A generalized Poisson and Poisson-Boltzmann solver for electrostatic environments

The computational study of chemical reactions in complex, wet environments is critical for applications in many fields. It is often essential to study chemical reactions in the presence of applied electrochemical potentials, taking into account the non-trivial electrostatic screening coming from the solvent and the electrolytes. As a consequence the electrostatic potential has to be found by solving the generalized Poisson and the Poisson-Boltzmann equation for neutral and ionic solutions, respectively. In the present work solvers for both problems have been developed. A preconditioned conjugate gradient method has been implemented to the generalized Poisson equation and the linear regime of the Poisson-Boltzmann, allowing to solve iteratively the minimization problem with some ten iterations of a ordinary Poisson equation solver. In addition, a self-consistent procedure enables us to solve the non-linear Poisson-Boltzmann problem. Both solvers exhibit very high accuracy and parallel efficiency, and allow for the treatment of different boundary conditions, as for example surface systems. The solver has been integrated into the BigDFT and Quantum-ESPRESSO electronic-structure packages and will be released as an independent program, suitable for integration in other codes

Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.Mark B. Nottle, Evelyn J. Salvaris, Nella Fisicaro, Stephen McIlfatrick, Ivan Vassiliev, Wayne J. Hawthorne, Philip J. O’Connell, Jamie L. Brady, Andrew M. Lew and Peter J. Cowa

The impact of currently licensed therapies on viral and immune responses in Chronic Hepatitis B: considerations for future novel therapeutics.

Despite the availability of a preventative vaccine, chronic hepatitis B (CHB) remains a global healthcare challenge with the risk of disease progression due to cirrhosis and hepatocellular carcinoma. Although current treatment strategies, interferon and nucleos(t)ide analogues have contributed to reducing morbidity and mortality related to CHB, these therapies are limited in providing functional cure. The treatment paradigm in CHB is rapidly evolving with a number of new agents in the developmental pipeline. However, until novel agents with functional cure capability are available in the clinical setting, there is a pressing need to optimize currently licensed therapies. Here, we discuss current agents used alone and/or in combination strategies along with the impact of these therapies on viral and immune responses. Novel treatment strategies are outlined, and the potential role of current therapies in the employment of pipeline agents is discussedWellcome Trust Clinical Research Training Fellowship (107389/Z/15/Z)NIHR Academic Clinical LectureshipBarts Charity Project Grants (723/1795 and MGU/0406NIHR Research for patient benefit award (PB‐PG‐0614‐34087) to PTF

Characterization of transgenic pigs expressing an anti-Cd2 monoclonal antibody. A continuing quest for the ideal islet donor

Background: We have restored long-term blood glucose control in diabetic immunosuppressed baboons by transplantation of GalT KO- human CD55-human CD59 neonatal porcine islets. Following with- drawal of immunosuppression, T cell infiltration, islet cell destruction, and loss of glucose control were observed. To combat this, we gener- ated transgenic pigs expressing diliximab, a chimeric anti-CD2 mono- clonal antibody that depletes and inhibits human and baboon T cells. CRISPR was used to integrate a single copy of the transgene into exon 10 of the GGTA1 gene, thus concurrently eliminating expression of the major xenoantigen αGal. Transgene expression was under the control of either (1) a mouse M HC class I (MHC-I) promoter for widespread expressionor (2) the pig insulin promoter (PIP) for islet-specific expres- sion. We hypothesize that local expression of diliximab will protect the islet xenograft from invading T cells, eliminating the need for contin- uous systemic immunosuppression and its associated risks and side effects.Aim: To evaluate the pattern and level of expression of diliximab in the transgenic pigs. Methods: Serum expression was detected by FACS of human T cells incubated with pig serum followed by anti-human IgG. Transgene mRNA expression was assessed by quantitative RT-PCR using TaqMan. Diliximab expression in spleen, kidney, and pancreas was assessed by immunohistochemistry and immunofluorescence. Results: MHC-I promoter transgenic pigs expressed diliximab in serum and tissues. Tissue expression was the strongest in a subset of spleen cells, with weaker expression throughout the kidney, and patchy pan- creatic staining, with minimal expression in islets as shown by IHC and IF. Transgene mRNA expression was detected in spleen and kidney. However, diliximab was undetectable in the PIP transgenic pigs Discussion/Conclusion: Our preliminary analysis suggests that the basal level of diliximab expression by native islets in the MHC-I promoter transgenic pigs is marginal at best. However, the MHC-I promoter is known to be induced by proinflammatory cytokines. Therefore, it is possible that transgenic islet xenografts will upregulate diliximab expression in response to cytokines produced by activated T cells recruited to the transplant site after withdrawal of immunosup pression, protecting them from rejection. To further investigate this possibility, we plan to isolate islets from the MHC-I promoter trans genic pigs and examine diliximab secretion following (1) in vitro culture in the presence and absence of proinflammatory cytokines, and (2) transplantation into diabetic immunodeficient mice. We will continue analysis of the PIP transgenic pigs as the lack of expression from the pig insulin promoter was surprising, given that this promoter had been reported to drive efficient islet-specific expression of another secreted immunomodulatory molecule (LEA29Y) in pigsEvelyn Salvaris, Adwin Thomas, Nicole Byrne, Nella Fisicaro, Mark B. Nottle, Wayne J. Hawthorne, Peter J. Cowa

FokI-dCas9 mediates high-fidelity genome editing in pigs

Friday, October 11, 2019 Oral Abstracts Session 200 on PERV. Abstract #326.5Nella Fisicaro, Evelyn J. Salvaris, Gayle K. Philip, Matthew J. Wakefield, Mark B. Nottle, Wayne J. Hawthorne, Peter Cowa

Synthesis and properties of cationic surfactants with tuned hydrophylicity

A series of pyridinium-based cationic surfactants has been synthesised and their amphiphilic properties have been studied by conductivity and surface tension measurements. The modification of the substitution pattern on the pyridinium ring by hydrophobic moieties (methyl vs. hydrogen and presence or not of condensed benzene ring) gave the opportunity to investigate structure–activity relationships. Characterization by conductivity and surface tension measurements shed light on the behaviour at the air/water interface and in the micellar environment. In particular, the tendency to form ion pairs at very low concentration was evidenced for all the surfactants substituted on the ring, but not for the simple pyridinium ones. The formation of ion pairs affects both the conductivity and the surface tension plots, showing that a series of steps is involved during the adsorption to the air/water surface. An attempt was made to qualify the single steps in the adsorption at the surface layer. Those steps were attributed to different chemical species (free surfactant ions or ion pairs) and to different arrangements of the surfactant. This work also represents a contribution of investigation at very low surfactant concentrations and high surface tension values

FokI-dCas9 mediates high-fidelity genome editing in pigs

Brief communicationGene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.Nella Fisicaro, Evelyn J. Salvaris, Gayle K. Philip, Matthew J. Wakefield, Mark B. Nottle, Wayne J. Hawthorne, Peter J. Cowa