抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.

Regulation of microRNA biogenesis

To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.

Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes

Small RNAs of 20-30 nucleotides can target both chromatin and transcripts, and thereby keep both the genome and the transcriptome under extensive surveillance. Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs of distinctive characteristics have been found and can be classified into three classes based on their biogenesis mechanism and the type of Argonaute protein that they are associated with: microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs) and Piwi-interacting RNAs (piRNAs). This Review summarizes our current knowledge of how these intriguing molecules are generated in animal cells.

/pdf/biogenesis-of-small-rnas-in-animals-1m2f3gqoz1.pdf

Biogenesis of small RNAs in animals.

Complete deficiency ofhypoxanthine-guanine phosphoribosyltransferase (HPRT)causes theLesch-Nyhan syndrome. Previous characterization ofa mutantformofHPRT, HPRTyaie, fromasubject withtheLesch-Nyhan syndrome revealed normal mRNA andprotein concentrations, noresidualcatalytic activity, andcathodal migration uponPAGE.We havecloned andsequenced HPRTy.ie cDNA.Thenucleotide sequence offull-length HPRTyk, cDNArevealed asingle nucleotide substitution compared withnormalHPRT cDNA: G Catnucleotide position 211.Thistransversion predicts substitution ofarginine forglycine ataminoacidposition 71, explaining thecathodal migration ofHPRTyafr. Chou-Fasman secondary structure analysis predicts achange intheprobabilityofj-turn formation intheregion containing themutation. Inclusion ofthebulky arginine side chain inplace ofglycine probably disrupts protein folding aswell. Cloning mutant formsofcDNA allows identification ofspecific mutations, provides insight into mutational mechanisms, andfacilitates structure-function analysis ofmutant proteins.

Identification ofa Single Nucleotide ChangeintheHypoxanthine-Guanine Phosphoribosyltransferase Gene(HPRTyaie) Responsible forLesch-Nyhan Syndrome

To date, gene therapies for human application rely on engineered promoters that cannot be finely controlled. Here, we report a universal switch element that allows precise control for gene silencing or gene replacement after exposure to a small molecule. Importantly, these small molecule inducers are in human use, are orally bioavailable when given to animals or humans, and can reach both peripheral tissues and the brain. Moreover, the switch system (Xon) does not require the co-expression of any regulatory proteins. Using Xon, translation of desired elements for gene knockdown or gene replacement occurs after a single oral dose, and expression levels can be controlled by drug dose or in waves with repeat drug intake. This universal switch can provide temporal control of gene editing machinery and gene addition cassettes that can be adapted to cell biology applications and animal studies. Additionally, due to the oral bioavailability and safety of the drugs employed, the Xon switch provides an unprecedented opportunity to refine gene therapies for more appropriate human application.

/pdf/regulated-control-of-gene-therapies-with-a-drug-induced-4bikyv1vo0.pdf

Regulated control of gene therapies with a drug induced switch

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, specifically to RNA interference molecules (RNAi) targeted against the nucleotide sequence in Huntington disease, and methods of using said RNAi molecules for treating Huntington's disease. Described is a nucleic acid for use in treating a subject with Huntington's disease, said nucleic acid encoding an artificial primary miRNA transcript (pri-miRNA), consisting of, in order of location, 5'-flanking region, a non-guiding region, a loop region, a guide region and 3'-flanking region, wherein the guide area consists of SEQ ID NO: 37 (miHDss3), SEQ ID NO: 6 (miHDS1v5U) or SEQ ID NO: 7 (miHDS1v6A) and non-guiding region is at least 80 % complementary to the guide region.EFFECT: invention can be used in treating Huntington's disease.74 cl, 6 dwg, 1 ex

Therapeutic compounds for treating huntington's disease

Huntington’s Disease (HD) is a dominantly inherited neurodegenerative disease for which the major causes of mortality are neurodegeneration-associated aspiration pneumonia followed by cardiac failure. mTORC1 pathway perturbations are present in HD models and human tissues. Amelioration of mTORC1 deficits by genetic modulation improves disease phenotypes in HD models, is not a viable therapeutic strategy. Here, we assessed a novel small molecule mTORC1 pathway activator, NV-5297, for its improvement of the disease phenotypes in the N171-82Q HD mouse model. Oral dosing of NV-5297 over 6 weeks activated mTORC1, increased striatal volume, improved motor learning and heart contractility. Further, the heart contractility, heart fibrosis, and survival were improved in response to the cardiac stressor isoprenaline when compared to vehicle-treated mice. Cummulatively, these data support mTORC1 activation as a therapeutic target in HD and consolidates NV-5297 as a promising drug candidate for treating central and peripheral HD phenotypes and, more generally, mTORC1-deficit related diseases.

Beverly L. Davidson

Papers

Identification ofa Single Nucleotide ChangeintheHypoxanthine-Guanine Phosphoribosyltransferase Gene(HPRTyaie) Responsible forLesch-Nyhan Syndrome

Regulated control of gene therapies with a drug induced switch

Therapeutic compounds for treating huntington's disease

Huntington’s disease phenotypes are improved via mTORC1 modulation by small molecule therapy

VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF