Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10-30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.

Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation.

The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.

Temperature Sensitive Mutations in Influenza A Viral Ribonucleoprotein Complex Responsible for the Attenuation of the Live Attenuated Influenza Vaccine.

Live attenuated influenza vaccines (LAIV) have prevented morbidity and mortality associated with influenza viral infections for many years and represent the best therapeutic option to protect against influenza viral infections in humans. However, the development of LAIV has traditionally relied on empirical methods, such as the adaptation of viruses to replicate at low temperatures. These approaches require an extensive investment of time and resources before identifying potential vaccine candidates that can be safely implemented as LAIV to protect humans. In addition, the mechanism of attenuation of these vaccines is poorly understood in some cases. Importantly, LAIV are more efficacious than inactivated vaccines because their ability to mount efficient innate and adaptive humoral and cellular immune responses. Therefore, the design of potential LAIV based on known properties of viral proteins appears to be a highly appropriate option for the treatment of influenza viral infections. For that, the viral RNA synthesis machinery has been a research focus to identify key amino acid substitutions that can lead to viral attenuation and their use in safe, immunogenic, and protective LAIV. In this review, we discuss the potential to manipulate the influenza viral RNA-dependent RNA polymerase (RdRp) complex to generate attenuated forms of the virus that can be used as LAIV for the treatment of influenza viral infections, one of the current and most effective prophylactic options for the control of influenza in humans.

Immune response against the coiled coil domain of Sjögren's syndrome associated autoantigen Ro52 induces salivary gland dysfunction.

OBJECTIVES: The structural domains of Ro52, termed the RING, B-box, coiled coil (CC) and B30.2/SPRY are targets of anti-Ro52 in multiple autoimmune disorders. In Sjögren's syndrome patients, the presence of anti-Ro52 is associated with higher disease severity, and in mice, they induce salivary gland hypofunction. This study was undertaken to investigate whether immune responses against different domains of Ro52, influences salivary gland disease in mice. METHODS: Female NZM2758 mice were immunised with Ro52 domains expressed as recombinant fusion proteins with maltose binding protein (MBP) [MBP-RING-B-box, MBP-CC, MBP-CC(ΔC19), MBP-B30.2/SPRY]. Sera from immunised mice were studied for IgG antibodies to Ro52 by immunoprecipitation, and to salivary gland cells by immunofluorescence. Pilocarpine-induced saliva production was measured to evaluate salivary gland function. Submandibular glands were investigated by histopathology for inflammation and by immune-histochemistry for IgG deposition. RESULTS: Mice immunised with different Ro52-domains had comparable reactivity to Ro52 and to salivary gland cells. However, only mice immunised with the CC domain and its C-terminal truncated version CC(ΔC19) showed a significant drop in saliva production. None of the mice developed severe salivary gland inflammation. The salivary gland hypofunction significantly correlated with increased intra-lobar IgG deposits in the submandibular salivary glands. CONCLUSIONS: Our data demonstrate that epitope specificity of anti-Ro52 antibodies plays a critical role in the induction of glandular dysfunction. Clearly, screening Sjögren's syndrome patients for relative levels of Ro52 domain specific antibodies will be more informative for associating anti-Ro52 with clinical measures of the disorder.

Microinjection of Antisense Morpholinos, CRISPR/Cas9 RNP, and RNA/DNA into Zebrafish Embryos.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

A thermostable Cas9 with increased lifetime in human plasma.

CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

Surveying the Delivery Methods of CRISPR/Cas9 for ex vivo Mammalian Cell Engineering.

The simplicity of the CRISPR/Cas9 technology has been transformative in making targeted genome editing accessible for laboratories around the world. However, due to the sheer volume of literature generated in the past five years, determining the best format and delivery method of CRISPR/Cas9 components can be challenging. Here, we provide a brief overview of the progress that has been made in the ex vivo genome editing of mammalian cells and summarize the key advances made for improving efficiency and delivery of CRISPR/Cas9 in DNA, RNA, and protein form. In particular, we highlight the delivery of Cas9 components to human cells for advanced genome editing applications such as large gene insertion.

Síndrome de lupus neonatal inducido por anticuerpos antirribonucleoproteína / Neonatal lupus syndrome induced by anti-ribonucleoprotein antibodies

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High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.

Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.

Induction of anti-Ro60/anti-La by immunisation with spectrin and induction of anti-spectrin by immunisation with Ro60 and 4-hydroxy-2-nonenal-modified Ro60 immunisation.

OBJECTIVES: The Ro ribonucleoprotein particle, targeted in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS), includes Ro60 (SSA) and La (SSA) autoantigens. Anti-Ro60 occurs in SLE and SS. The importance of α-fodrin and spectrin as well as anti-Ro and anti-fodrin/spectrin antibodies in SS and SLE, led us to hypothesise that rabbit immunisation with Ro60 or 4-hydroxy-2-nonenal-modified Ro60 would induce anti-spectrin. In addition, we hypothesised that antibodies to Ro60 and La will develop in animals immunised with spectrin. METHODS: Two NZW rabbits each were immunised with 4-hydroxy-2-nonenal-modified Ro60 or unmodified Ro60. Methods used included ELISA, including an inside-out RBC membrane ELISA, and Crithidia lucilae assays. RESULTS: Commercial anti-spectrin sera bound significantly to Ro60 (OD 2.6 ± 0.1), Ro60 multiple antigenic peptides (MAPs) (3 out of 21 Ro60 MAPs), La (OD 4.4±0.5), and La fragments as well as to double stranded DNA but not to BSA (OD 0.6±0.1). Anti-spectrin binding to purified spectrin could be inhibited by spectrin (>95%), and Ro60 or La (70%). When the binding of anti-spectrin was tested against a nested set of La fragments we found that a N4 fragment representing the C-terminal 250 aa (aa 159 to 408) bound the strongest (OD=4.12) followed by a N9 fragment (the C-terminal 36aa; aa373 to 408 (OD=1.36). Also, significant anti-spectrin antibody levels were induced by Ro60 and HNE-modified Ro60 immunisation. CONCLUSIONS: We found intermolecular epitope spreading from Ro60/La to spectrin and vice versa, and this may have pathological significance in these animal models of autoimmunity.

Maternal MHC regulates generation of pathogenic antibodies and fetal MHC-encoded genes determine susceptibility in congenital heart block.

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1(av1) (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1(l)). Different anti-Ro52 Ab fine specificities were generated in RT1(av1) versus RT1(l) animals. Maternal and fetal influence was determined in an F(2) cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1(l) than RT1(av1) pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1(l) pups more frequently developed heart block than RT1(av1) pups after passive transfer of RT1(av1) anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52-positive pregnancies.

Neo-epitopes are required for immunogenicity of the La/SS-B nuclear antigen in the context of late apoptotic cells.

Mechanisms responsible for the induction of anti-nuclear autoantibodies (ANA) following exposure of the immune system to an excess of apoptotic cells are incompletely understood. In this study, the immunogenicity of late apoptotic cells expressing heterologous or syngeneic forms of La/SS-B was investigated following subcutaneous administration to A/J mice, a non-autoimmune strain in which the La antigenic system is well understood. Immunization of A/J mice with late apoptotic thymocytes taken from mice transgenic (Tg) for the human La (hLa) nuclear antigen resulted in the production of IgG ANA specific for human and mouse forms of La in the absence of foreign adjuvants. Preparations of phenotypically healthy cells expressing heterologous hLa were also immunogenic. However, hLa Tg late apoptotic cells accelerated and enhanced the apparent heterologous healthy cell-induced anti-La humoral response, while non-Tg late apoptotic cells did not. Subcutaneous administration of late apoptotic cells was insufficient to break existing tolerance to the hLa antigen in hLa Tg mice or to the endogenous mouse La (mLa) antigen in A/J mice immunized with syngeneic thymocytes, indicating a requirement for the presence of heterologous epitopes for anti-La ANA production. Lymph node dendritic cells (DC) but not B cells isolated from non-Tg mice injected with hLa Tg late apoptotic cells presented immunodominant T helper cell epitopes of hLa. These studies support a model in which the generation of neo-T cell epitopes is required for loss of tolerance to nuclear proteins after exposure of the healthy immune system to an excess of cells in late stages of apoptosis.

Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren's syndrome.

Sjögren's syndrome is a poorly understood autoimmune inflammatory illness that affects the salivary and lacrimal glands as well as other organ systems. We undertook the present study to determine whether mice immunized with short peptides from the 60-kDa Ro (or SSA) Ag, which is a common target of the autoimmunity of Sjögren's syndrome, develop an illness similar to Sjögren's syndrome. BALB/c mice were immunized with one of two short peptides from 60-kDa Ro that are know to induce epitope spreading. The animals were analyzed for the presence of anti-Ro and anti-La (or SSB) in the sera by immunoblot and ELISA. Salivary glands were collected and examined by histology after H&E staining. Salivary lymphocytes were purified and studied for cell surface makers by fluorescence-activated cell sorting. Timed stimulated salivary flow was measured. As reported previously, BALB/c mice immunized with 60-kDa Ro peptides developed an immune response directed against the entire Ro/La ribonucleoprotein particle that was similar to that found in humans with lupus or Sjögren's syndrome. Functional studies showed a statistical decrease in salivary flow in immunized mice compared with controls. Furthermore, there were lymphocytic infiltrates in the salivary glands of immunized animals that were not present in controls. The infiltrates consisted of both CD4- and CD8+ T lymphocytes as well as B lymphocytes. BALB/c mice immunized with 60-kDa Ro peptides develop anti-Ro, salivary gland lymphocyte infiltrates, and salivary dysfunction that is highly reminiscent of human Sjögren's syndrome.

Inhibitors of respiratory syncytial virus replication target cotranscriptional mRNA guanylylation by viral RNA-dependent RNA polymerase.

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5' and 3' ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5' cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

Induction of oral tolerance in experimental Sjogren's syndrome autoimmunity.

Previous studies have showed that immunization with peptides from Ro 60 results in Sjogren's syndrome (SS)-like condition in BALB/c mice. We hypothesized that oral feeding with Ro 60 peptide or Ro 60 would prevent the disease. Four groups (each consisting of 10) of BALB/c mice were used. Group I-III were immunized with Ro 274 peptide. Group IV mice were administered adjuvant only. Group II mice were fed orally with Ro 274 peptide and Group III with Ro 60 for 5 days before immunization. There was a significant reduction in the binding of sera from both Group II and Group III mice to most of the Ro multiple antigenic peptides bound by Group I mice. In Group III mice, salivary flow was maintained above that of the Group I mice (average: 117.5 versus 58.6 microl; t = 2.7; P = 0.02). Salivary infiltrates were drastically decreased in the Ro peptide or Ro 60-fed groups, compared to non-tolerized group. Two of eight mice in Group II and 3/6 mice in Group III had no infiltrates, whereas all eight mice studied in Group I had a significant number of infiltrates. Thus, epitope spreading was prevented, lymphocytic infiltration was blocked and saliva flow was restored by means of oral feeding of either Ro 274 or Ro 60 in this animal model of SS.

Immune responses to small nuclear ribonucleoproteins: antigen-dependent distinct B cell epitope spreading patterns in mice immunized with recombinant polypeptides of small nuclear ribonucleoproteins.

Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.

The angiogenesis inhibitor vasostatin does not impair wound healing at tumor-inhibiting doses.

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair.

Down-regulation of L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits.

Congenital heart block is considered a model of passively acquired autoimmune disease in which the mother generates anti-SSA/Ro and/or anti-SSB/La antibodies that cross the placenta and presumably injure the heart of developing fetus. CHB is accompanied by ECG abnormalities including AV block, sinus bradycardia, and ventricular dysfunction. Our previous data indicate that these abnormalities are caused by maternal autoantibody-mediated disturbance of L-type Ca channels. To investigate the consequence of chronic exposure of L-type Ca channels in newborn pups to maternal autoantibodies during pregnancy, we immunized female rabbits with human 52 kDa-SSA/Ro (Ro52) recombinant protein. ECG revealed that pups from the immunized group had varying degrees of conduction defects. In addition, I(CaL) density and protein were reduced in hearts of pups from the immunized group. Sera and purified IgG from immunized rabbits inhibited I(Ba) recorded from oocytes with expressed alpha(1C) and beta(2a) subunits of L-type Ca channel. Pups born to Ro52 immunized mothers exhibited down-regulation of L-type calcium channels in heart. The data provide new insight into the pathogenesis of congenital heart block.