Transgenomic (United States)

CRISPR-Cas3 represents a mechanistically distinct genome-editing system compared to Cas9 that generates long-range deletions rather than small indels, thereby reducing the risk of residual protein function from in-frame mutations. Here we evaluated CRISPR-Cas3 to correct mutations in the TTR gene causing transthyretin amyloidosis, a systemic proteinopathy where loss of mutant TTR in the liver offers therapeutic benefit. Through CRISPR RNA optimization we achieved 58.9% ± 0.5% editing at the TTR locus in vitro, inducing large deletions that abolished TTR expression. Cas3 generated mostly directional deletions up to 75 kb without reproducible off-target mutations, in contrast to Cas9, which induced indels at several off-target sites. In vivo, a single lipid-nanoparticle-based treatment achieved 48.7% ± 1.1% hepatic editing and reduced serum TTR levels by 80.1% ± 4.6%. Deletion size was limited to 21 kb. In TTR exon-humanized mice, Cas3 editing reduced serum TTR without in-frame mutations and attenuated macrophage-associated TTR deposition. These findings highlight Cas3 as an efficient and distinct sytem for in vivo genome editing.

The NADase sterile alpha and TIR motif containing 1 (Sarm1) protein drives axon degeneration after injury. Loss or inhibition of Sarm1 structurally protects axons after sciatic nerve transection (SNT) in vivo but whether Sarm1 also drives functional loss after nerve injury is less clear. We established compound muscle action potential (CMAP) as a novel functional correlate of Sarm1 activation in a SNT mouse model and evaluated its relationship with biochemical and a novel Cellpose-based histological axon detection measure. CMAP amplitudes were elicited 8 h post-SNT but reached near-floor levels by 24 h. Decreases in CMAP amplitude are delayed in a gene dose-dependent manner in Sarm1 knockout mice or by pharmacological Sarm1 inhibition. Myelinated axon density, the NAD hydrolysis product cyclic adenosine diphosphate ribose (cADPR), and the axon degeneration plasma biomarker neurofilament light (NfL) were all altered in a Sarm1-dependent manner. In wild type mice, axon density and NfL were altered at time points after that of cADPR and functional loss, indicating that functional deficits preceded structural deficits. We conclude that functional and structural declines after injury are delayed by Sarm1 inhibition and that CMAP measures after SNT can serve as a novel, preclinical, functional, pharmacodynamic readout for Sarm1 inhibition.

Aims: Biological studies have shown that some condensed derivatives of pyrano[3,4- c]pyridines 6 exhibited pronounced biological activity. Considering these results, the principal aim of this work is to study the regiochemistry of the synthesis of pyrano[3,4-c]pyridines 6, to optimize the reaction conditions and thus to increase the previously observed low yields of pyrano[ 3,4-c]pyridines. Background: Within this research line, some of us, several years ago, developed a method for the preparation of 6-oxopyrano[3,4-c]pyridines 6 starting from 2,2-dimethyltetrahydro-4Hpyran- 4-one 1. In these studies, we separated and identified only the most expected reaction products 6-oxopyrano[3,4-c]pyridines 6. On the basis of this datum, we suggested that the enamines 2 and 3, reacting with acyl chlorides, were not acylated at C-3 and that 5-acylpyran-4- ones 4 were the only products of the reaction. We have justified this result by considering the steric effects exerted by the two methyl groups present in the pyran ring. Moreover, we did not identify the products at the second reaction center: that is, the isomeric compounds 7 and this result was justified considering the different reactivity of aliphatic and cyclic ketone groups. Objectives: The main objectives of this work were: • implementation of the reaction of 2,2-dimethyltetrahydro-4H-pyran-4-one 1 with morpholine; • acylation of the obtained enamines 2 and 3 with acyl chlorides under Stork conditions; • synthesis of pyranopyridines 6-8 based on β-diketones: 3-acylpyran-4-ones 4 and 5-acylpyran-4-ones 5; • confirmation of the structure of the obtained compounds. Methods: For the synthesis of pyrano[3,4-c]pyridines known methods were used. Thus, the reaction of starting 2,2- dimethyltetrahydro-4H-pyran-4-one 1 with morpholine in benzene led to the formation of isomeric enamines 2 and 3. After, they were acylated with acyl chlorides under Stork conditions with formation of two β -diketones: 3- acylpyran-4-ones 4 and 5-acylpyran-4-ones 5. Finally, in order to obtain the aimed pyrano[3,4-c]pyridines 6, the obtained β-dicarbonyl compounds 4 and 5 (as a mixture of isomers) were reacted with 2-cyanoacetamide in ethanol in the presence of diethylamine, according to the Knoevenagel condensation. The structure of the obtained compounds has been unambiguously confirmed by using a wide spectrum of physico-chemical methods (NMR, IR, Xray structural and elemental analysis) and, in the instance of compounds 7, also by an alternative synthesis. Results: Starting from the 2,2-dimethyltetrahydro-4H-pyran-4-one 1 a series of new and already known 6- oxopyrano[3,4-c]pyridines 6 were synthesized. As a result of the study of the regiochemistry in the synthesis of pyrano[3,4-c]pyridines, of the four possible isomer pyranopyridines 6-9, we have succeeded in identifying three of them (6-8). Thus, isomer pyranopyridines 7 and 8 were identified in the mixture with the main compounds 6. Moreover, isomeric pyrano[3,4-c]pyridines 8 were detected when alkyl groups are present in the starting compounds 4 and 5, while isomeric pyrano[4,3-b]pyridines 7 were detected in the case of the presence of aromatic groups. Unfortunately, we have not been able to isolate compounds 7 and 8 in the pure state from the reaction mixtures. At now, we have not been able to detect and identify isomeric pyrano[4,3-b]pyridines 9. On the whole, we have been able to better the effectiveness of the synthesis of pyrano[3,4-c]pyridines 6, increasing their yields by ≈ 5-15%. Conclusion: As a result of our investigation, we have found that the acylation reaction of enamines 2 and 3 and the cyclization reaction of β-diketones 4 and 5 are not regioselective. Therefore, we can state that enamines 2 and 3 can be acylated at both C-3 and C-5 with the formation of a mixture of 3-acylpyran-4-ones 4 and of 5-acylpyran-4-ones 5. Their condensation with 2-cyanoacetamide led to the formation of mixtures of regioisomeric pyranopyridines 6- 8. In conclusion, as a result of our present research, we can say that we have been able to increase the effectiveness of the synthesis of pyranopyridines, largely improving our previous results. Other: Now, we are working to look for the fourth isomeric pyrano[4,3-b]pyridines 9 by using the most modern and fine methods. Moreover, we hope that we shall be able to separate the mixtures of pyranopyridines 6-8: any way they can be used for further syntheses as they are.

Background and Objective: Continuing our research in the field of heterocyclic compounds and exploiting our experience in the synthesis of pyridothieno(furo)[3,2- d]pyrimidine derivatives decorated by suitable reactive groups (in turn able to give rise to the formation of new heterocyclic rings), we now carried out a research with the aim of building in different position of the pyrimidine some new hetero-rings: the 1,3,4-triazole, thiazole and thiazine. Moreover their possible antimicrobial activity against some grampositive and gram-negative bacilli strains has been evaluated. Methods: Starting from the hydrazino derivative of pyrido[3',2':4,5]thieno(furo)[3,2- d]pyrimidines 2 (7) by reflux with triethyl orthoformate or formic acid, some substituted isomeric pyrido[3',2':4,5]thieno(furo)[2,3-e][1,2,4]triazolopyrimidines 3 and 4 (10) have been synthesized. By alkylation of compounds 8 with alkyl dichlorides, the cyclization to a thiazoline or to a thiazine ring on the [a] side of the pyrimidine ring occurs, with the formation of the new pentacyclic systems: pyrido[3',2':4,5]thieno[ 3,2-d][1,3]thiazolo[3,2-a]pyrimidin-7(8)-one 15 and pyrido[3'',2'':4',5']furo(thieno)[3',2':4,5]pyrimido[2,1- b][1,3]thiazin-7(8)-ones 16. Results: 11-Alkyl(aryl)-8,8-dimethyl-7,10-dihydro-8H-pyrano[4'',3'':4',5']pyrido[3',2':4,5]thieno[2,3-e][1,2,4] triazolo[4,3-c]pyrimidines 3 gave a Dimroth rearrangement in acidic media. It was observed that the cyclization of compounds 7 and 8 occurred linearly with the expected formation of pyrido[3',2':4,5]thieno(furo)[3,2- d][1,2,4]triazolo[4,3-a]pyrimidin-7(8)-ones 10 and pyrido[3',2':4,5]thieno[3,2-d][1,3]thiazolo[3,2-a]pyrimidin- 7(8)-one 15 and pyrido[3'',2'':4',5']furo(thieno)[3',2':4,5]pyrimido[2,1-b][1,3]thiazin-7(8)-ones 16: that is triazole, thiazole and thiazine ring condensation occurs at the [b] side of the pyrimidine ring. During the alkylation of compounds 7, the unusual cleavage of the hydrazino group took place with the formation of 8(9)- methylpyrido[3',2':4,5]thieno(furo)[3,2-d]pyrimidin-7(8)-ones 12. Conclusion: The synthesis of some new ‘complex’ pentaheterocyclic systems containing triazolopyrimidine, thiazolo[3,2-a]pyrimidine and pyrimido[2,1-b]thiazine moiety based on the pyrido[3',2':4,5]thieno(furo)[3,2- d]pyrimidines is described. During the alkylation of 9(10)-hydrazinopyrido[3',2':4,5]thieno(furo)[3,2- d]pyrimidin-7(8)-ones 7 an unexpected cleavage of the hydrazino group has been observed with the formation of 8(9)-methylpyrido[3',2':4,5]thieno(furo)[3,2-d]pyrimidin-7(8)-ones 12. Biological tests on the newly synthesized compounds evidenced that some of them showed promising antimicrobial activity. Keywords: Triazolopyrimidine, thiazolo[3, 2-a]pyrimidine, pyrimido[2, 1-b]thiazine, cyclization, alkylation, antimicrobial activity.

This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without using crosslinking agents, such as formaldehyde, which compromise DNA integrity and extraction efficiency. Blood was drawn from pregnant donors into K3EDTA and Blood Exo DNA ProTeck® (ProTeck) tubes. Blood drawn into both tubes were aliquoted and stored at three different temperatures. At indicated times sample aliquots were processed for cell-free DNA (cfDNA) extraction. Plasma cfDNA and cffDNA quantified by droplet digital PCR (ddPCR) assay which amplify RASSF1A gene promoter region. ProTeck reagent is formaldehyde free and inhibits blood cell metabolism in blood samples during storage. Cell-free DNA concentration increased over time in blood plasma stored in K3EDTA tubes at 4, 22 and 30°C. Blood stored in ProTeck tubes, cfDNA concentration was stable at 4, 22 and 30°C for 21, 28 and 7 days, respectively. In K3EDTA tubes cffDNA proportion decreases steadily over time whereas in ProTeck tubes cffDNA proportion remained stable. This novel technology stabilizes cffDNA proportion in maternal blood samples at 4, 22 and 30°C for 21, 28 and 7 days, respectively.

BackgroundPlasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. MethodsAssays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. ResultsThe relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. ConclusionNon-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA.

Pathogenic RYR2 variants account for ≈60% of clinically definite cases of catecholaminergic polymorphic ventricular tachycardia. However, the rate of rare benign RYR2 variants identified in the general population remains a challenge for genetic test interpretation. Therefore, we examined the results of the RYR2 genetic test among patients referred for commercial genetic testing and examined factors impacting variant interpretability. Frequency and location comparisons were made for RYR2 variants identified among 1355 total patients of varying clinical certainty and 60 706 Exome Aggregation Consortium controls. The impact of the clinical phenotype on the yield of RYR2 variants was examined. Six in silico tools were assessed using patient- and control-derived variants. A total of 18.2% (218/1200) of patients referred for commercial testing hosted rare RYR2 variants, statistically less than the 59% (46/78) yield among clinically definite cases, resulting in a much higher potential genetic false discovery rate among referrals considering the 3.2% background rate of rare, benign RYR2 variants. Exclusion of clearly putative pathogenic variants further complicates the interpretation of the next novel RYR2 variant. Exonic/topologic analyses revealed overrepresentation of patient variants in exons covering only one third of the protein. In silico tools largely failed to show evidence toward enhancement of variant interpretation. Current expert recommendations have resulted in increased use of RYR2 genetic testing in patients with questionable clinical phenotypes. Using the largest to date catecholaminergic polymorphic ventricular tachycardia patient versus control comparison, this study highlights important variables in the interpretation of variants to overcome the 3.2% background rate that confounds RYR2 variant interpretation.

MLL4 is an essential subunit of the H3K4 methylation complexes. We report that MLL4 deficiency compromised regulatory T (Treg) cell development and resulted in substantial decreases in H3K4me1 and chromatin interaction at putative enhancers, a remarkable portion of which were not direct targets of MLL4 but were enhancers that interact with MLL4-bound sites. The decrease in H3K4me1 and chromatin interaction at the MLL4-unbound enhancers correlated with MLL4 binding at distant-interacting regions. Deletion of an upstream MLL4 binding site reduced H3K4me1 at the Foxp3 regulatory elements looped to the MLL4 binding site and compromised both thymic Treg and inducible Treg cell differentiation. We show that MLL4 catalyzed H3K4 methylation at distant unbound enhancers via chromatin looping, thus providing a new mechanism of regulating T cell enhancer landscape and impacting Treg cell differentiation.