Enhancing prime editor flexibility with coiled-coil heterodimers.

BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol. CONCLUSIONS: Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase.

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Generation of a genetically modified pig model with CREBRFR457Q variant.

Obesity is among the strongest risk factors for type 2 diabetes (T2D). The CREBRF missense allele rs373863828 (p. Arg457Gln, p. R457Q) is associated with increased body mass index but reduced risk of T2D in people of Pacific ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation R457Q into the porcine genome. The CREBRFR457Q pigs displayed dramatically increased fat deposition, which was mainly distributed in subcutaneous adipose tissue other than visceral adipose tissue. The CREBRFR457Q variant promoted preadipocyte differentiation. The increased differentiation capacity of precursor adipocytes conferred pigs the unique histological phenotype that adipocytes had a smaller size but a greater number in subcutaneous adipose tissue (SAT) of CREBRFR457Q variant pigs. In addition, in SAT of CREBRFR457Q pigs, the contents of the peroxidative metabolites 4-hydroxy-nonenal and malondialdehyde were significantly decreased, while the activity of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, was increased, which was in accordance with the declined level of the reactive oxygen species (ROS) in CREBRFR457Q pigs. Together, these data supported a causal role of the CREBRFR457Q variant in the pathogenesis of obesity, partly via adipocyte hyperplasia, and further suggested that reduced oxidative stress in adipose tissue may mediate the relative metabolic protection afforded by this variant despite the related obesity.

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy.

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Zika Virus Envelope Protein induces G2/M Cell Cycle Arrest and Apoptosis via an Intrinsic Cell Death Signaling Pathway in Neuroendocrine PC12 Cells.

Clinical evidence suggests that there exists a strong correlation between Zika virus (ZIKV) infection and abnormal development of the nervous system. However, the underlying mechanisms remain to be elusive. In this study, recombinant lentiviral vectors coding for ZIKV structural proteins and truncations (prM-Env, M-Env and Env) were transduced into PC12 cells. Envelope (Env) overexpression induced significant inhibition of proliferation and triggered G2/M cell cycle arrest and apoptosis in PC12 cells. Flow cytometry and western blot analysis showed that the apoptosis was associated with up-regulation of both p53 and p21Cip1/Waf1 and down-regulation of cyclin B1. Presence of aberrant nuclei clusters were confirmed by immunofluorescence staining analysis. The data indicate that overexpression of prM-Env, M-Env or Env led to apoptosis via an intrinsic cell death signaling pathway that is dependent on the activation of caspase-9 and caspase-3 and accompanied by an increased ratio of Bax to Bcl-2 in transduced PC12 cells. In summary, our results suggest that ZIKV Env protein causes apoptosis in PC12 cells via an intrinsic cell death signaling pathway, which may contribute to ZIKV-induced abnormal development of the nervous system.

Construction and Identification of Recombinant HEK293T Cell Lines Expressing Non-structural Protein 1 of Zika Virus.

Background: Zika virus (ZIKV) infection has become a major public health problem all around the world. Early diagnosis of Zika infection is important for better management of the disease. Non-structural protein 1 (NS1) is a potential biomarker for ZIKV infections. The purpose of this study was to produce the ZIKV NS1 protein for establishing serological diagnostic methods for ZIKV. Methods: The cDNA fragment encoding a chimeric protein composed of murine Igκ signal peptide, NS1 and histidine tag was synthesized and cloned into the lentiviral expression vector pLV-eGFP. The resulting expression vector pLV-eGFP-ZIKV-NS1 was packaged and transduced into human embryonic kidney (HEK) 293T cells and clonal cell lines with NS1 gene were generated from the tranduced cells by limiting dilution. Over expressed recombination NS1 (rNS1) fusion protein was purified by nickel affinity chromatography. Mice immunization and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate the immunogenicity of rNS1. Results: Western blot analysis revealed that the reconstituted cells stably expressed and secreted high levels of approximately 45-kDa NS1, and no significant changes were observed in green fluorescent protein (GFP) fluorescence ratio and fluorescence intensity. The scanned gels showed that the purity of the purified rNS1 was 99.42%. BALB/c mice were then immunized with purified rNS1 and a high level of antibodies against NS1 was elicited in the mice. Conclusion: Overall, recombinant NS1 proteins were successfully purified and their antigenicity was assessed. Immunization of mice with recombinant proteins demonstrated the immunogenicity of the NS1 protein. Thus, the generated recombinant NS1 can be potentially used in the development of serological diagnostic methods for ZIKV.

Molecular detection of rifabutin-susceptible Mycobacterium tuberculosis.

Rapid assays are still needed to detect rifabutin (RFB) susceptibility for proper tuberculosis treatment. To assess the use of the GenoType MTBDRplus assay and subsequent rpoB gene sequencing on detection of RFB susceptibility, we analyzed 800 multidrug-resistant Mycobacterium tuberculosis isolates, and 13% (104/800) were RFB susceptible. Of the 104 RFB-susceptible isolates, 71 (68.3%) isolates were rapidly identified using two molecular assays, while the remaining isolates could be determined using conventional drug-susceptibility testing according to the clinician's decision.

Evaluation of the rapid MGIT TBc identification test for culture confirmation of Mycobacterium tuberculosis complex strain detection.

A culture confirmation test for the detection of Mycobacterium tuberculosis complex strains that uses a lateral-flow immunochromatographic assay to detect the MPB64 antigen, the MGIT TBc identification (TBc ID) test, has been developed. We evaluated the performance of the TBc ID test in the detection of the M. tuberculosis complex in 222 primary-positive liquid cultures. We compared these results to those of nucleic acid-based identification and conventional biochemical tests. The validity of the TBc ID test was determined, and all of the nontuberculous mycobacteria (NTM) and Nocardia species tested were found to be negative. The detection limit of the TBc ID test was 5 × 10(5) CFU/ml, and for IS6110 real-time PCR it was 5 CFU/ml. All of the M. tuberculosis and M. africanum cultures were found to be positive, while M. bovis and M. bovis BCG cultures were negative. With the exception of 1 contaminated culture, the 221 culture-positive isolates contained 171 (77.5%) M. tuberculosis isolates, 39 (17.6%) NTM species, and 11 (5.0%) unidentified species. Two culture-positive isolates harbored a 63-bp deletion at position 196 of the mpb64 gene. The sensitivity, specificity, positive predictive values, and negative predictive values of the TBc ID test were 98.8, 100, 100, and 95.1%, respectively. Furthermore, the approximate turnaround time for real-time PCR was 4 h (including buffer and sample preparation), while for the TBc ID test it was less than 1 h. We suggest an algorithm for the primary identification of M. tuberculosis in liquid culture using the TBc ID test as an alternative to conventional subculture followed by identification using biochemical methods.