Biochemistry (Moscow) (v.81, #7)

Special issue: Genome editing and gene therapy by A. A. Zamyatnin Jr. (651-652).
Gene therapy is one of the most rapidly developing fields of molecular medicine. Gene therapy allows simple transfer of genetic methods aimed at correcting pathological processes into clinical practice. However, a number of technical problems still exists limiting broad use of gene therapy approaches. This special issue discusses modern methods and approaches used for the development of novel, effective, and safe agents for gene therapy.
Keywords: gene therapy; genome editing; CRISPR-Cas; viral vectors

CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology.
Keywords: CRISPR array; cas genes; CRISPR adaptation; CRISPR interference

Methods of genome engineering: a new era of molecular biology by A. A. Chugunova; O. A. Dontsova; P. V. Sergiev (662-677).
Genome sequencing now progressing much faster than our understanding of the majority of gene functions. Studies of physiological functions of various genes would not be possible without the ability to manipulate the genome. Methods of genome engineering can now be used to inactivate a gene to study consequences, introduce heterologous genes into the genome for scientific and biotechnology applications, create genes coding for fusion proteins to study gene expression, protein localization, and molecular interactions, and to develop animal models of human diseases to find appropriate treatment. Finally, genome engineering might present the possibility to cure hereditary diseases. In this review, we discuss and compare the most important methods for gene inactivation and editing, as well as methods for incorporation of heterologous genes into the genome.
Keywords: transgenosis; knockout; knockin; genome engineering; homologous recombination; integration; zinc finger nuclease (ZFN); TALEN; CRISPR

Genome- and cell-based strategies in therapy of muscular dystrophies by Y. Bou Saada; Carla Dib; M. Lipinski; Y. S. Vassetzky (678-690).
Muscular dystrophies are a group of heterogeneous genetic disorders characterized by progressive loss of skeletal muscle mass. Depending on the muscular dystrophy, the muscle weakness varies in degree of severity. The majority of myopathies are due to genetic events leading to a loss of function of key genes involved in muscle function. Although there is until now no curative treatment to stop the progression of most myopathies, a significant number of experimental gene- and cell-based strategies and approaches have been and are being tested in vitro and in animal models, aiming to restore gene function. Genome editing using programmable endonucleases is a powerful tool for modifying target genome sequences and has been extensively used over the last decade to correct in vitro genetic defects of many single-gene diseases. By inducing double-strand breaks (DSBs), the engineered endonucleases specifically target chosen sequences. These DSBs are spontaneously repaired either by homologous recombination in the presence of a sequence template, or by nonhomologous-end joining error prone repair. In this review, we highlight recent developments and challenges for genome-editing based strategies that hold great promise for muscular dystrophies and regenerative medicine.
Keywords: muscular dystrophies; gene editing; cell therapy

Better understanding of phosphoinositide 3-kinase (PI3K) pathways in vasculature: Towards precision therapy targeting angiogenesis and tumor blood supply by D. Tsvetkov; A. Shymanets; Yu Huang; K. Bucher; R. Piekorz; E. Hirsch; S. Beer-Hammer; C. Harteneck; M. Gollasch; B. Nürnberg (691-699).
The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kβ, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg –/–/Pik3cd –/–) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 μM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10 μM) and PI-103 (1 μM), but not by the PI3Kβ isoform inhibitor TGX 221 (100 nM). Pik3cg –/–/Pik3cd –/–-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α1-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.
Keywords: p110α; phenylephrine; α1-adrenergic receptor; PI3K/Akt

Viral vectors for gene therapy: Current state and clinical perspectives by A. N. Lukashev; A. A. Zamyatnin Jr. (700-708).
Gene therapy is the straightforward approach for the application of recent advances in molecular biology into clinical practice. One of the major obstacles in the development of gene therapy is the delivery of the effector to and into the target cell. Unfortunately, most methods commonly used in laboratory practice are poorly suited for clinical use. Viral vectors are one of the most promising methods for gene therapy delivery. Millions of years of evolution of viruses have resulted in the development of various molecular mechanisms for entry into cells, long-term survival within cells, and activation, inhibition, or modification of the host defense mechanisms at all levels. The relatively simple organization of viruses, small genome size, and evolutionary plasticity allow modifying them to create effective instruments for gene therapy approaches. This review summarizes the latest trends in the development of gene therapy, in particular, various aspects and prospects of the development of clinical products based on viral delivery systems.
Keywords: DNA; RNA; viral vector; molecular targeting; smart drugs

mRNA-based therapeutics–Advances and perspectives by O. V. Sergeeva; V. E. Koteliansky; T. S. Zatsepin (709-722).
In this review we discuss features of mRNA synthesis and modifications used to minimize immune response and prolong efficiency of the translation process in vivo. Considerable attention is given to the use of liposomes and nanoparticles containing lipids and polymers for the mRNA delivery. Finally we briefly discuss mRNAs which are currently in the clinical trials for cancer immunotherapy, vaccination against infectious diseases, and replacement therapy.
Keywords: mRNA; in vivo delivery; therapy; nucleic acids; nanoparticles

Binding of DNA with Abf2p increases efficiency of DNA uptake by isolated mitochondria by E. O. Samoilova; I. A. Krasheninnikov; E. N. Vinogradova; P. A. Kamenski; S. A. Levitskii (723-730).
Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA–protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome.
Keywords: mitochondria; nucleic acid import; Abf2p; binding site; dissociation constant; mitochondrial diseases; gene therapy

Cancer is a complex system. Tumor complexity is determined not only by genetic and epigenetic heterogeneity, but also by a huge number of interactions between cancer and normal cells. The heterogeneity and complexity of a tumor causes failure of molecular targeting therapy as a tool for fighting cancer. This review considers the concepts of malignant tumors as organisms that have common characteristics despite all heterogeneity. This leads to the idea that one of the most promising strategies for fighting cancer is the use of the patient’s immune system.
Keywords: cancer; pancreas; complex system; heterogeneity; targeted therapy; irreproducibility

Pseudogene PTENP1 functions as a competing endogenous RNA (ceRNA) to regulate PTEN expression by sponging miR-499-5p by Lei Wang; Ning Zhang; Zun Wang; Dong-mei Ai; Zhen-yu Cao; Hua-ping Pan (739-747).
Increasing evidence has shown that pseudogenes can widely regulate gene expression. However, little is known about the specific role of PTENP1 and miR-499-5p in insulin resistance. The relative transcription level of PTENP1 was examined in db/db mice and high fat diet (HFD)-fed mice by real-time PCR. To explore the effect of PTENP1 on insulin resistance, adenovirus overexpressing or inhibiting vectors were injected through the tail vein. Bioinformatics predictions and a luciferase reporter assay were used to explore the interaction between PTENP1 and miR-499-5p. The relative transcription level of PTENP1 was largely enhanced in db/db mice and HFD-fed mice. Furthermore, the overexpression of PTENP1 resulted in impaired Akt/GSK activation as well as glycogen synthesis, while PTENP1 inhibition led to the improved activation of Akt/GSK and enhanced glycogen contents. More importantly, PTENP1 could directly bind miR-499-5p, thereby becoming a sink for miR-499-5p. PTENP1 overexpression results in the impairment of the insulin-signaling pathway and may function as a competing endogenous RNA for miR-499-5p, thereby contributing to insulin resistance.
Keywords: PTENP1; ceRNA; PTEN; miR-499-5p

Previously unclassified mutation of mtDNA m.3472T>C: Evidence of pathogenicity in Leber’s hereditary optic neuropathy by N. L. Sheremet; T. A. Nevinitsyna; N. V. Zhorzholadze; I. A. Ronzina; Y. S. Itkis; T. D. Krylova; P. G. Tsygankova; V. A. Malakhova; E. Y. Zakharova; A. V. Tokarchuk; A. A. Panteleeva; E. M. Karger; K. G. Lyamzaev; S. E. Avetisov (748-754).
Leber’s hereditary optic neuropathy (LHON) refers to a group of mitochondrial diseases and is characterized by defects of the mitochondrial electron transport chain and decreased level of oxidative phosphorylation. The list of LHON primary mtDNA mutations is regularly updated. In this study, we describe the homoplasmic nucleotide substitution m.3472T>C in the MT-ND1 (NADH-ubiquinone oxidoreductase chain 1) gene and specific changes in cell metabolism in a patient with LHON and his asymptomatic sister. To confirm the presence of mutation-related mitochondrial dysfunction, respiration of skin fibroblasts and platelets from the patient and his sister was studied, as well as the mitochondrial potential and production of reactive oxygen species in the skin fibroblasts. In addition, based on characteristics of the toxic effect of paraquat, a new approach was developed for detecting the functional activity of complex I of the mitochondrial respiratory chain.
Keywords: Leber’s hereditary optic neuropathy; mtDNA mutations; respiratory chain complex I; fibroblasts

Peptide Aβ(16-25) forms nanofilms in the process of its aggregation by O. M. Selivanova; E. Yu. Gorbunova; L. G. Mustaeva; E. I. Grigorashvili; M. Yu. Suvorina; A. K. Surin; O. V. Galzitskaya (755-761).
A method for the synthesis and high purification of fragments of Aβ(1-42) peptide has been elaborated. We have synthesized the amyloidogenic fragment Aβ(16-25) predicted by us and studied the process of its aggregation by electron microscopy and X-ray analysis. Electron microscopy images show that the peptide forms a film, which is not characteristic of amyloid fibrils. At the same time, according to the X-ray diffraction data, its preparations display the presence of two main reflections (4.6-4.8 and 8-12 Å) characteristic of cross-β structure of amyloid fibrils. Thus, the fragment Aβ(16-25) that we predicted is a promising object not only for studying the process of polymerization of the peptides/proteins, but also for using it as a nanomaterial to study a number of biological processes.
Keywords: amyloid fibril; protofibril; oligomer; Aβ peptide; Alzheimer’s disease; electron microscopy

Determination of regions involved in amyloid fibril formation for Aβ(1-40) peptide by A. K. Surin; E. I. Grigorashvili; M. Yu. Suvorina; O. M. Selivanova; O. V. Galzitskaya (762-769).
The studies of amyloid structures and the process of their formation are important problems of biophysics. One of the aspects of such studies is to determine the amyloidogenic regions of a protein chain that form the core of an amyloid fibril. We have theoretically predicted the amyloidogenic regions of the Aβ(1-40) peptide capable of forming an amyloid structure. These regions are from 16 to 21 and from 32 to 36 amino acid residues. In this work, we have attempted to identify these sites experimentally by the method of tandem mass spectrometry. As a result, we show that regions of the Aβ(1-40) peptide from 16 to 22 and from 28 to 40 amino acid residues are resistant to proteases, i.e. they are included in the core of amyloid fibrils. Our results correlate with the results of the theoretical prediction.
Keywords: Aβ peptide; amyloid fibril; amyloidogenic regions; mass spectrometry; Alzheimer’s disease

Phylogenomic analysis of type 1 NADH:Quinone oxidoreductase by G. E. Novakovsky; D. V. Dibrova; A. Y. Mulkidjanian (770-784).
We performed phylogenomic analysis of the catalytic core of NADH:quinone oxidoreductases of type 1 (NDH-1). Analysis of phylogenetic trees, as constructed for the core subunits of NDH-1, revealed fundamental differences in their topologies. In the case of four putatively homologous ion-carrying membrane subunits, the trees for the NuoH and NuoN subunits contained separate archaeal clades, whereas subunits NuoL and NuoM were characterized by multiple archaeal clades spread among bacterial branches. Large, separate clades, which united sequences belonging to different archaeal subdomains, were also found for cytoplasmic subunits NuoD and NuoB, homologous to the large and small subunits of nickel-iron hydrogenases. A smaller such clade was also shown for subunit NuoC. Based on these data, we suggest that the ancestral NDH-1 complex could be present already at the stage of the Last Universal Cellular Ancestor (LUCA). Ancestral forms of membrane subunits NuoN and NuoH and cytoplasmic subunits NuoD, NuoB, and, perhaps NuoC, may have formed a membrane complex that operated as an ion-translocating membrane hydrogenase. After the complex attained the ability to reduce membrane quinones, gene duplications could yield the subunits NuoL and NuoM, which enabled translocation of additional ions.
Keywords: proton bioenergetics; molecular evolution; respiration; protein complexes; quinone reduction; phylogenetic analysis