Gene-Based Therapeutics for Inherited Retinal Diseases

Inherited retinal diseases (IRDs) are a heterogenous group of orphan eye diseases that typically result from monogenic mutations and are considered attractive targets for gene-based therapeutics. Following the approval of an IRD gene replacement therapy for Leber’s congenital amaurosis due to RPE65 mutations, there has been an intensive international research effort to identify the optimal gene therapy approaches for a range of IRDs and many are now undergoing clinical trials. In this review we explore therapeutic challenges posed by IRDs and review current and future approaches that may be applicable to different subsets of IRD mutations. Emphasis is placed on five distinct approaches to gene-based therapy that have potential to treat the full spectrum of IRDs: 1) gene replacement using adeno-associated virus (AAV) and nonviral delivery vectors, 2) genome editing via the CRISPR/Cas9 system, 3) RNA editing by endogenous and exogenous ADAR, 4) mRNA targeting with antisense oligonucleotides for gene knockdown and splicing modification, and 5) optogenetic approaches that aim to replace the function of native retinal photoreceptors by engineering other retinal cell types to become capable of phototransduction.

Translation-dependent mRNA localization to Caenorhabditis elegans adherens junctions

mRNA localization is an evolutionarily widespread phenomenon that can facilitate subcellular protein targeting. Extensive work has focused on mRNA targeting through “zip-codes” within untranslated regions (UTRs), while much less is known about translation-dependent cues. Here, we examine mRNA localization in Caenorhabditis elegans embryonic epithelia. From an smFISH-based survey, we identified mRNAs associated with the cell membrane or cortex, and with apical junctions in a stage- and cell type-specific manner. Mutational analyses for one of these transcripts, dlg-1/discs large, revealed that it relied on a translation-dependent process and did not require its 5’ or 3'UTRs. We suggest a model in which dlg-1 transcripts are co-translationally localized with the nascent protein: first the translating complex goes to the cell membrane using sequences located at the carboxy-terminus/3’ end, and then apically using amino-terminal/5’ sequences. These studies identify a translation-based process for mRNA localization within developing epithelia and determine the necessary cis-acting sequences for dlg-1 mRNA targeting.

Molecular Strategies to Target Protein Aggregation in Huntington’s Disease

Huntington’s disease (HD) is a neurodegenerative disorder caused by the aggregation of the mutant huntingtin (mHTT) protein in nerve cells. mHTT self-aggregates to form soluble oligomers and insoluble fibrils, which interfere in a number of key cellular functions. This leads to cell quiescence and ultimately cell death. There are currently still no treatments available for HD, but approaches targeting the HTT levels offer systematic, mechanism-driven routes towards curing HD and other neurodegenerative diseases. This review summarizes the current state of knowledge of the mRNA targeting approaches such as antisense oligonucleotides and RNAi system; and the novel methods targeting mHTT and aggregates for degradation via the ubiquitin proteasome or the autophagy-lysosomal systems. These methods include the proteolysis-targeting chimera, Trim-Away, autophagosome-tethering compound, autophagy-targeting chimera, lysosome-targeting chimera and approach targeting mHTT for chaperone-mediated autophagy. These molecular strategies provide a knowledge-based approach to target HD and other neurodegenerative diseases at the origin.

Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.

Dcp2 C-terminal Cis-Binding Elements Control Selective Targeting of the Decapping Enzyme by Forming Distinct Decapping Complexes

A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5′ to 3′ decay, but the molecular mechanisms controlling selective mRNA targeting by the enzyme remain elusive. Through extensive genetic analyses we uncover cis-regulatory elements in the Dcp2 C-terminal domain that control selective targeting of the decapping enzyme by forming distinct decapping complexes. Two Upf1-binding motifs target the decapping enzyme to NMD substrates, and a single Edc3-binding motif targets both Edc3 and Dhh1 substrates. Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Xrn1 also has a specific Dcp2 binding site, allowing it to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its targeting specificity and enzymatic activation.

Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.

Translation-dependent mRNA localization to Caenorhabditis elegans adherens junctions

mRNA localization is an evolutionarily widespread phenomenon that facilitates sub-cellular protein targeting. Extensive work has focused on mRNA targeting through “zip codes” within untranslated regions (UTRs), while much less is known about translation-dependent cues. Here, we examine mRNA localization in Caenorhabditis elegans embryonic epithelia. From an smFISH-based survey, we identified mRNAs associated with the cell membrane or cortex, and with apical junctions in a stage- and cell type-specific manner. Mutational analyses for one of these transcripts, dlg-1/discs large, revealed that it relied on a translation-dependent process and did not require its 5′ or 3′ UTR. We suggest a model in which dlg-1 transcripts are co-translationally colocalized with the encoded protein: first the translating complex goes to the cell membrane through sequences of the SH3 domain, and then to the apical junction by the L27 and PDZ sequences. In addition, the Hook and GuK sequences contribute to the second step: they are required for mRNA, but not protein, to accumulate at the apical junctions from locations at or near the membrane. These studies identify a translation-based process for mRNA localization within developing epithelia and determine the necessary cis-acting sequences for dlg-1 mRNA targeting.

Bioinformatics Analysis Revealed Novel 3′UTR Variants Associated with Intellectual Disability

MicroRNAs (or miRNAs) are short nucleotide sequences (~17–22 bp long) that play important roles in gene regulation through targeting genes in the 3′untranslated regions (UTRs). Variants located in genomic regions might have different biological consequences in changing gene expression. Exonic variants (e.g., coding variant and 3′UTR variant) are often causative of diseases due to their influence on gene product. Variants harbored in the 3′UTR region where miRNAs perform their targeting function could potentially alter the binding relationships for target pairs, which could relate to disease causation. We gathered miRNA–mRNA targeting pairs from published studies and then employed the database of microRNA Target Site single nucleotide variants (SNVs) (dbMTS) to discover novel SNVs within the selected pairs. We identified a total of 183 SNVs for the 114 pairs of accurate miRNA–mRNA targeting pairs selected. Detailed bioinformatics analysis of the three genes with identified variants that were exclusively located in the 3′UTR section indicated their association with intellectual disability (ID). Our result showed an exceptionally high expression of GPR88 in brain tissues based on GTEx gene expression data, while WNT7A expression data were relatively high in brain tissues when compared to other tissues. Motif analysis for the 3′UTR region of WNT7A showed that five identified variants were well-conserved across three species (human, mouse, and rat); the motif that contains the variant identified in GPR88 is significant at the level of the 3′UTR of the human genome. Studies of pathways, protein–protein interactions, and relations to diseases further suggest potential association with intellectual disability of our discovered SNVs. Our results demonstrated that 3′UTR variants could change target interactions of miRNA–mRNA pairs in the context of their association with ID. We plan to automate the methods through developing a bioinformatics pipeline for identifying novel 3′UTR SNVs harbored by miRNA-targeted genes in the future.

Coupling of translation quality control and mRNA targeting to stress granules

Stress granules are dynamic assemblies of proteins and nontranslating RNAs that form when translation is inhibited in response to diverse stresses. Defects in ubiquitin–proteasome system factors including valosin-containing protein (VCP) and the proteasome impact the kinetics of stress granule induction and dissolution as well as being implicated in neuropathogenesis. However, the impacts of dysregulated proteostasis on mRNA regulation and stress granules are not well understood. Using single mRNA imaging, we discovered ribosomes stall on some mRNAs during arsenite stress, and the release of transcripts from stalled ribosomes for their partitioning into stress granules requires the activities of VCP, components of the ribosome-associated quality control (RQC) complex, and the proteasome. This is an unexpected contribution of the RQC system in releasing mRNAs from translation under stress, thus identifying a new type of stress-activated RQC (saRQC) distinct from canonical RQC pathways in mRNA substrates, cellular context, and mRNA fate.