First-in-human in vivo genome editing via AAV-zinc-finger nucleases for mucopolysaccharidosis I/II and hemophilia B.

Zinc-finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis I (MPS I) (n = 3), MPS II (n = 9), and hemophilia B (n = 1). Treatment was well tolerated with no serious treatment-related adverse events. At the 1e13 vg/kg dose, evidence of genome editing was detected through albumin-transgene fusion transcripts in liver for MPS II (n = 2) and MPS I (n = 1) subjects. The MPS I subject also had a transient increase in leukocyte iduronidase activity to the lower normal range. At the 5e13 vg/kg dose, one MPS II subject had a transient increase in plasma iduronate-2-sulfatase approaching normal levels and one MPS I subject approached mid-normal levels of leukocyte iduronidase activity with no evidence of genome editing. The hemophilia B subject was not able to decrease use of factor IX concentrate; genome editing could not be assessed. Overall, ZFN in vivo editing therapy had a favorable safety profile with evidence of targeted genome editing in liver, but no long-term enzyme expression in blood.

The posterior pituitary expresses the serotonin receptor 2C.

The serotonin receptor 2C (5HT2C) is an important drug target to treat obesity and depression. Its pre-mRNA undergoes alternative splicing, encoding a short RNA1 isoform that is localized intracellularly and a full-length isoform (RNA2) that can reach the cell membrane. These splicing isoforms are deregulated in Prader-Willi syndrome (PWS), due to the loss of a trans-acting regulatory RNA, SNORD115. Here we show that the 5HT2C mRNA is expressed in the posterior pituitary, suggesting that 5HT2C mRNA is generated in the hypothalamus and subsequently conveyed by axonal transport. In the pituitary, the ratio of 5HT2C isoforms is regulated by feeding, and can be manipulated using a splice-site changing oligonucleotide injected into the blood. The pituitary expression of the 5HT2C mRNA may constitute a previously unknown mechanism whereby serotonin in the circulation or drugs targeting the 5HT2C might induce side-effects. Finally, the deregulation of 5HT2C splicing isoforms in PWS could contribute to the known hormonal imbalances.

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35 kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD+ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.

C/D-box snoRNAs form methylating and non-methylating ribonucleoprotein complexes: Old dogs show new tricks.

C/D box snoRNAs (SNORDs) are an abundantly expressed class of short, non-coding RNAs that have been long known to perform 2'-O-methylation of rRNAs. However, approximately half of human SNORDs have no predictable rRNA targets, and numerous SNORDs have been associated with diseases that show no defects in rRNAs, among them Prader-Willi syndrome, Duplication 15q syndrome and cancer. This apparent discrepancy has been addressed by recent studies showing that SNORDs can act to regulate pre-mRNA alternative splicing, mRNA abundance, activate enzymes, and be processed into shorter ncRNAs resembling miRNAs and piRNAs. Furthermore, recent biochemical studies have shown that a given SNORD can form both methylating and non-methylating ribonucleoprotein complexes, providing an indication of the likely physical basis for such diverse new functions. Thus, SNORDs are more structurally and functionally diverse than previously thought, and their role in gene expression is under-appreciated. The action of SNORDs in non-methylating complexes can be substituted with oligonucleotides, allowing devising therapies for diseases like Prader-Willi syndrome.

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing.

C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.

SNORD116 and SNORD115 change expression of multiple genes and modify each other's activity.

The loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116 contribute to Prader-Willi syndrome (PWS), the most common syndromic form of obesity in humans. SNORD115 and SNORD116 are considered to be orphan C/D box snoRNAs (SNORDs) as they do not target rRNAs or snRNAs. SNORD115 exhibits sequence complementarity towards the serotonin receptor 2C, but SNORD116 shows no extended complementarities to known RNAs. To identify molecular targets, we performed genome-wide array analysis after overexpressing SNORD115 and SNORD116 in HEK 293T cells, either alone or together. We found that SNORD116 changes the expression of over 200 genes. SNORD116 mainly changed mRNA expression levels. Surprisingly, we found that SNORD115 changes SNORD116's influence on gene expression. In similar experiments, we compared gene expression in post-mortem hypothalamus between individuals with PWS and aged-matched controls. The synopsis of these experiments resulted in 23 genes whose expression levels were influenced by SNORD116. Together our results indicate that SNORD115 and SNORD116 influence expression levels of multiple genes and modify each other activity.

Direct cloning of double-stranded RNAs.

Most annotated genomes show a large number of sense-antisense transcripts that can generate double-stranded RNAs. We describe a method to clone these dsRNAs from total RNA preparations.

Transcription of the Streptococcus pyogenes hyaluronic acid capsule biosynthesis operon is regulated by previously unknown upstream elements.

The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

Molecular characterization of a patient presumed to have prader-willi syndrome.

Prader-Willi syndrome (PWS) is caused by the loss of RNA expression from an imprinted region on chromosome 15 that includes SNRPN, SNORD115, and SNORD116. Currently, there are no mouse models that faithfully reflect the human phenotype and investigations rely on human post-mortem material. During molecular characterization of tissue deposited in a public brain bank from a patient diagnosed with Prader-Willi syndrome, we found RNA expression from SNRPN, SNORD115, and SNORD116 which does not support a genetic diagnosis of Prader-Willi syndrome. The patient was a female, Caucasian nursing home resident with history of morbid obesity (BMI 56.3) and mental retardation. She died at age of 56 from pulmonary embolism. SNORD115 and SNORD116 are unexpectedly stable in post mortem tissue and can be used for post-mortem diagnosis. Molecular characterization of PWS tissue donors can confirm the diagnosis and identify those patients that have been misdiagnosed.

The 5' untranslated region of the serotonin receptor 2C pre-mRNA generates miRNAs and is expressed in non-neuronal cells.

The serotonin receptor 2C (HTR2C) gene encodes a G protein-coupled receptor that is exclusively expressed in neurons. Here, we report that the 5' untranslated region of the receptor pre-mRNA as well as its hosted miRNAs is widely expressed in non-neuronal cell lines. Alternative splicing of HTR2C is regulated by MBII-52. MBII-52 and the neighboring MBII-85 cluster are absent in people with Prader-Willi syndrome, which likely causes the disease. We show that MBII-52 and MBII-85 increase expression of the HTR2C 5' UTR and influence expression of the hosted miRNAs. The data indicate that the transcriptional unit expressing HTR2C is more complex than previously recognized and likely deregulated in Prader-Willi syndrome.

Processing of snoRNAs as a new source of regulatory non-coding RNAs: snoRNA fragments form a new class of functional RNAs.

Recent experimental evidence suggests that most of the genome is transcribed into non-coding RNAs. The initial transcripts undergo further processing generating shorter, metabolically stable RNAs with diverse functions. Small nucleolar RNAs (snoRNAs) are non-coding RNAs that modify rRNAs, tRNAs, and snRNAs that were considered stable. We review evidence that snoRNAs undergo further processing. High-throughput sequencing and RNase protection experiments showed widespread expression of snoRNA fragments, known as snoRNA-derived RNAs (sdRNAs). Some sdRNAs resemble miRNAs, these can associate with argonaute proteins and influence translation. Other sdRNAs are longer, form complexes with hnRNPs and influence gene expression. C/D box snoRNA fragmentation patterns are conserved across multiple cell types, suggesting a processing event, rather than degradation. The loss of expression from genetic loci that generate canonical snoRNAs and processed snoRNAs results in diseases, such as Prader-Willi Syndrome, indicating possible physiological roles for processed snoRNAs. We propose that processed snoRNAs acquire new roles in gene expression and represent a new class of regulatory RNAs distinct from canonical snoRNAs.

Function of alternative splicing.

Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in 'splicing programs', which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.

Factors influencing RNA degradation by Thermus thermophilus polynucleotide phosphorylase.

At the optimal temperature (65 degrees C), Thermus thermophilus polynucleotide phosphorylase (Tth PNPase), produced in Escherichia coli cells and isolated to functional homogeneity, completely destroys RNAs that possess even a very stable intramolecular secondary structure, but leaves intact RNAs whose 3' end is protected by chemical modification or by hybridization with a complementary oligonucleotide. This allows individual RNAs to be isolated from heterogeneous populations by degrading unprotected species. If oligonucleotide is hybridized to an internal RNA segment, the Tth PNPase stalls eight nucleotides downstream of that segment. This allows any arbitrary 5'-terminal fragment of RNA to be prepared with a precision similar to that of run-off transcription, but without the need for a restriction site. In contrast to the high Mg(2+) requirements of mesophilic PNPases, Tth PNPase retains significant activity when the free Mg(2+) concentration is in the micromolar range. This allows minimization of the Mg(2+)-catalysed nonenzymatic hydrolysis of RNA when phosphorolysis is performed at a high temperature. This capability of Tth PNPase for fully controlled RNA phosphorolysis could be utilized in a variety of research and practical applications.

Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification.

A universal procedure that permits the whole human blood to be tested for the presence of single molecules of DNA and RNA targets is described. The procedure includes a novel protocol for the isolation of total nucleic acids from the guanidinium thiocyanate lysate of unfractionated blood in which, prior to phenol/chloroform extraction, the sample is deproteinized by precipitation with isopropanol. The procedure results in a nearly 100% yield of DNA and RNA, preserves the integrity of RNA, and removes any polymerase chain reaction (PCR) inhibitors. Following reverse transcription (RT), target molecules are counted after having been amplified as molecular colonies by carrying out PCR in a polyacrylamide gel. The entire procedure was checked by assaying viral DNA and RNA in 100-microl aliquots of the whole blood and was found to be capable of detecting 100% molecules of DNA target and 50% molecules of RNA target. Unexpectedly, nucleic acids at relatively high concentrations (1 ng/microl) were found to selectively inhibit the RT activity of Thermus thermophilus DNA polymerase without affecting its DNA-dependent polymerization activity. It follows that the popular single-enzyme RT-PCR format, in which this DNA polymerase serves for both RT and PCR, is not appropriate for assaying rare RNA targets.