Full-Length Transcript Phasing with Third-Generation Sequencing.

Haplotyping individual full-length transcripts can be important in diagnosis and treatment of certain genetic diseases. One set of diseases, repeat expansions of simple tandem repeat sequences are the cause of over 40 neurological disorders. In many of these conditions, expanding a polymorphic repeat beyond a given threshold has been strongly associated with disease onset and severity. Given that most repeat expansions are inherited in an autosomal dominant pattern, repeat expansion disorders are typically characterized by a heterozygous expansion locus associated with a single haplotype. Precision genetic medicines can be used to selectively target expansion-containing sequences in a haplotype-specific manner.However, repeat expansion lengths often exceed the capacity of next-generation sequencing (NGS) reads. Therefore, the accurate length and haplotype determination of repeat expansions requires special considerations and requires the development of custom methods. Here we highlight a method for targeted haplotype phasing of the HTT gene, which can be adopted for use with other full-length transcripts and in other repeat expansion disorders.

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.

Investigational Assay for Haplotype Phasing of the Huntingtin Gene.

Novel treatments for Huntington's disease (HD), a progressive neurodegenerative disorder, include selective targeting of the mutant allele of the huntingtin gene (mHTT) carrying the abnormally expanded disease-causing cytosine-adenine-guanine (CAG) repeat. WVE-120101 and WVE-120102 are investigational stereopure antisense oligonucleotides that enable selective suppression of mHTT by targeting single-nucleotide polymorphisms (SNPs) that are in haplotype phase with the CAG repeat expansion. Recently developed long-read sequencing technologies can capture CAG expansions and distant SNPs of interest and potentially facilitate haplotype-based identification of patients for clinical trials of oligonucleotide therapies. However, improved methods are needed to phase SNPs with CAG repeat expansions directly and reliably without need for familial genotype/haplotype data. Our haplotype phasing method uses single-molecule real-time sequencing and a custom algorithm to determine with confidence bases at SNPs on mutant alleles, even without familial data. Herein, we summarize this methodology and validate the approach using patient-derived samples with known phasing results. Comparison of experimentally measured CAG repeat lengths, heterozygosity, and phasing with previously determined results showed improved performance. Our methodology enables the haplotype phasing of SNPs of interest and the disease-causing, expanded CAG repeat of the huntingtin gene, enabling accurate identification of patients with HD eligible for allele-selective clinical studies.

Genotyping single nucleotide polymorphisms for allele-selective therapy in Huntington disease.

BACKGROUND: The huntingtin gene (HTT) pathogenic cytosine-adenine-guanine (CAG) repeat expansion responsible for Huntington disease (HD) is phased with single nucleotide polymorphisms (SNPs), providing targets for allele-selective treatments. OBJECTIVE: This prospective observational study defined the frequency at which rs362307 (SNP1) or rs362331 (SNP2) was found on the same allele with pathogenic CAG expansions. METHODS: Across 7 US sites, 202 individuals with HD provided blood samples that were processed centrally to determine the number and size of CAG repeats, presence and heterozygosity of SNPs, and whether SNPs were present on the mutant HTT allele using long-read sequencing and phasing. RESULTS: Heterozygosity of SNP1 and/or SNP2 was identified in 146 (72%) individuals. The 2 polymorphisms were associated only with the mHTT allele in 61% (95% high density interval: 55%, 67%) of individuals. CONCLUSIONS: These results are consistent with previous reports and demonstrate the feasibility of genotyping, phasing, and targeting of HTT SNPs for personalized treatment of HD.

Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement.

UNLABELLED: RNA interference (RNAi) is a potent and specific mechanism for regulating gene expression. Harnessing RNAi to silence genes involved in disease holds promise for the development of a new class of therapeutics. Delivery is key to realizing the potential of RNAi, and lipid nanoparticles (LNP) have proved effective in delivery of siRNAs to the liver and to tumors in animals. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated a trial of ALN-VSP, an LNP formulation of siRNAs targeting VEGF and kinesin spindle protein (KSP), in patients with cancer. Here, we show detection of drug in tumor biopsies, siRNA-mediated mRNA cleavage in the liver, pharmacodynamics suggestive of target downregulation, and antitumor activity, including complete regression of liver metastases in endometrial cancer. In addition, we show that biweekly intravenous administration of ALN-VSP was safe and well tolerated. These data provide proof-of-concept for RNAi therapeutics in humans and form the basis for further development in cancer. SIGNIFICANCE: The fi ndings in this report show safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel fi rst-in-class LNP-formulated RNAi therapeutic in patients with cancer. The ability to harness RNAi to facilitate specifi c multitargeting, as well as increase the number of druggable targets, has important implications for future drug development in oncology.

Next-generation sequencing to generate interactome datasets.

Next-generation sequencing has not been applied to protein-protein interactome network mapping so far because the association between the members of each interacting pair would not be maintained in en masse sequencing. We describe a massively parallel interactome-mapping pipeline, Stitch-seq, that combines PCR stitching with next-generation sequencing and used it to generate a new human interactome dataset. Stitch-seq is applicable to various interaction assays and should help expand interactome network mapping.

In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR).

BACKGROUND: While increasing numbers of small interfering RNA (siRNA) therapeutics enter into clinical trials, the quantification of siRNA from clinical samples for pharmacokinetic studies remains a challenge. This challenge is even more acute for the quantification of chemically modified and formulated siRNAs such as those typically required for systemic delivery. RESULTS: Here, we describe a novel method, heating-in-Triton quantitative reverse transcription PCR (HIT qRT-PCR) that improves upon the stem-loop RT-PCR technique for the detection of formulated and chemically modified siRNAs from plasma and tissue. The broad dynamic range of this assay spans five orders of magnitude and can detect as little as 70 pg duplex in 1 g of liver or in 1 ml of plasma. We have used this assay to quantify intravenously administrated siRNA in rodents and have reliably correlated target reduction with tissue drug concentrations. We were able to detect siRNA in rat liver for at least 10 days post injection and determined that for a modified factor VII (FVII) siRNA, on average, approximately 500 siRNA molecules per cell are required to achieve a 50% target reduction. CONCLUSIONS: HIT qRT-PCR is a novel approach that simplifies the in vivo quantification of siRNA and provides a highly sensitive and reproducible tool to measure the silencing efficiency of chemically modified and formulated siRNAs.

An antibiotic selection marker for nematode transgenesis.

We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene (NeoR) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae. This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic).

'Edgetic' perturbation of a C. elegans BCL2 ortholog.

Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

Large-scale RACE approach for proactive experimental definition of C. elegans ORFeome.

Although a highly accurate sequence of the Caenorhabditis elegans genome has been available for 10 years, the exact transcript structures of many of its protein-coding genes remain unsettled. Approximately two-thirds of the ORFeome has been verified reactively by amplifying and cloning computationally predicted transcript models; still a full third of the ORFeome remains experimentally unverified. To fully identify the protein-coding potential of the worm genome including transcripts that may not satisfy existing heuristics for gene prediction, we developed a computational and experimental platform adapting rapid amplification of cDNA ends (RACE) for large-scale structural transcript annotation. We interrogated 2000 unverified protein-coding genes using this platform. We obtained RACE data for approximately two-thirds of the examined transcripts and reconstructed ORF and transcript models for close to 1000 of these. We defined untranslated regions, identified new exons, and redefined previously annotated exons. Our results show that as much as 20% of the C. elegans genome may be incorrectly annotated. Many annotation errors could be corrected proactively with our large-scale RACE platform.

High-quality binary protein interaction map of the yeast interactome network.

Current yeast interactome network maps contain several hundred molecular complexes with limited and somewhat controversial representation of direct binary interactions. We carried out a comparative quality assessment of current yeast interactome data sets, demonstrating that high-throughput yeast two-hybrid (Y2H) screening provides high-quality binary interaction information. Because a large fraction of the yeast binary interactome remains to be mapped, we developed an empirically controlled mapping framework to produce a "second-generation" high-quality, high-throughput Y2H data set covering approximately 20% of all yeast binary interactions. Both Y2H and affinity purification followed by mass spectrometry (AP/MS) data are of equally high quality but of a fundamentally different and complementary nature, resulting in networks with different topological and biological properties. Compared to co-complex interactome models, this binary map is enriched for transient signaling interactions and intercomplex connections with a highly significant clustering between essential proteins. Rather than correlating with essentiality, protein connectivity correlates with genetic pleiotropy.

Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.

Bloom syndrome ortholog HIM-6 maintains genomic stability in C. elegans.

Bloom syndrome is caused by mutation of the Bloom helicase (BLM), a member of the RecQ helicase family. Loss of BLM function results in genomic instability that causes a high incidence of cancer. It has been demonstrated that BLM is important for maintaining genomic stability by playing a role in DNA recombination and repair; however, the exact function of BLM is not clearly understood. To determine the mechanism by which BLM controls genomic stability in vivo, we examined the phenotypes caused by mutation of the C. elegans BLM helicase ortholog, HIM-6. We find that the loss of HIM-6 leads to genomic instability as evidenced by an increased number of genomic insertions and deletions, which results in visible random mutant phenotypes. In addition to the mutator phenotype, him-6 mutants have a low brood size, a high incidence of males, a shortened life span, and an increased amount of germ line apoptosis. Upon exposure to high temperature, him-6 mutants that are serially passed become sterile demonstrating a mortal germ line phenotype. Our data suggest a model in which loss of HIM-6 results in genomic instability due to an increased number of DNA lesions, which either cannot be repaired and/or are introduced by low fidelity recombination events. The increased level of genomic instability that leads to him-6(ok412) mutants having a shortened life span.

JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.

DAF-16/forkhead transcription factor, the downstream target of the insulin-like signaling in Caenorhabditis elegans, is indispensable for both lifespan regulation and stress resistance. Here, we demonstrate that c-Jun N-terminal kinase (JNK) is a positive regulator of DAF-16 in both processes. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate lifespan and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into the nucleus. Our findings define an interaction between two well conserved proteins, JNK-1 and DAF-16, and provide a mechanism by which JNK regulates longevity and stress resistance.