We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats ...(CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10–50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.
Over the past decade, the Sleeping Beauty (SB) transposon system has been developed as the leading non-viral vector for gene therapy. This vector combines the advantages of viruses and naked DNA. ...Here we review progress over the last 2 years in vector design, methods of delivery and safety that have supported its use in the clinic. Currently, the SB vector has been validated for ex vivo gene delivery to stem cells, including T-cells for the treatment of lymphoma. Progress in delivery of SB transposons to liver for treatment of various systemic diseases, such as hemophilia and mucopolysaccharidoses types I and VII, has encountered some problems, but even here progress is being made.
We present a detailed report on sterile neutrino oscillation and 235Uν¯e energy spectrum measurement results from the PROSPECT experiment at the highly enriched High Flux Isotope Reactor (HFIR) at ...Oak Ridge National Laboratory. In 96 calendar days of data taken at an average baseline distance of 7.9 m from the center of the 85 MW HFIR core, the PROSPECT detector has observed more than 50,000 interactions of νe produced in beta decays of 235U fission products. New limits on the oscillation of ν¯e to light sterile neutrinos have been set by comparing the detected energy spectra of ten reactor-detector baselines between 6.7 and 9.2 meters. Measured differences in energy spectra between baselines show no statistically significant indication of ν¯e to sterile neutrino oscillation and disfavor the reactor antineutrino anomaly best-fit point at the 2.5σ confidence level. The reported 235U ν¯e energy spectrum measurement shows excellent agreement with energy spectrum models generated via conversion of the measured 235U beta spectrum, with a χ2/d.o.f. of 31/31. PROSPECT is able to disfavor at 2.4σ confidence level the hypothesis that 235U ν¯e are solely responsible for spectrum discrepancies between model and data obtained at commercial reactor cores. A data-model deviation in PROSPECT similar to that observed by commercial core experiments is preferred with respect to no observed deviation, at a 2.2σ confidence level.
Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction ...limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disorder that causes syndromes characterized by physiological dysfunction in many organs and tissues. Despite the recognizable ...morphological and behavioral deficits associated with MPS I, neither the underlying alterations in functional neural connectivity nor its restoration following gene therapy have been shown. By employing high-resolution resting-state fMRI (rs-fMRI), we found significant reductions in functional neural connectivity in the limbic areas of the brain that play key roles in learning and memory in MPS I mice, and that adeno-associated virus (AAV)-mediated gene therapy can reestablish most brain connectivity. Using logistic regression in MPS I and treated animals, we identified functional networks with the most alterations. The rs-fMRI and statistical methods should be translatable into clinical evaluation of humans with neurological disorders.
This Letter reports the first measurement of the 235U $\bar{ν}$e energy spectrum by PROSPECT, the Precision Reactor Oscillation and Spectrum experiment, operating 7.9 m from the 85 MWth highly ...enriched uranium (HEU) High Flux Isotope Reactor. With a surface-based, segmented detector, PROSPECT has observed 31678±304(stat) $\bar{ν}$e-induced inverse beta decays, the largest sample from HEU fission to date, 99% of which are attributed to 235U. Despite broad agreement, comparison of the Huber 235U model to the measured spectrum produces a χ2/ndf=51.4/31, driven primarily by deviations in two localized energy regions. The measured 235U spectrum shape is consistent with a deviation relative to prediction equal in size to that observed at low-enriched uranium power reactors in the $\bar{ν}$e energy region of 5–7 MeV.
We previously developed a
Zr-labeled antibody-based immuno-positron emission tomography (immunoPET) tracer targeting interferon gamma (IFNγ), a cytokine produced predominantly by activated T and ...natural killer (NK) cells during pathogen clearance, anti-tumor immunity, and various inflammatory and autoimmune conditions. The current study investigated
ZrZr-DFO-anti-IFNγ PET as a method to monitor response to immune checkpoint inhibitors (ICIs).
BALB/c mice bearing CT26 colorectal tumors were treated with combined ICI (anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death 1 (PD-1)). The
ZrZr-DFO-anti-IFNγ PET tracer, generated with antibody clone AN18, was administered on the day of the second ICI treatment, with PET imaging 72 hours later. Tumor mRNA was analyzed by quantitative reverse-transcribed PCR (qRT-PCR).
We detected significantly higher intratumoral localization of
ZrZr-DFO-anti-IFNγ in ICI-treated mice compared to untreated controls, while uptake of an isotype control tracer remained similar between treated and untreated mice. Interestingly,
ZrZr-DFO-anti-IFNγ uptake was also elevated relative to the isotype control in untreated mice, suggesting that the IFNγ-specific tracer might be able to detect underlying immune activity
in this immunogenic model. In an efficacy experiment, a significant inverse correlation between tracer uptake and tumor burden was also observed. Because antibodies to cytokines often exhibit neutralizing effects which might alter cellular communication within the tumor microenvironment, we also evaluated the impact of AN18 on downstream IFNγ signaling and ICI outcomes. Tumor transcript analysis using interferon regulatory factor 1 (IRF1) expression as a readout of IFNγ signaling suggested there may be a marginal disruption of this pathway. However, compared to a 250 µg dose known to neutralize IFNγ, which diminished ICI efficacy, a tracer-equivalent 50 µg dose did not reduce ICI response rates.
These results support the use of IFNγ PET as a method to monitor immune activity
after ICI, which may also extend to additional T cell-activating immunotherapies.
Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge ...of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) P(MAG-b-MAEMt)-1, -2, -3. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.
Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing ...fluorescent fusion proteins of beta-Actin, alpha-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.
Members of the
Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to ...construct a synthetic transposon,
Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations.
Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells.
Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.
PDF
