Application of a split-Cre system for high-capacity adenoviral vector amplification

Background and aims: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. Methods and results: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. Conclusions: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production

Application of a split-Cre system for high-capacity adenoviral vector amplification

Background and aims: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. Methods and results: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. Conclusions: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production

Working Algorithms and Detection Methods of Autoantibodies in Autoimmune Liver Disease: A Nationwide Study

Autoantibody detection is the cornerstone of autoimmune liver diseases (AILD) diagnosis. Standardisation of working algorithms among autoimmunity laboratories, as well as being aware of the sensitivity and specificity of various commercial techniques in daily practice, are still necessary. The aim of this nationwide study is to report the results of the 2020 Autoimmunity Workshop organised by the Autoimmunity Group of the Spanish Society of Immunology and to provide useful information to clinicians and laboratory specialists to improve the management of autoantibody detection in AILD diagnoses. Serum samples from 17 patients with liver diseases were provided by the organisers of the 2020 Autoimmunity Workshop and were subsequently analysed by the 40 participating laboratories. Each laboratory used different techniques for the detection of autoantibodies in each patients’ serum sample, according to their working algorithm. Thus, almost 680 total complete patient reports were obtained, and the number of results from different autoantibody detection techniques was >3000. Up to eight different working algorithms were employed, including indirect immunofluorescence assays (IFA) and antigen-specific techniques (AgST). The IFA of HEp-2 cells was more sensitive than IFA of rat triple tissue for the study of anti-nuclear autoantibodies (ANA) associated with AILD. The IFA of a human neutrophil study for the analysis of anti-neutrophil cytoplasmic autoantibodies was not carried out systemically in all patients, or by all laboratories. AgSTs were the most sensitive methods for the detection of anti-smooth muscle/F-actin, soluble liver antigen, liver cytosol-1, M2-mitochondrial autoantibodies, and ANA associated with primary biliary cholangitis. The main differences in AMA detection were due to patients with autoantibodies against the non-dominant epitope of pyruvate dehydrogenase complex. Given that they are complementary, IFA and AgST should be performed in parallel. If there is high suspicion of AILD, AgST should always be performed

Characterization of novel pathogenic variants leading to caspase-8 cleavage-resistant RIPK1-induced autoinflammatory syndrome

Pathogenic RIPK1 variants have been described as the cause of two different inborn errors of immunity. Biallelic loss-of-function variants cause the recessively inherited RIPK1 deficiency, while monoallelic variants impairing the caspase-8-mediated RIPK1 cleavage provoke a novel autoinflammatory disease (AID) called cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome. The aim of this study was to characterize the pathogenicity of two novel RIPK1 variants located at the cleavage site of caspase-8 detected in patients with dominantly-inherited, early-onset undefined AID. RIPK1 genotyping was performed by Sanger and next-generation sequencing. Clinical and analytical data were collected from medical charts, and in silico and in vitro assays were performed to evaluate the functional consequences. Genetic analyses identified two novel heterozygous RIPK1 variants at the caspase-8 cleavage site (p.Leu321Arg and p.Asp324Gly), which displayed a perfect intrafamilial phenotype-genotype segregation following a dominant inheritance pattern. Structural analyses suggested that these variants disrupt the normal RIPK1 structure, probably making it less accessible to and/or less cleavable by caspase-8. In vitro experiments confirmed that the p.Leu321Arg and p.Asp324Gly RIPK1 variants were resistant to caspase-8-mediated cleavage and induced a constitutive activation of necroptotic pathway in a similar manner that previously characterized RIPK1 variants causing CRIA syndrome. All these results strongly supported the pathogenicity of the two novel RIPK1 variants and the diagnosis of CRIA syndrome in all enrolled patients. Moreover, the evidences here collected expand the phenotypic and genetic diversity of this recently described AID, and provide interesting data about effectiveness of treatments that may benefit future patients.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been partially funded by the following: Grants from the Spanish Ministry of Science, Innovation and Universities and co-funded by the European Regional Development Fund / Agencia Estatal de Investigación: RTI2018-096824-B-C21 (JIA); RTI2018-096824-B-C22 (FC); SAF2017-88276-R (PP). Grant from the Spanish Ministry of Science and Innovation / Agencia Estatal de Investigación (10.13039/501100011033): PID2020-116709RB-I00 (PP). Grant from Instituto de Salud Carlos III and co-funded by the European Union: PI19/01567 (AM-V). Grants from Fundación Séneca: 20859/PI/18 (PP); 21081/PDC/19 (PP); 0003/COVI/20 (PP). Grants from European Research Council: ERC-2013-CoG 614578 (PP); ERC-2019-PoC 899636 (PP