M. Yu. Skoblov

Therapeutic siRNAs and nonviral systems for their delivery

Gene-directed therapy with small interfering RNA (siRNA) has a tremendous potential and will undoubtedly occupy one of the leading positions among other therapeutic methods in the future. The lack of efficient and targeted delivery vectors delays a successful implementation of this method in medicine. To develop such systems, one needs a comprehensive insight into the interactions of siRNAs, its delivery systems, and an organism. The review covers the properties of therapeutic siRNAs and nonviral systems for their delivery.

Prospects of antisense therapy technologies

Three variants of antisense technologies are presently known: antisense oligonucleotides, RNA interference, and ribozymes. In spite of the difference in the mechanisms of action, all of them are based on a common principle: an antisense preparation works after binding with an RNA target to form a duplex. All of the three variants are intensely used in experiments in vivo. The review considers the current situation in the field of using antisense technologies to treat various diseases. Key words: antisense therapy, antisense oligonucleotides, RNA interference, ribozymes

Mutations in the DNA polymerase and thymidine kinase genes of herpes simplex virus clinical isolates resistant to antiherpetic drugs

Primary structures of DNA polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates di ffering in sensitivity to several antiherpetic drugs were determined and compared to those of two laboratory HSV-1 strains one of which (L2) was sensitive and the other (L2/R) was resistant to acyclovir. The phylogenetic sequence analysis showed that the ul30 and ul23 sequences of clinical isolates were close to those of L2, and that ul30 conserved regions differed between HSV-1 isolates and L2 only in point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA polymerase and thymidine kinase functional domains were identified as substitutions associated with strain resistance to ACV and other antiherpetic drugs.

Nonviral delivery systems for small interfering RNAs

RNA interference is now considered to be the most powerful and promising tool for gene-targeted therapy. Several problems are still to be solved for its successful use in medicine. One of the main issues is efficient siRNA delivery. The review considers various types of nonviral siRNA delivery systems.

Cloning and expression of the IGA-specific endopeptidase genes from Neisseria meningitides

IgA1-specific proteinases (Igase) are known as a pivotal pathogenicity factor in meningococcus (Neisseria meningitides) and in some related bacteria. These enzymes belong to the trypsin-like clan of serine proteases. They exhibit high substrate selectivity, being able to discriminate between IgA1 and IgA2 and to distinguish the human IgA1 from IgA1 of nonprimate mammalian species [8]. According to recent data, in addition to the conventional IgA1-processing enzymes, meningococci contain alternative enzymes. However, the substrate specificity of the alternative Igase, its role in pathogenesis, and the ability to complement the functionality of the conventional Igase remains obscure. In this study, we investigated the structure of the Igase genes and their products in two highly virulent strains M9 and A208 of N. meningitides serogroup A. In particular, we found both conventional and alternative Igase genes in each genome; the nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the accession numbers AY770504, AY558158, and AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in both strains, whereas the recently discovered alternative Igase (formerly known as type 1 meningococcal adhesin) contained a hypervariable region approximately 900 bp long in the 5′-terminal part of the gene. The conventional genes from both strains were expressed in E. coli in the form of inclusion bodies. The recombinant products were used for immunization of rabbits. The antibodies obtained efficiently reacted with both recombinant and native antigens from the N. meningitides culture medium.

[Antisense regulation of human gene MAP3K13: true phenomenon or artifact].

Antisense regulation of gene expression is a widespread but poorly understood mechanism of gene expression regulation. The potential role of antisense transcripts in tumorigenesis is most intriguing for functional research. Here we experimentally characterize an antisense mRNA asLZK overlapping the human MAP3K13/LZK gene that is involved in the mitogenesis-related JNK/SAPK signal transduction pathway. According to the functional annotation of the human genome, asLZK transcript (LOC647276) is expressed at the relatively high level and overrepresented in tumor samples. To our surprise, experimental study of human asLZK revealed that this sequence is not expressed, but represents a silent pseudogene of ribosomal protein L4 encoding gene RPL4. This pseudogene resulted from relatively recent retroposition of RPL4 mRNA into the first intron of MAP3K13 gene and does not participate in the regulation of MAP3K13 expression. This study stresses that, after initial in silico mapping efforts, experimental verification of the expression landscape is warranted.

Sodium Channelopathies: From Molecular Physiology towards Medical Genetics

Voltage-gated sodium channels are heteromeric transmembrane proteins involved in the conduction of sodium ion currents in response to membrane depolarization. In humans, nine homologous genes, SCN1A–11A, which encode different isoforms of the voltage-gated sodium channel family, are known. Sodium channel isoforms exhibit different kinetic properties that determine different types of neurons. Mutations in different channels are described in patients with various congenital disorders, from epilepsy to congenital insensitivity to pain. This review presents an analysis of the current literature on the properties of different isoforms of voltage-gated sodium channels and associated diseases.

Clinical and genetic characteristics and diagnostic features of Landouzy–Dejerine facioscapulohumeral muscular dystrophy

Landouzy–Dejerine facioscapulohumeral muscular dystrophy (FSHD) is one of the most common hereditary myodystrophies. A study of the genetic nature of the disease, which has an autosomal dominant mode of inheritance, is extremely interesting and revealing. A unique structure of D4Z4 macrosatellite repeats found in the 4q35 region was originally characterized by a decrease in the number of repeats in patients with Landouzy–Dejerine muscular dystrophy, which resulted in the activation of neighboring genes, in particular, the DUX4 transcription factor. Later, it was found that the epigenetic mechanisms responsible for the chromatin condensation of this region underlie the activation. To date, additional participants leading to pathogenesis of the disease, such as SMCHD1 methylation regulator and DBE-T regulatory long noncoding RNA, have been identified. The revealed complexity of the disease mechanisms is in good agreement with the observed pattern of the disease inheritance. The study of the Landouzy–Dejerine muscular dystrophy pathogenesis is a good example of how monogenic diseases can possess a more complex nature of inheritance.

The genome nucleotide sequence of herpes simplex virus 1 strain L2

The genome nucleotide sequence of the reference strain of herpes simplex virus type 1 was obtained using the technique of full size sequencing. For the virus genome structure determination, 402444 reads with an average length of 202 bp were performed, which corresponded to the 542-fold genome coverage. The data were collected to 52 contigs with N50-4518 and the total contig length of 120929 bp. The sequence obtained was deposited into the GenBank database.

RANDTRAN: Random transcriptome sequence generator that accounts for partition specific features in eukaryotic mRNA datasets

The generation of true random and pseudorandom control sequences is an important problem of computational biology. Available random sequence generators differ in underlying probabilistic models that often remain undisclosed to users. Random sequences produced by differing probabilistic models substantially differ in their outputs commonly used as baselines for evaluations of the motif frequencies. Moreover, modern bioinformatics studies often require generation of matching control transcriptome with emulated partitions into ORFs, 5- and 3-UTRs, as well as the proportion of non-coding RNAs within model transcriptome rather than relatively simple continuous control sequences. Here we describe novel random sequence generating tool RANDTRAN that accounts for the length distribution of 5′ and 3′ non-translated regions in given transcriptome and the partition-specific di- and trinucleotide compositions in translated and non-translated regions. RANDRAN presents matching control transcriptomes in ready-to-use UCSC genome browser-compatible input files. These features may be useful for generating of control sequence sets for common types of computational analysis of various sequence motifs within various sets of RNA. RANDTRAN is available for free download at http://www.generesearch.ru/images/Randtran.rar