ISSN 19950780, Nanotechnologies in Russia, 2009, Vol. 4, Nos. 11–12, pp. 776–789. © Pleiades Publishing, Ltd., 2009. Original Russian Text © I.L. Tutykhina, M.M. Shmarov, D.Yu. Logunov, B.S. Naroditsky, A.L. Gintsburg, 2009, published in Rossiiskie nanotekhnologii, 2009, Vol. 4, Nos. 11–12.
REVIEWS
Recombinant Adenoviral Nanostructures: Construction and Prospects of Use in Medicine I. L. Tutykhina, M. M. Shmarov, D. Yu. Logunov, B. S. Naroditsky, and A. L. Gintsburg Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Gamaleya Street 18, Moscow, 123098 Russia email:
[email protected] Received May 22, 2009
Abstract—A widespread investigation is currently being conducted in various fields of science and technol ogy regarding the possibilities of using nanoparticles created on the basis of viruses. This review deals with nanotechnologies that are concerned with engineering and the use of recombinant adenoviral nanoparticles (RAVNs) in medicine. RAVNs are containers consisting of adenoviral coat proteins used for delivering the genetic information included in it to different cells. On the basis of recombinant adenoviruses by means of their genetic and/or physical and chemical modifications, it is possible to create different nanoparticles for various purposes: from targeted drug delivery in specific cells to the creation of systems of noninvasive diag nostics for various diseases. DOI: 10.1134/S1995078009110032
INTRODUCTION Nanotechnology is an interdisciplinary area of sci ence and technology. In connection with this, nano technological methods and techniques can be used in all spheres of human activities. Medicine is a promis ing field of the application of nanotechnology. The basic conditions that provide the opportunity of using nanostructures for medical purposes are their func tionality (ability to have therapeutic, prophylactic, or other effects), biocompatibility, and biodegradability. The biocompatibility of materials is based on the absence of toxic effects on the organism; biodegrad ability is ensured by excretion from the organism in the form of nontoxic decay products. The nanostructures of natural origin meet these conditions in the best pos sible way. In recent years many natural biomolecules have been investigated for the purpose of researching the possibilities of creating nanomaterials on their basis. Both distinct biomolecules (DNA, RNA, and proteins (peptides)) and their complexes (ferritins, heat shock proteins, shaperones, carboxysomes, enzyme complexes, and phospholipids (liposomes)) were analyzed. Viruses are natural heterogeneous complexes of biomolecules. The simplest viruses con sist of nucleic acid—the genetic material (genome) of a virus—and a protein coat covering the nucleic acid. Some viruses also contain carbohydrates and lipids. As a result of longterm research of viruses, their biological, genetic, and physical properties were stud ied in great detail. The size of most viral particles varies from 20 to 100 nm. The exceptions are those listed in families with larger size (hyperviruses, paramyxovi ruses, and pox viruses). Developing technologies of
recombinant DNA provided a methodological foun dation for different manipulations with the viral genomes—introduction of various genetic modifica tions (insertions, deletions) in them, depending on research purposes. The unique ability of virus coat proteins for selfassembly into higherorder homoge neous or heterogeneous structures allowed the cre ation of their recombinant analogues on the basis of natural viruses (recombinant viral nanostructures (RVNs)), with or without modified genomes. Knowl edge of the structure of the virion on the molecular level allows one to make a change in the virus coat pro teins and makes it possible to create composites. Thus, RVNs are considered to be a good foundation for engi neering and the production of nanomaterials with a preset structure and predictable properties that can be used in medicine. This is determined by the following RVN characteristics: (i) the relative simplicity of structural organization; (ii) the homogeneity of one type particles in size and form; (iii) the high degree of symmetry and multivalency of particles; (iv) the possibility of controlling the size, form, and properties of RVNs on the genome level using meth ods of genetic engineering; (v) using the physical and chemical genetic meth ods of modifying the RVN surface structure to infuse it with the new properties; (vi) the simplicity of the largescale production technology of RVN; (vii) the maintenance of stable structures over long periods of time;
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Linear double stranded DNA Surface capsid proteins Hexon
Penton
Fiber
Fiber knob domain
protein pIX
Fig.1. The adenovirus virion structure. E1A E1B pLX
pVIII E3 Fiber 52K
ITR Ψ
IIIa PB
pVII
pV pX(mu) pVI Hexon
II 100K
Polymerase
ITR E2A
IVa2
E4
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E2B Genes of major capsid proteins
Genes of virion core proteins
Genes of minor capsid proteins
Fig. 2. The scheme of adenovirus genome. Explanation: ITR is the inverted terminal repeat; P is the protease; Ψ is the packaging signal; PB is the penton base.
(viii) the natural mechanism of genetic informa tion carrier packaging into the protein coat; (ix) the natural process of cell penetration. The listed properties make it possible to use RVNs as the basis for the development of gene therapeutic and vaccine drugs and to use new technologies of non invasive diagnostics of various diseases. Currently, RVNs for the purpose of medical use (and containing various characteristics) are being cre ated on the basis of recombinant adenoviruses [1, 3, 4], retroviruses [2, 3, 4], adenoassociated viruses [3, 4], herpes virus [3, 4], and some others [3, 4]. This review deals with an analysis of the basic characteristics, means of creation, and prospects for the use of various adenovirusbased RVNs in medicine. RVN produced on the basis of recombinant adenoviruses will be called recombinant adenoviral nanostructures (RAVNs). MEANS OF CREATING RECOMBINANT ADENOVIRAL NANOSTRUCTURES The virions of adenoviruses consist of double stranded DNA enclosed by a protein coat (capsid) NANOTECHNOLOGIES IN RUSSIA
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(Fig. 1). Virions represent isometric particles in the form of icosahedron 70–90 nm in [5]. The molecular weight of a virion is 170–175 Mda with a sedimenta tion constant 560S. The structure of adenoviral virions remains the same if there is an increase in the ionic force of a solution, a decline in pH to the acid side (to pH 4), and a temperature change within the range of from 10 to 85°C. Adenoviral virion contains about 2700 polypeptide molecules of 13 types [6]. A DNA molecule and 6 types of proteins (proteins pV, pVII, Mu, pIVa2, ter minal protein and 23K protease) form the inner DNA core of a virion. The other seven types of proteins form the virion coat (Fig. 1). The main components of a capsid are hexones (240 capsomers). Apical capsomers (pentons) carry one or two 10–37nmlong thread like processes (fibers) each. Viral capsid also contains several minor proteins: pIIIa, pVI, pVIII, and pIX. Adenoviruses have been described in great detail on the genetic level as well (Fig.2). The genomes of most of them are completely sequenced. The mechanisms of DNA replication and gene expression are determined, and most of the functions of virus proteins are well
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Genes responsible for RAVN replication and/or the insertion of the target gene
Physical and chemical modifications of the coat of RAVN
Genes of surface proteins of a coat of RAVN
Surface proteins of a coat of RAVN
The whole RAVN
Fiber
Hexon
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Replicationcompetent and replicationincompetent RAVN
Fig. 3. Means of RAVN creation.
known. The main mechanisms of interaction between adenoviruses and hosts at the molecular and cellular level are investigated [7]. The availability of such detailed information on the structure, physicalchemical, and biological properties of adenoviruses makes it possible to implement manipulations not only with whole virions of adenoviruses, but also with their parts: particular genes and coat protein domains. This provides the opportunity to create RAVNs, which possess, on the basis of adenovi ruses, new predictable characteristics.
In terms of the structure of an adenoviral virion, an RAVN can be considered a “container” consisting of adenoviral capsid proteins which delivers the genetic information contained in it to different cells. There are two basic ways for modifying adenoviral virions (Fig. 3) for RAVN production: genetic (manip ulations with the DNA molecule) and physicochemi cal (manipulations with the molecules of coat pro teins, their groups, or the whole nanostructure).
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RECOMBINANT ADENOVIRAL NANOSTRUCTURES WITH MODIFIED GENOME RAVNs with modified genomes are produced by means of intentionally altering the original adenovirus DNA. Using the methods of genetic engineering, var ious foreign nucleotide sequences are inserted into it, including the change in nucleotide structure or dele tion of alreadyexisting elements. The DNA of aden oviruses contains genes responsible for virus reproduc tion (genome replication, viral genes expression, and protection from antivirus organism activity), as well as genes of structural coat proteins of a virus. It is possible to control the structure and properties of the nanocon tainer itself, genetically modifying the genes of capsid proteins during the production of RAVNs. RAVNs with modified coat proteins are called capsidmodi fied (Fig. 3). The choice of foreign genetic material, which will be delivered by a nanocontainer to cells in the composition of modified adenoviral DNA, is made in accordance with the purposes of the RAVN use. At the same time, the genes responsible for virus repro duction are deleted from the composition of adenovi ral DNA for safety purposes. As a result, replication incompetent RAVNs are produced (Fig. 3) that are able only to deliver and express foreign genetic infor mation in cells. In some cases the genes responsible for the genome replication are not deleted on purpose. Regulatory elements controlling their expression are replaced by other elements, whose function depends on the presence of the specific inductor molecule. This is the way replicationcompetent RAVNs (Fig. 3) that are replicable only in strictly determined conditions are produced. REPLICATIONCOMPETENT RAVNS Life cycles of viruses are carried out only inside cells. Having penetrated into a cell, adenoviruses cause active infection, which leads in the end to the lysis of the infected cell and the emergence of the virus progeny. The basis of the infection lies in the repro duction of a virus in a cell, i.e. the replication (copy ing) of its genome with the further packaging of each copy in a protein coat. The property of adenoviruses to kill the infected cell was used for the creation of the adenovirusbased RAVNs, the goal of which is to destruct tumor cells in an organism. The main prop erty of such RAVNs is that they are reproducible only in tumor cells (Fig. 4). Thus, for example, there were designed RAVNs which reproduce in and are able to destroy the tumor cells of a prostate gland. To produce them, the condi tions of expression of one of the genes necessary for the replication of genome (gene E1A) were altered. Instead of a natural promoter of this gene, a prostate specific antigen (PSA) promoter was inserted into the RAVN genome, the concentration of which greatly increases in the case of prostate tumors. As a result, the NANOTECHNOLOGIES IN RUSSIA
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reproduction of RAVNs in a cell would depend entirely on the presence of those factors which stimu late the expression of PSA. Thus RAVNs that are able to cause the infection of only prostate tumor cells were produced; those RAVNs penetrating a healthy cell would be rapidly eliminated from the organism [8]. RAVNs that destroy tumor cells with a malfunc tioning p53 gene were also created. This gene is responsible for the selfdestruction of a cell if certain mechanisms of its functioning are violated, including viral infection. The ingress of a virus into a normal cell leads to the increase in the expression level of gene p53 and the consequent destruction of the cell together with the infectious agent. One of the adenoviral pro teins (product of gene E1B55 kDa) can bind with the promoter of gene p53 and suppress its expression. RAVNs that can not produce an infection in cells with normalfunctioning p53 are created by means of elim inating gene E1B55 kDa. However, in cells with mal functioning p53, these RAVNs can successfully repli cate and form new particles which are also able to destroy tumor cells [9]. REPLICATIONINCOMPETENT RAVNS As was previously mentioned, the basis of the ade novirus infection lies in the replication of the viral genome, which is accompanied by the expression of all genes coded in it. The property of adenoviruses to express genes included in the composition of the viral genome provided the basis for the creation of RAVNs, its aim being to deliver and express foreign genetic information in cells. RAVNs created for this purpose must be safe and should not be able to cause the devel opment of infectious process. Replicationincompe tent RAVNs, which were produced by means of gene elimination from the original adenovirus DNA responsible for its reproduction (Fig. 4), meet this requirement. It should be emphasized that, without the deletion of certain segments of the adenoviral genome, it is possible to include only small amounts of DNA (5% of the genome size) in its composition. If the volume of foreign genetic information increases during the delivery process into cells by RAVNs, it is necessary to eliminate all large parts of it from aden oviral DNA. For example, replicationincompetent RAVNs are created by means of eliminating genes E1 from the segment of adenoviral genome (Fig. 5). RAVNs pro duced in this way are not replicable in the absence of E1 products in natural host cells in vivo. For their pro duction and accumulation, cell lines such as 293 [99], 911 [80], and PerC6 (Crucell, Netherlands) were cre ated. The segment that was deleted from the genome DNA of adenovirus was built into their genome DNA. An effective expression of builtin adenoviral genes takes place, and their products complement their absence in the DNA of RAVNs (Fig. 5). RAVNs may carry deletions in another segment as well (E3), which
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Presence of inducer in cell
Cell destruction by a RAVN
Absence of inducer in cell
Elimination of RAVN
(a)
The binding site of inducer of expression of genes replicators of a RAVN
Protein
Protein
(b)
Elimination of the DNA of a RAVN
DNA of RAVN
mRNA
Expression of target gene
(c)
The cell with a ligand specific receptor
Ligand on the surface of RAVN
Fig. 4. The action circuit of RAVN with modified genomes, (a) replicationcompetent RAVN, (b) replicationincompetent RAVN, and (c) capsidmodified RAVN.
is responsible for the protecting the cell infected by adenovirus from the antiviral activity of the Organisms immune system, to increase the size of the builtin segment [10].
RAVNs, lacking all viral genes and consisting of two inverted terminal repeats (left and right) and the signal of DNA packaging into a capsid, have been cre ated on the basis of the human adenovirus serotype 5.
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Genomic DNA of adenovirus Elimination of the domain responsible for virus replication
Insertion of foreign DNA
Producing of cell line, stably expressing the gene, responsible for the replication of RAVN
DNA of RAVN
Transformation of DNA cells of RAVN
Product of gene responsible for the reproduction of RAVN
Replication of the DNA of RAVN during the interaction with the product of gene responsible for the reproduction of RAVN
Selfassembly of RAVN
RAVN outlet from a cell
Fig. 5. The production process layout of a replicationincompetent RAVN.
In theory such RAVNs may contain up to 35 000 base pairs of foreign DNA [11]. The accumulation of min imal vectors is carried out using helper adenovirus that are able to complement in trans all genes necessary for the replication and assembling of virions. However, such RAVNs have not received widespread application NANOTECHNOLOGIES IN RUSSIA
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because of the fact that drugs usually contain admix tures of helper viruses. CAPSIDMODIFIED RAVNS Adenoviruses are able to penetrate into a variety of cells: endothelial, muscular, respiratory epithelium,
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primary tumor, hemopoietic, dendritic, and other types of cells in an organism [12–21]. Different sero types of adenoviruses differ according to the type of cell which they can penetrate into. For example, ade noviruses of serotypes 2, 4, 5, and 7 have a greater affinity to the cells of pulmonary epithelium and other cells of the respiratory tract and adenoviruses 40 and 41 are similar to those of the gastrointestinal tract. The interactions between adenoviruses and certain cells of an organism, as well as their penetration into these cells, are determined by the structure of the capsid proteins surface. The main participants of these pro cesses are the major proteins of the viral coat (hexon, penton base, and fiber). The creation of target RAVNs is the main purpose for modifications in the structure of capsid proteins of adenoviruses. Target RAVNs are capable of narrowtargeted interactions and penetrat ing certain cells in the organism (Fig. 4). Nowadays there are several ways of creating target RAVNs. The first is the restructuring of fiber. Fiber plays an important role in the expression of the speci ficity of a virus towards one or another type of cells, since it is fiber that is responsible for the primary bind ing with the cell. All adenoviruses are characterized by a high degree of homology of fiber structure except for their terminal domains. This opens up the opportunity to create chimeric RAVNs, in which the whole fiber or just its terminal domain is substituted for an analogous structure of the adenovirus of another serotype. Thus, RAVNs were created with a higher specificity to the cells with the receptor CD34 on their surface (the sub stitution of the Ad5 fiber for the Ad35 fiber), smooth myocytes (the substitution of the Ad5 fiber for that of Ad16), and the upper airways epithelial cells (the sub stitution of the Ad2 fiber the Ad17 fiber). Substituting an Ad5 fiber knob domain for an analogous domain of Ad3 leads to an increase in the affinity of the produced chimeric RAVNs to the ovarian carcinoma cells and the squamous carcinoma cells [22]. The second strategy of the creation of target RAVNs is to insert ligands (mainly peptides) to certain endorgans in the structure of fiber. It was shown that the insertion of integrinbinding RGD motif and seven lysines into the fiber terminal structure lead to the effective interaction of RAVNs with cells unchar acteristic for the unmodified precursor. For example, RAVNs based on Ad5 with RGDmotif were able to penetrate to the ovarian carcinoma cells, pancreas car cinoma, and some head and neck cancer cells; those RAVNs containing the sequence of seven lysines in the structure of fiber penetrated macrophages, smooth muscle cells, endothelial cells, Tcells, and glioma and myeloma cells [22]. Inserting RGDmotif in the fiber structure of avian adenovirus serotype 1 led to similar results [23]. The peptides produced from bacterioph ages (peptide SIGYLPLP) were also inserted in the terminal structure of fiber. This resulted in the rise in specificity of such RAVNs to the vascular endothelial
cells, transferring receptors, and smooth muscle cells [22]. In spite of the diversity of adenoviruses and peptide ligands of viral origin, their combination can not pro vide for the creation of a variety of necessary target specific RAVNs. The solution to this problem lies in the development of a third strategy based on the full substitution of fiber for the structural components of other viruses, bacteria, and whole ligands. For exam ple, it was previously shown that substituting fiber for the ferritin of bacteriophage T4 composed of a sequence of six histidines results in RAVN penetration into cells that express a receptor for this sequence of histidines. In another work the ligand was inserted to the CD40 receptor in the sequence of ferritin. As a result, RAVNs effectively interacted with the dendritic cells, which expressed the corresponding receptor [24]. For the purpose of creating target RAVNs, in addi tion to fiber, other capsid proteins, such as hexon, penton base, and pIX are modified. The protein of adenoviral capsid pIX stabilizes hexon–hexon inter actions, thus maintaining the integrity of a virion. Since the Cterminal domain of pIX is located on the surface of a capsid, RAVNs are produced mainly by inserting ligands into this vary domain. Besides the introduction of RGDmotif into pIX, the peptide consisting of seven lysines was also inserted into this protein. It was shown that RAVNs that have been modified in this way are able to penetrate cells con taining heparinsulfate on their surface [24]. PHYSICAL AND CHEMICAL MODIFICATIONS OF RECOMBINANT ADENOVIRAL NANOSTRUCTURES The infusion of RAVNs in the organism is often accompanied by their rapid elimination from it by hepatic cells (Kupffer’s cells), macrophages, and monocytes [25]. This effect manifests itself to a greater extent in the case of the systematic insertion of RAVNs. In connection with this, RAVNs are often infused in a large amount, which can lead to negative results e.g., the ingress of most of the RAVNs into the liver hepatocytes [26]. One more negative effect of the RAVN infusion in the organism is the immune response to coat proteins, which may cause a toxic state [27]. Several approaches have been developed for elimi nating undesirable effects and the reduction of the infused RAVN dose, including the previously described methods of creating genetically modified target RAVNs. Another approach that can solve this problem is the creation of RAVNs with physical and chemical modifications of capsid proteins. RAVNs that have been modified with the polymeric molecules are the most efficient and safe. For example, N(2hydroxypropyl)methacrylamide (HPMA) and polyethylene glycol (PEG) are widely used for this purpose [28]. Polymers based on HPMA have been
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used for more than ten years for the purpose of deliv ering chemotherapeutical agents to an organism with out showing immunogenicity or causing any toxic side effects. Modifications by means of polymer molecules result in the reduction of the immune response to the RAVN coat proteins due to the decrease in the effi ciency of their interaction with the immune system cells. At the same time, the efficiency of the interac tion between RAVNs and other cells is reduced as well. To ensure the ability of polymermodified RAVNs to interact with cells of an organism, different ligands (peptides, proteins, and antibodies) are inserted into the polymer structure [30, 31, 32]. For example, the integrinbinding RGD motif would be covalently inserted into the structure of PEGmodified RAVNs. As a result of experiments with RAVNs having double modification (PEG and RGDmotif) in vivo, it was proven that they interact more effectively with specific cells than geneticallymodified RAVNs containing RGDmotif in the structure of fiber [32]. Interaction between the polymer particles and the RAVN coat is achieved with the help of lysine ami noacid residuals, which are present in the structure of all capsid major proteins. The modification of RAVNs by means of other molecules—such as peptides, car bohydrates, biotin, and fluorophors—is also possible through these aminoacid residuals. Using the combined strategies of genetic and phys ical and chemical modification allows RAVNs that carry metal nanoparticles on the surface of protein coat to be created. For example, a geneticallymodi fied RAVN that contains six aminoacid residuals in the structure of hexon or protein pIX can effectively inter act with gold nanoparticles. Gold nanoparticles are composed of an artificially produced reaction group of nickel nitrileacetate which can effectively interact with the histidine residuals on the surface of RAVNs [33]. These RAVNs can be used in oncology for the delivery of gold particles to cancerous cells because it is well known fact that this metal potentizes radio therapy. Thus, RAVNs are considered a promising founda tion for the creation of multipurpose medical drugs. The properties of such nanoparticles can be modified, depending on the objects of research, by manipulating the molecules of the original adenovirus, as well as by inserting other structures of biogenic and nonbio genic origin into the structure of the RAVN. USING RAVNS IN GENE THERAPY Contemporary methods of medical treatment of various human and animal diseases are connected with inserting missing products of the metabolism in the recipient’s organism. Insertion a product of deficit gene expression, namely ferment or peptide hormone, in the organism is now a common practice. New treat ment methods are based on the insertion of one or more functional genes that code the synthesis of cor NANOTECHNOLOGIES IN RUSSIA
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responding therapeutic proteins into an organism. RAVNs have been used for several decades to deliver such genes into organisms. Currently RAVNbased therapeutic drugs are being developed by inserting the expression cassette carrying the target gene—the gene of therapeutic intervention—into its structure. The key factors determining the efficiency of this or other RAVNbased medication are as following: (i) the degree of expression of the target gene; (ii) the duration of circulation of the target gene product in the organism; (iii) the specificity of RAVN to those cells, tissues, and organs where penetration makes it possible to achieve the best therapeutic effects Replicationdeficit targeted RAVNs are generally used for the development of genetic therapeutic drugs. The rise in the degree of expression of the target gene in the structure of RAVNs is generally achieved by selecting and inserting the optimum set of regula tory elements into the structure of the expression cas sette (gene + regulatory elements). One of the basic regulatory elements influencing the degree of expres sion is the promoter. The promoter determines whether the gene will effectively express itself only in certain cells or in most cells of an organism. The most widely used promoter is the one that was produced from the genome of the human cytomegalovirus. It provides a high expression level in practically all cells. To increase gene expression, some additional 5' and 3' untranslated regulatory elements (such as polypu rine(A)rich sequence, posttranscriptional regulatory element of the woodchuck hepatitis virus, and the Internal ribosome entry site) are inserted into the structure of the RAVN expression cassette [34]. The late transcription region of an adenoviral genome con tains its own 5' untranslated element—tripartite leader (TPL) of Mastadenoviridae or bipartite leader (BPL) of Aviadenoviridae [35, 36]. Using TPL and BPL as addi tional regulatory elements of expression cassettes in the structure of RAVNs results in a 2–3time increase in transgene expression [37]. The previously described physical and chemical modifications of RAVN coat, which serve for the reduction of the RAVN interaction with the cells of the immune system of the organism, promote the longer circulation of the target gene product in the organism. However, the residual expression of adenoviral genes, which are in the DNA of RAVNs, can also have a cyto toxic effect and produces the immune response of the host [38, 39], leading to the reduction in the time of the delivered transgene expression. The deletions of different segments of viral genome can help overcome these side effects. For example, the elimination of the region responsible for replication and the region pro viding the viral genes expression (E1 and E2 regions) prolongs the transient expression of transgenes by pre venting the synthesis of RAVN coat proteins.
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Table 1. Clinical trials of Advexin for treating various tumor diseases Disease Head and neck tumors Nonsmallcell lung cancer Breast cancer Cancer of esophagus Prostate cancer Ovarian cancer Bladder cancer Brain cancer Bronchioloalveolar cancer
The phase of clinical trials III II II II I I I I I
When the disease is caused, for example, by genetic damage, it is necessary to prolong the gene expression during therapeutic intervention as long as possible (up to several years). Since the DNA of adenoviruses does not integrate in the cell genome, this DNA and the transgene along with it are slowly eliminated from the organism (over a period of several weeks). It is possible to solve this problem by inserting the DNA of RAVN into the cell genome. For this purpose, hybrid recom binant nanoparticles were created on the basis of ade novirus and the adenoassociated virus. The adeno associated virus integrates in the cell genome, thus prolonging the expression of the therapeutic gene coded in its DNA. On the basis of this, we can assume that the RAVNs that are most effective in genetic therapy can contain the following set of structural elements: (i) DNA carrying the expressing cassette with a set of regulatory elements providing a sufficient level of the target gene expression to achieve a therapeutic effect; (ii) a coat (of viral or nonviral origin), which pro tects RAVNs from the influence of immune system cells and provides the delivery of the target gene; Works on the creation of such RAVNs are being conducted in many research institutions around the world. The spectrum of diseases which this engineer ing is aimed to cure is extremely wide. It includes both hereditary genetic and acquired (including infectious) diseases [40, 41]. For example, RAVNs that are unable to replicate and that also contain an expressing cas sette with the human lactoferrin gene in the structure of their DNA were created on the basis of human ade novirus of serotype 5. Lactoferrin has an antibacterial immunestimulated effect and shows antiphlogistic, detoxicant, and antioxidant properties. When inserted into an organism, these RAVNs are able to penetrate into a wide variety of cell types and effectively express the target gene [42].
RAVNs that were created for use in genetic therapy have good prospects in clinical use. By now two drugs have been created on their basis and have been already admitted for use in oncology in China: Gendicine (Sibiono GeneTech Co.) and H101 (Sunway Biotech Co.) [43, 44]. The efficiency of two other similar drugs based on RAVNs is now being investigated in the United States. Advexin (Introgen) and ONYX015 (Onyx Pharmaceuticals) are now at different stages of clinical trials. Advexin made up of RAVNs, the DNA structure of which contains the expressing cassette with the standard set of regulatory elements (a pro moter with the genome of human cytomegalovirus of early transcription region and the signal of polyadeni lation of the Simian virus SV40) and with the thera peutic gene (the gene of the oncosuppressor protein p53). Protein p53 responds to different violations in the cell genome by starting the chain of reactions that lead to its destruction. Moreover, it functions as a sup pressor of the development of malignant tumors. Mutations of gene p53 are found in the cells of 50% of cancerous growths. The insertion of unmutated gene p53 into the malignant cells using RAVNs has a thera peutic effect. The Advexin drug has successfully passed the first stage of clinical trials on five types of tumor and is in the third stage of trials on head and neck tumors (Table 1). Twoyear clinical trials of the neurotrophic influ ence of the vascular endothelium growth factor and angiogenine, expressed in the composition of RAVNs during the amyotrophic lateral sclerosis, showed the good tolerance and safety of their use while increasing the patient’s life span [45]. Clinical results using angiogeninegenecarrying RAVNs in treating chronic ischemia of the lower extremities on an exper imental subject (rat) and volunteer patients showed a visible decrease in the degree of ischemia, which was confirmed by instrumental research [46, 47]. THE USE OF RAVN FOR VACCINATION RAVNs expressing the antigen of a pathogen in an organism can induce the formation of both a cellular immune (Th1dependent) and humoral (Th2depen dent) response to the corresponding pathogen at the same time. This property of RAVN is crucial during the development of vaccines against the intracellular pathogens. Nowadays, vaccines against many patho gens that cause diseases such as tularemia, tuberculo sis, and brucellosis, as well as viruses (influenza virus, human immunodeficiency virus, human papilloma virus, rabies virus, Ebola virus, etc.), are being devel oped on the basis of RAVNs. The efficiency of RAVNs has been proven in works that were dedicated to the creation of candidate vaccines against different sero types of influenza virus (including the avian influenza A virus H5N1). The candidate RAVNbased vaccine against the Influenza A virus has successfully passed the first stage of clinical trials in the United States.
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Twentyfour volunteers participated in the research; the RAVNbased drugs were introduced intranasaly or subcutaneously. As a result, it was shown that RAVNs containing the hemagglutinin gene of the influenza virus are harmless to humans, have a high immunoge nicity towards the given pathogen, and can be used as vaccines [48]. Positive results were obtained in experiments on the use of RAVNs containing the antigen determinant of the influenza virus hemagglutinin in the composi tion of capsid proteins (fiber, hexon and pIX). On lab oratory mice it was shown that introducing RAVNs containing different coating proteins of hemagglutinin epitope in the structure of the polypeptide chain causes the formation of a highlevel antibody response in animals [49]. The analysis of the possibility of using target RAVNs which can specifically interact with the anti genpresenting (dendritic) cells as a vaccine is now at the stage of laboratory research [50, 51]. The ability to induce the formation of Tcell immunity determined the development of a new type of RAVNbased drugs and a new method for treating oncology diseases: vaccine therapy. This approach has much in common with the mechanism of RAVN action against intracellular pathogens. Tumor cells produce specific antigens, presenting epitopes on their surface in a complex with the MNC I molecules. Such cells can be recognized and destroyed by correspond ing cytotoxic lymphocytes. The introduction of RAVNs expressing the tumor antigen into the organ ism leads, as in the case with antiinfective vaccines, to the formation of cytotoxic lymphocytes. Currently, over 50 clinical trials of the first and second phases are being conducted worldwide within the framework of researching the use of such vaccines in medicine (http://www.wiley.co.uk\genmed\clinical\) for treat ing bladder cancer, melanoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, chronic lymphocytic lymphoma, prostate cancer, etc. Perhaps in the future a new generation of vaccines will be created on the basis of RAVNs, and their effi cacy and safety will be provided by a combination of the genetic, physical, and chemical means of their engineering. For example, such vaccines can represent the DNA containing the expressing cassette with the genome of the infectious agent antigen in its structure; this cassette is enclosed by the coat of not only viral but also synthetic origin which (1) carries on its surface the antibodies against the corresponding pathogen, (2) prevents the rapid elimination of RAVN from the organism, and (3) promotes the specific interaction and penetration of RAVNs in antigenpresenting cells. The creation of vaccines with such properties can contribute to the intensification of the immune response to the corresponding pathogenic organisms. NANOTECHNOLOGIES IN RUSSIA
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CREATING RAVNS FOR NONINVASIVE DIAGNOSTICS Noninvasive diagnostic has a great potential. It can serve both for early detection and for the research of a clinical course of different diseases. It is of great importance, particularly with respect to the early find ing of various tumors and cardiovascular diseases. Today, in light of the discovery of a number of mole cules and ligands which can provide high specificity during the diagnostic measures, it became possible to investigate different processes in the organism on the molecular level in realtime. Many of these methods have been already introduced into medical practice: computed tomography, magnetic resonance, etc. RAVNs can be a perspective platform for the cre ation of materials and methods of noninvasive diag nostics. The purpose of engineering diagnosticori ented RAVNs is to detect certain type of cells in the organism. For example, RAVNs where one of the coat proteins is additionally modified with a fluorescent agent are generally used for this purpose. As a result, the entire RAVN is labeled. The first work dedicated to this strategy is based on the creation of RAVNs on the basis of human adenovirus serotype 5 with chimeric protein pIX containing the introduced green fluores cent protein gene [52]. The green fluorescent protein (GFP) is used as an intravital marker, making it possi ble to investigate different processes within living cells and organisms which were practically impossible to observe before. The in vivo results can be visualized using a cooled CCDcamera (CooledChargedCou pled Device Imaging). Target RAVNs carrying the DNA containing the expressing cassette with the gene coding the probe (for example, fluorescent protein) can also be used for diagnostic purposes. Currently the gene of fluorescent protein can be controlled both by the constitutive pro moter, which makes it possible to detect all the cells RAVNs were inserted into, and the inducible pro moter, which allows detection not only of a certain type of cells, but also the violations taking place in them. This method has been proven on laboratory ani mals. As a result, it was shown that this method helps visualize a tumor and trace the process of its develop ment [53]. Another approach of creating RAVNs for noninva sive diagnostics lies not only in the specific detection of cellstargets, but also in delivering the necessary therapeutic agents to them. This approach can be widely applied in oncology for the selective influence of chemotherapeutic drugs on malignant tissues in combination with noninvasive diagnostics of the effi ciency of such an influence. Chemotherapeutic drugs currently in use affect not only malignant neoplasm cells, but also healthy tissues and organs. Currently RAVNbased drugs which make it possible to over come this problem are being clinically tested. For example, RAVNs that have been created for the treat
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Table 2. The statistics of clinical trials in the area of genetic therapy according to the data of The Journal of Gene Medicine (http://www.wiley.co.uk/genmed/clinical/). (a) The number of clinical trials including RAVNs and (b) total amount of clinical trials Diseases
The phase of clinical trials
Cancer
Cardiovascular
Infectious
Monogenic
Neurological
Others
I I/II II II/III III
169a/580b 44/177 43/163 6/7 9/33
25/59 6/30 15/36 4/6 2/6
7/63 0/27 2/16 0/0 0/6
16/85 2/32 0/3 0/0 0/0
1/7 0/5 0/5 0/0 0/0
4/31 0/4 1/12 0/0 0/2
ment of the prostate cancer state T1c are now going through the first phase of clinical trials. The genes of two ferments—thymidine kinase of herpes simplex virus (HSV1TK) and natrium iodine symporter (hNIS)—were inserted into the DNA of these RAVNs. The gene of natrium iodine symporter is used for to visualize the therapy results. When the radioac tively labeled substrate (sodium pertechnetate 99m Tc) is inserted into the organism, the symporter expressed in the cell that RAVN penetrated helps 99mTc penetrate into it. The localization of sodium pertechnetate in the organism is detected by Single Photon Emission Computed Tomography (Spect). Thymidine kinase is the ferment phosphorylating the thymidine and transforming it into a monophosphate, which is further phosphorylated by other kinases until triphosphate and used in the process of DNA synthe sis. RAVNs expressing HSV1TK and hNIS are intro duced directly into the tumor and penetrate into the cells, where the expression of target genes occurs. Two days after that, patients orally take a drug (gencyclo vir). Gencyclovir is a substrate for thymidine kinase of the herpes simplex virus; cellular thymidine kinases can not phosphorylate it. After gencyclovir phospho rylation by the ferment HSV1TK, the additional phosphorylation of its monophosphate by other intra cellular ferments takes place and it is inserted into the DNA structure, leading to the termination of the nucleotide chain formation at the time of cell division. The chain opening accompanied by the DNA synthe sis leads to cell destruction. It is known that tumor cells have a higher divisibility. Thus, the selective destruction of tumor cells is achieved. To visualize the therapy results with the help of RAVNs (coding HSV1TK and hNIS), patients intravenously take sodium pertechnetate directly before analysis. As a result of clinical trials on the use of RAVNs, which car ries both the therapeutic (HSV1TK) gene and the gene for visualizing therapy results (hNIS) in their DNA, the inhibition of the development of a prostate tumor at stage T1c was detected by the Spect method. It was also shown that this method of noninvasive diagnostics is effective and safe [54].
CONCLUSIONS In conclusion, on the basis of foregoing material concerning the engineering of different RAVNs, we can name their common characteristics. RAVNs are nanoparticles created on the basis of an adenovirus by means of its genetic and/or chemical modification. They are incapable of independent existence during the full life cycle (which is typical for original adenovi rus in nature) for the maintenance of a given structure. All RAVNs (with rare exceptions) possess the follow ing properties: (i) their introduction into the organism does not lead to the development of adenovirus infection with its typical symptoms; (ii) the genetic material of a RAVN (or its part) does not integrate in a cell genome; (iii) RAVN are completely eliminated from the organism in 4–5 weeks. The listed properties of this type of RAVN makes it possible to say that they are safe for humans and, con sequently, that they are highly prospective for use in medicine. This has been proven by the statistical data of clinical trials in the area of genetic therapy. A quar ter of all research work is carried out with the use of RAVNs (Table 2). Today, many different RAVNs have been created. Engineering a new type of RAVN is always strictly dic tated by the purpose of its further application. The combination of the physical, chemical, and genetic modification methods during engineering makes it possible to create more effective RAVNs for achieving different medical purposes. In the future perhaps it will be possible to create polyfunctional RAVNs with a whole complex of properties, including not only the ability to diagnose and treat different diseases, but also making it possible to observe the course of therapy and its efficiency. REFERENCES 1. R. Singh and K. Kostarelos, “Designer Adenoviruses for Nanomedicine and Nanodiagnostics,” Trends Bio technol. 27 (4), 220–229 (2009). 2. T. Rodrigues, M. J. Carrondo, P. M. Alves, and P. E. Cruz, “Purification of Retroviral Vectors for Clin
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