Use of cell-penetrating-peptides in oligonucleotide splice switching therapy

The hydrophobic plasma membrane constitutes an indispensable barrier for cells, allowing influx of essential molecules while preventing access to other macromolecules. Although pivotal for the maintenance of cells, the inability to cross the plasma membrane is one of the major obstacles toward current drug development. Oligonucleotides (ONs) are a group of substances that display great therapeutic potential to interfere with gene expression. Several classes of ONs have emerged either based on double stranded RNAs, such as short interfering RNAs that are utilized to confer gene silencing, or single stranded ONs of various chemistries for antisense targeting of small regulatory micro RNAs or mRNAs. In particular the use of splice switching oligonucleotides (SSOs) to manipulate alternative splicing, by targeting pre-mRNA, has proven to be a highly promising therapeutic strategy to treat various genetic disorders, including Duchenne muscular dystrophy and spinal muscular atrophy. Despite being efficient compounds to alter splicing patterns, their hydrophilic macromolecular nature prohibits efficient cellular internalization.Various chemical drug delivery vehicles have been developed aiming at improving the bioavailability of nucleic acid-based drugs. In the context of SSOs, one group of peptidebased delivery vectors, i.e. cell-penetrating peptides (CPPs), display extremely high potency. CPPs have a remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This review provides insight into the application of CPPs and ONs in gene regulation with particular focus on CPP-assisted delivery of therapeutic SSOs.     

Developmental regulation of SR protein phosphorylation and activity

Serine/arginine-rich splicing factors (SR proteins) are substrates for serine phosphorylation that can regulate SR protein function. We have observed gross changes in SR protein phosphorylation during early development coincident with major zygotic gene activation in the nematode Ascaris lumbricoides. These differences correlate with large-scale changes in SR protein activity in promoting both trans- and cis-splicing. Importantly, inactive early stage extracts can be made splicing competent on addition of later stage SR proteins. These data suggest that changes in SR protein phosphorylation have a role in the activation of pre-mRNA splicing during early development.     

A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment

We previously described a novel homozygous point mutation (FGB c.115-600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50-bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre-mRNA splicing factor (SF2)/arginine/serine-rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bbeta chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re-established normal pre-mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein-RNA interaction and successfully restored normal splicing ( approximately 50% at 2 microM and approximately 90% at 10 microM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation.     

Surmounting limited gene delivery into primary immune cell populations: Efficient cell type‐specific adenoviral transduction by CAR

Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication‐defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: 1614–1620] report the generation of transgenic mice that enable conditional Cre/loxP‐mediated expression of human CAR. The authors demonstrate that this R26/CAG‐CAR?1^StopF mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge.     

Prp8蛋白在前体mRNA剪接中的作用

前体mRNA的剪接是基因表达的关键一步,发生在蛋白质的转录之后与合成之前。在前体mR- NA剪接加工过程中需要将转录本中的内含子切除,因为它会干扰基因的转录。前体mRNA的剪接发生在细胞核中,是在一个大的RNA与蛋白质的复合物即剪接体的催化下完成的。Prp8(precursor mRNA process- ing)是参与前体mRNA剪接的最大的蛋白,其序列从酵母到人类是高度保守的。Prp8同时也是细胞核内一个最重要的剪接因子。在剪接过程中,Prp8组成剪接体的催化中心。有人推断Prp8是剪接体的支架蛋白,很可能在催化中心起到锚定RNA的作用,同时也调节着激活剪接体所必需的构象变化。Prp8还与色素性视网膜炎的发生密切相关。     

Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism

Functional peptides that transfer biomaterials, such as semiconductor quantum dots (QDs), into cells in biomaterial research have been developed in recent years. Delivery of QDs conjugated with cell-penetrating peptides (CPPs) into cells by the endocytic pathway was problematic in biomedical applications because of lysosomal trapping. Here, we demonstrate that histidine- and arginine-rich CPPs (HR9 peptides) stably and noncovalently combined with QDs are able to enter into cells in an extremely short period (4 min). Interrupting both F-actin polymerization and active transport did not inhibit the entry of HR9/QD complexes into cells, indicating that HR9 penetrates cell membrane directly. Subcellular colocalization studies indicated that QDs delivered by HR9 stay in cytosol without any organelle capture. Dimethyl sulphoxide, ethanol and oleic acid, but not pyrenebutyrate, enhanced HR9-mediated intracellular delivery of QDs by promoting the direct membrane translocation pathway. HR9 and HR9/QDs were not cytotoxic. These findings suggest that HR9 could be an efficient carrier to deliver drugs without interfering with their therapeutic activity.     

Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum

Antisense morpholino oligonucleotides (AMOs) can reprogram pre-mRNA splicing by complementary binding to a target site and regulating splice site selection, thereby offering a potential therapeutic tool for genetic disorders. However, the application of this technology into a clinical scenario has been limited by the low correction efficiency in vivo and inability of AMOs to efficiently cross the blood brain barrier and target brain cells when applied to neurogenetic disorders such as ataxia-telangiecatasia (A-T). We previously used AMOs to correct subtypes of ATM splicing mutations in A-T cells; AMOs restored up to 20% of the ATM protein and corrected the A-T cellular phenotype. In this study, we demonstrate that an arginine-rich cell-penetrating peptide, (RXRRBR)(2)XB, dramatically improved ATM splicing correction efficiency when conjugated with AMOs, and almost fully corrected aberrant splicing. The restored ATM protein was close to normal levels in cells with homozygous splicing mutations, and a gene dose effect was observed in cells with heterozygous mutations. A significant amount of the ATM protein was still detected 21 days after a single 5 μm treatment. Systemic administration of an fluorescein isothiocyanate-labeled (RXRRBR)(2)XB-AMO in mice showed efficient uptake in the brain. Fluorescence was evident in Purkinje cells after a single intravenous injection of 60 mg/kg. Furthermore, multiple injections significantly increased uptake in all areas of the brain, notably in cerebellum and Purkinje cells, and showed no apparent signs of toxicity. Taken together, these results highlight the therapeutic potential of (RXRRBR)(2)XB-AMOs in A-T and other neurogenetic disorders.     

Characterization of primary cell cultures as potential target cells for analysis of bovine cytotoxic T lymphocytes

In domestic animal species, assessment of cell-mediated immune responses to virus infection is hampered by the requirement for class I MHC compatibility between target and effector cells. Additional complicating factors can include an inability to infect target cells in vitro, or virus-induced lysis of infected target cells. One way to circumvent these problems is to use virus-mediated gene transfer to deliver individual viral genes to autologous primary target cells. Several primary bovine cell cultures were assessed as potential target cells for cytotoxic T lymphocyte (CTL) assays by measuring their levels of class I MHC expression and susceptibilities to retroviral gene delivery. High levels in both class I MHC expression and susceptibility to gene delivery were seen in adherent cell cultures isolated from peripheral blood (PBAC). PBAC, which arose as an outgrowth of adherent peripheral blood mononuclear cell cultures, had morphology, protein expression patterns, and response to functional assays characteristic of high endothelial cells. Expression of viral vector-delivered genes in PBAC cells was confirmed with a recombinant retrovirus carrying the green fluorescent protein (GFP) gene. The use of vector-mediated delivery of viral genes to bovine high endothelial cells is a promising method for assessment of cell-mediated immunity in cattle.     

The Development of Animal Models of Inflammatory Bowel Disease

Alternative splicing is a fine-tuned process known for generating multiple functional variants from individual genes leading to protein diversity. The immune system utilizes pre-mRNA splicing to expand its gene function. Numerous immunologically relevant genes have been found to undergo alternative splicing, thus revealing a new source of complexity in the immune gene network. This review attempts to summarize the general features of alternative splicing and its role in the immune system along with a special focus on the available reports of alternative splicing in cytokines, mainly interleukins and their receptors and their regulatory significance.