vaccines are perfect examples to highlight the role played by drug delivery systems in advancing human health. The fundamentals of LNPs for the delivery of nucleic acid- and mRNA-based therapeutics, are well established. Thus, future research on LNPs will focus on addressing the following: expanding the scope of drug delivery to different constituents of the human body, expanding the number of diseases that can be targeted, and studying the change in the pharmacokinetics of LNPs under physiological and pathological conditions. This review article provides an overview of recent advances aimed at expanding the application of LNPs, focusing on the pharmacokinetics and advantages of LNPs. In addition, analytical techniques, library construction and screening, rational design, active targeting, and applicability to gene editing therapy have also been discussed.     

Experimental determination of the recombination correction factor kS for SNC 125c,SNC 350p and SNC 600c ionization chambers in pulsed photon beams

Accurate ionization chamber measurements of the absorbed dose to water require the correction of incomplete collection of charges created within the chamber volume. According to current dosimetry protocols such as the TRS-398 or the DIN 6800-2, incomplete charge collection is accounted for by the correction factor k_s, which can be determined numerically or experimentally. The method proposed by Burns & McEwen (Phys. Med. Biol., 1998) was used in this study to determine the coefficients γ and δ used for the calculation of the correction factor k_s of three ionization chambers, the SNC 125c, the SNC 600c and the SNC 350p (all Sun Nuclear Corp., Melbourne, Florida) for an absorbed dose to water range of 0.2 mGy to 1.6 mGy per pulse in pulsed photon beams. The shift of the effective point of measurement from the reference point Δz and the correction factor k_r were determined for the SNC 350p according to the draft DIN 6800-2:2019-07.     

重组人表皮生长因子应用于肛裂手术的临床研究

目的：研究外源性表皮生长因子对于肛裂手术后的手术创面愈合情况的影响。方法：肛裂手术后，治疗组患者每日用重组人表皮生长因子敷于肛管创面，对照组患者采用常规的方式换药。比较两组患者术后伤口疼痛、出血情况，以及伤口肉芽生长愈合情况。结果：治疗组在减轻术后伤口疼痛、伤口水肿和出血等方面均显著优于对照组，其平均住院时间和伤口愈合时间也显著短于对照组。结论：外源性表皮生长因子有助于肛裂术后创面的快速修复，减轻患者的痛苦。     

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss,<Emphasis Type="Italic"> Physcomitrella patens</Emphasis>

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1,000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.Communicated by U. Kück     

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.     

Gene conversion,unequal crossing-over and mispairing at a non-tandem duplication during meiosis of Saccharomyces cerevisiae

Summary We have developed a novel system to examine conversion, exchange and mispairing involving a nontandem duplication of the ade8 locus in yeast by monitoring the segregation of heterozygous markers between the duplicated sequence. Plasmid Yrp 17 carries the yeast selectable markers URA3 ⁺ and TRP1 ⁺. Yrpl7 derivatives with a 4 kb insert carrying ade8-18 were used to clone the mutations trpl-1 and ura3-1 by gap repair. Integrants of the resulting plasmids at the Ade8 locus were crossed to yield diploid hybrids with a non-tandem duplication of Ade8 and heterozygosity for the plasmid markers between the duplicated sequences. 1192 complete, unselected asci were analyzed and 270 exhibiting recombination of the markers contributed by the plasmid were analyzed by Southern transfers to detect changes in plasmid sequences. Twenty-seven tetrads had unequal homologous exchanges and five had unequal sister-chromatid exchanges. Seven tetrads carry an additional copy of the integrated plasmid and ten are missing one. We propose that these two classes represent conversions of the entire 11 kb plasmid, which occur after misalignment and formation of an unpaired loop. Mispairing is a frequent event, and occurs in approximately fifty percent of all meioses. The system described provides a means to determine the meiotic rules of conversion, exchange and pairing for duplicated DNA sequences.     

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones

Summary Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.     

Homologous recombination in the fission yeast Schizosaccharomyces pombe: different requirements for the rhp51+, rhp54+ and rad22+ genes

The Schizosaccharomyces pombe rhp51 ⁺ , rad22 ⁺ and rhp54 ⁺ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Δ and rhp54Δ strains are extremely sensitive to ionizing radiation; the rad22Δ mutant turned out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Δ and rhp54Δ are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Δ mutant is only reduced by a factor of two. Blot-analysis demonstrated that the majority of the leu⁺ transformants of the wild-type and rad22Δ strains have integrated one or more copies of the vector. Gene conversion events were observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Δ and a rhp54Δ background. Meiotic recombination is hardly affected in the rad22Δ mutant. The rhp51Δ and rhp54Δ strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Δ strain and 27% in the rhp54Δ strain, as compared with wild-type cells. However, in the rhp51Δ mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe. Received: 20 July 1996     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

RAD58 (XRS4)—A new gene in the RAD52 epistasis group

The RAD58 (XRS4) gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that RAD58 also encodes an essential meiotic function. The spore inviability of rad58 strains is not rescued by a spo13 mutation. The rad50 mutation suppresses spore inviability of a spo13 rad58 strain suggesting that RAD58 acts after RAD50 in meiotic recombination. The rad58-4 mutation does not prevent mitotic recombination events. Haploid rad58 cells fail to carry out G₂-repair of gamma-induced lesions, whereas rad58/rad58 diploids are able to perform some diploid-specific repair of these lesions.