Secretion of functionally active complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma Multiforme patients.

The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis.

Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors.

Ribozymes and siRNA for the treatment of diseases of the nervous system.

Recent advances in our understanding of RNA biology have focused attention on the potential of developing RNA-based strategies to treat human disease. Naturally occurring catalytic RNA molecules (ribozymes), their synthetic DNA counterparts (deoxyribozymes or DNAzymes), as well as the exciting, emerging technology of small interfering RNA which utilizes the highly conserved cellular RNA interference pathway, are being developed for therapeutic gene silencing purposes. The challenges for the application of this technology to neurological disease will be to identify appropriate disease targets, and to optimize the function, and particularly delivery of these RNA-based therapeutic molecules within the complex environment of the nervous system. This review will assess the potential of these RNA-based therapeutic strategies and the challenges ahead in their application to the treatment of neurological disease.

Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells.

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.

Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype.

Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration. Since no treatment exists for this chronic disease, novel therapies such post-transcriptional RNA interference-based gene silencing are under investigation, in particular those that might enable constitutive and tissue-specific silencing, such as expressed hairpins. Given that this method of silencing can be abolished by the presence of nucleotide mismatches against the target RNA, we sought to identify expressed RNA hairpins selective for silencing the mutant ataxin-7 transcript using a linked SNP. By targeting both short and full-length tagged ataxin-7 sequences, we show that mutation-specific selectivity can be obtained with single nucleotide mismatches to the wild-type RNA target incorporated 3' to the centre of the active strand of short hairpin RNAs. The activity of the most effective short hairpin RNA incorporating the nucleotide mismatch at position 16 was further studied in a heterozygous ataxin-7 disease model, demonstrating significantly reduced levels of toxic mutant ataxin-7 protein with decreased mutant protein aggregation and retention of normal wild-type protein in a non-aggregated diffuse cellular distribution. Allele-specific mutant ataxin7 silencing was also obtained with the use of primary microRNA mimics, the most highly effective construct also harbouring the single nucleotide mismatch at position 16, corroborating our earlier findings. Our data provide understanding of RNA interference guide strand anatomy optimised for the allele-specific silencing of a polyglutamine mutation linked SNP and give a basis for the use of allele-specific RNA interference as a viable therapeutic approach for spinocerebellar ataxia 7.

Hairpin DNAzymes: a new tool for efficient cellular gene silencing.

BACKGROUND: RNA-based gene silencing is potentially a powerful therapeutic strategy. Catalytic 10-23 DNAzymes bind to target RNA by complimentary sequence arms on a Watson-Crick basis and thus can be targeted to effectively cleave specific mRNA species. However, for in vivo applications it is necessary to stabilise DNAzymes against nucleolytic attack. Chemical modifications can be introduced into the binding arms to increase stability but these may alter catalytic activity and in some cases increase cell toxicity. METHODS: We designed novel 10-23 DNAzyme structures that incorporate stem-loop hairpins at either end on the DNAzyme binding arms. The catalytic activity of hairpin DNAzymes (hpDNAzyme) were tested in vitro against 32P-labelled cRNA encoding the muscle acetylcholine receptor (AChR) alpha-subunit. Resistance of hpDNAzymes to nucleolytic degradation was tested by incubation of the hpDNAzymes with Bal-31, DNase1 or HeLa cell extract. Gene silencing by hpDNAzymes was assessed by measuring reduced fluorescence from DsRed2 and EGFP reporters in cell culture systems, and reduced 125I-alpha-bungarotoxin binding in cells transfected with cDNA encoding the AChR. RESULTS: We show that hpDNAzymes show remarkable resistance to nucleolytic degradation, and demonstrate that in cell culture systems the hpDNAzymes are far more effective than standard 10-23 DNAzymes in down-regulating protein expression from target mRNA species. CONCLUSION: hpDNAzymes provide new molecular tools that, without chemical modification, give highly efficient gene silencing in cells, and may have potential therapeutic applications.

Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes.

Slow channel congenital myasthenic syndrome (SCCMS) is a dominant disorder caused by missense mutations in muscle acetylcholine receptors (AChR). Expression from mutant alleles causes prolonged AChR ion-channel activations. This 'gain of function' results in excitotoxic damage due to excess entry of calcium ions that manifests as an endplate myopathy. The biology of SCCMS provides a model system to investigate the potential of catalytic nucleic acids for therapy in dominantly inherited disorders involving single missense mutations. Hammerhead ribozymes can catalytically cleave RNA transcripts in a sequence-specific manner. We designed hammerhead ribozymes to target transcripts from four SCCMS mutations, alphaT254I, alphaS226F, alphaS269I and epsilonL221F. Ribozymes were incubated with cRNA transcripts encoding wild type and mutant AChR subunits. The ribozymes efficiently cleaved the mutant allele cRNA transcripts but left the wild type cRNA intact. Cleavage efficiency was optimised for alphaS226F. We were able to demonstrate robust catalytic activity under simulated physiological conditions and at high Ca(2+) concentrations, which is likely to be accumulated at the endplate region of the SCCMS patient muscles. These results demonstrate the potential for gene therapy applications of ribozymes to specifically down-regulate expression of mutant alleles in dominantly inherited disorders.

Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm.

Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences.

Design of efficient DNAzymes against muscle AChR alpha-subunit cRNA in vitro and in HEK 293 cells.

DNAzymes are catalytic DNA which bind to target RNA by complementary sequence arms on a Watson-Crick basis and cleave RNA at specific sites. Potential therapeutic applications require DNAzymes that can efficiently cleave their target. Here we investigate factors affecting DNAzyme cleavage efficacy against the muscle acetylcholine receptor (AChR) alpha-subunit. The 10-23 DNAzymes cleave at Y-R nucleotide motifs, where R is A or G, and Y is U or C. Targeting a series of sites within different regions of the full-coding length cRNA under simulated physiological conditions found that the most efficient motifs for cleavage were in the hierarchy: GU >/= AU > GC >>> AC. This order is consistent with the kinetic analysis of short synthetic RNA substrates that have the same binding arms but different cleavage sites. DNAzymes with longer symmetric binding arms were more efficient than those with shorter arms, while asymmetric DNAzymes with a longer arm I were also more efficient, suggesting a dominant role for arm I in determining cleavage activity. Modification of one DNAzyme by inverted thymidine (iT) or locked nucleic acids (LNA) showed the LNA-modified DNAzyme gave efficient silencing of AChR expression in HEK 293 cells. Our data demonstrate the usefulness of screening in vitro for an efficient DNAzyme prior to cellular applications.

Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference.

Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125I-alpha-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of alphaS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders.

Therapeutic gene silencing in the nervous system.

Progress in the understanding of RNA biology has brought into focus the prospect of using RNA-based therapeutics as a novel approach to treat human disease. In particular, following the discovery of the RNA interference (RNAi) pathway, the emergence of technology based on small interfering RNA (siRNA) now offers a powerful and highly specific tool for therapeutic gene silencing. Many neurological diseases, including neurodegenerative disorders, tumours and retinal disease are likely candidates to benefit from such advances. The challenges ahead will be to identify appropriate disease gene targets and, crucially, to understand the biological parameters that determine safe, precise and effective delivery and function of RNA-based therapeutic molecules within the unique environment of the nervous system.

Mutations in congenital myasthenic syndromes reveal an epsilon subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR.

Many congenital myasthenic syndromes (CMS) are associated with mutations in the genes encoding the acetylcholine receptor (AChR), an oligomeric protein with the structure alpha(2)betadelta epsilon. AChR deficiency is frequently due to homozygous or heteroallelic mutations in the AChR epsilon subunit, most of which cause truncation of the polypeptide chain and loss of surface expression of AChR. Here we identified mutations epsilon 1369delG and epsilon Y458X, located in the 18 amino acid epsilon subunit C-terminus that lies extracellular to the M4 transmembrane domain. We then incorporated green fluorescent protein (GFP) into the intracellular loop between M3 and M4 of mutant or wild-type epsilon subunits and expressed the AChRs in RD or HEK 293 cells. AChR containing wild-type GFP-tagged epsilon subunits were incorporated into the surface membrane, whereas the GFP-tagged AChR mutant epsilon subunits co-localized with an endoplasmic reticulum (ER) marker and were not expressed on the cell surface. In addition, mutant AChRs did not reach the cell surface, as measured by labelling of intact cells with (125)I-alpha-bungarotoxin and precipitation with an epsilon-subunit-specific antiserum. Mutagenesis studies showed that cysteine 470, located four amino acids from the C-terminus, is essential for alpha/epsilon assembly and surface expression of adult AChR. Replacement of cysteine 470 by serine does not restore alpha/epsilon assembly or surface expression. Our results provide the first use of GFP-tagged AChR as a tool for investigation of CMS and demonstrate a previously undetermined role for a disulphide-bonded cystine in the epsilon subunit C-terminus, which plays a crucial role in expression of the adult AChR.