Oncolytic potential of autonomous parvoviruses

Key words: Background . In many countries, more than 40 oncolytic viruses from 10 different families are undergoing clinical trials (phase I, II, III) against various types of cancer; some of them have already been approved for use. The results of the experimental studies that deal with the development of oncolytic drugs based on autonomous parvoviruses deserve special attention. Purpose . The purpose of the study was to analyze and summarize literature sources that are concerned with the development of oncolytic drugs based on autonomous parvoviruses. Materials and Methods . Publications were selected using keywords in PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Google Scholar (https://scholar.google.com/) databases published over the period from 2010 to 2023. Results . Among autonomous parvoviruses, protoparvoviruses are the most promising for the development of anticancer drugs. They detect CPE mainly in tumor cells, CPE is absent or limited in non-transformed human and rodent cell lines. Many of them are capable of actively replicating in cancer cells without destroying normal body cells. They primarily block the pathways of activation of type I interferons. The main non-structural viral protein NS1 plays a significant role in the manifestations of oncolytic activity of protoparvoviruses. protoparvoviruses, erythroparvoviruses, cancer, oncolysis, vectors.

Parvoviruses are of great interest for the creation of highly effective oncolytic drugs.They are the smallest (18-22 nm) spherical DNA viruses of animals and humans, the reproduction of which depends on the transition of infected cells to the S-phase of the cell cycle [7,8].According to the latest data of the International Committee on Taxonomy of Viruses (ICTV), all of these viruses (134 species) belong to the Parvoviridae family which includes two subfamilies -Parvovirinae and Densovirinae [7].With the aim of developing oncolytic drugs for the treatment of patients among the existing eight genera of the Parvovirinae subfamily, oncologists and virologists are most interested in representatives of two genera of parvoviruses: Protoparvovirus (H-1 and other rodent viruses) and Erythroparvovirus (B-19), which, unlike the only genus in this subfamily (Dependoparvovirus), are autonomous, meaning that their reproduction does not depend on the auxiliary functions of the helper virus.Using metagenomic analysis, Chinese researchers described two new parvoviruses in California sea lions and wild rats.They propose to assign these parvoviruses to a new genera of the Parvovirinae subfamily under the names Marinoparvovirus and Chapparvovirus [9].However, the oncolytic potential of these new parvoviruses has not yet been established.
The objective of the study was to summarize the information of the use of autonomic parvoviruses as oncolytic drugs.
Publications were selected using keywords in PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Google Scholar (https://scholar.google.com/)databases published over the period from 2010 to 2023 that covered information on the development of oncolytic drugs based on autonomous parvoviruses.

Oncolytic protoparvoviruses
Most of the information about the oncolytic properties of parvoviruses was obtained on the model of protoparvoviruses.Although they were often isolated from tumor cells in the past, in vitro and in vivo studies have shown that they do not have any tumorigenic activity but, on the contrary, show the ability to inhibit tumor growth [10].The Protoparvovirus genus includes such rodent parvoviruses as minute virus of mice (MVM), rat parvovirus (H-1), murine parvovirus (MPV), Kilham rat virus, LuIII parvovirus, tumor virus X and other viruses that are oncolytic by their nature.The oncolytic effect of protoparvoviruses is not limited to rodents; canine transmissible venereal sarcoma (or Sticker's sarcoma) is inhibited by both virulent and vaccine strains of canine parvovirus (CPV), with virulent strains being more effective [11].Protoparvoviruses also include feline parvovirus (FPV) and several new protoparvoviruses isolated from horses, pigs, foxes, shrews, and sea otters [12].
The anti-cancer effect linked with parvoviruses was first discovered in the Tulane laboratory that reported in 1965 that hamsters persistently infected with protoparvovirus H-1 developed spontaneous tumors 5-25 times less often than control animals [13].Later studies showed that protoparvoviruses can affect various types of tumors that are induced experimentally in rodents and chickens both with the help of oncogenic viruses and chemical carcinogenesis [10].
Protoparvoviruses exert cytopathic effect (CPE) mainly in tumor cells; in non-transformed human and rodent cell lines, CPE is absent or limited.Many of them are capable of actively replicating in cancer cells without destroying normal body cells.They primarily block the pathways of activation of type I interferons, especially in transformed fibroblasts [14].Moreover, the main non-structural protein NS1 plays a significant role in the manifestations of oncolytic activity of protoparvoviruses [15].Compared to normal cells, the expression level of NS1 protoparvovirus protein is much higher in transformed cells.This protein affects various cell functions, and its cytotoxic effect may be caused by the influence on some regulatory proteins (P53, cyc-
Репродукція протопарвовірусів (H-1, MVM, та ін.) викликає імунну відповідь, яка пов'язана з виробленням інтерферонів I типу та блокує розмноження The parvovirus NS1 protein is a multifunctional phosphoprotein that contains 672 amino acids.It is mainly located in the nucleus, although a significant proportion of the protein remains in the cytoplasm.The C-end of this protein is responsible for the regulation of the P38 promoter, NS1 activity is modulated by post-translational modifications, such as phosphorylation and acetylation [18].Due to its ATPase and helicase activity, NS1 is the main regulator of viral DNA replication.For viral DNA replication, protoparvoviruses require cell proliferation because they are unable to induce the transition of cells from quiescence to S-phase entry.As soon as the cell enters S-phase, the single-stranded genome of protoparvoviruses is converted into an active double-stranded form available for transcription.The transformation of the singlestranded genome into an active double-stranded form depends on cellular factors, primarily the cyclin A/CDK2 complex which is specific for the S-phase and is usually expressed in proliferating cells.The virus finds the conditions for the successful implementation of genetic information in tumor tissues, i.e. protoparvoviruses are highly tumour-selective [16].One of the advantages of using oncolytic protoparvoviruses compared to other human oncolytic viruses is the absence of antibodies to these viruses in humans.Therefore, the later neutralizing antibodies appear, the better the result of virotherapy will be.Clinical studies have shown that treatment with protoparvoviruses is safe, well tolerated and does not cause undesirable side effects [16,17,19].
Reproduction of protoparvoviruses (H-1, MVM, etc.) triggers an immune response associated with the production of type I interferons and blocks the reproduction of these viruses in normal cells but not in

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Literature review cancer cells.Under the influence of protoparvoviruses, the presenting activity of dendritic cells increases, the antitumor activity of macrophages and cytotoxic lymphocytes is stimulated, and the activity of natural killer cells (NK) is significantly increased [1,3,8].
The oncolytic properties of protoparvoviruses are linked with many factors, primarily with apoptosis.In glioma cells, lysosome-dependent cell death under the influence of H-1 is linked with the movement of active cathepsins B and L (CTSB and CTSL) from lysosomes to the cytosol and the regression of two cathepsin inhibitors, cystatins B and C [8]. Cell death induced by protoparvovirus H-1 is mediated by NS1 through the accumulation of reactive oxygen species, which leads to oxidative stress, permeabilization of the outer mitochondrial membrane, DNA damage, cell cycle arrest, and activation of caspases [8].In addition, protoparvovirus-induced cell death is associated with several markers of immunogenic cell death, such as the release of high mobility group protein B1 (HMGB1) [18] and the immunogenic heat shock protein HSP72 [20].
Galectin-1 protein also plays a significant role in the process of oncolysis under the influence of protoparvovirus H-1 [21].
Cancer cell lines and primary cultures obtained from various tumors, including brain, pancreatic, breast, lung, cervical and colorectal cancer, melanoma, and osteosarcoma, are sensitive to oncolysis under the influence of protoparvoviruses [18].In recent years, clinical trials (phase I/II) of the oncolytic drug ParvOryx01, created on the basis of parvovirus H-1 at the German Cancer Research Center (DKFZ, Heidelberg) have shown its effectiveness against inoperable metastatic adenocarcinoma of the pancreas and recurrent glioblastoma multiforme [14,22].This drug proved to be safe for patients and showed immunogenic properties when administered intravenously, as well as when injected directly into the tumor.

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Literature review deacetylase (HDS) inhibitor, the oncolytic activity of protoparvovirus H-1 in cancer cell lines derived from cervical and pancreatic carcinomas is also increased [24].This synergistic anti-cancer effect is caused by the effect of VPA on increasing the degree of acetylation of the NS1 viral protein, which leads to an increase in protoparvovirus replication.Under the influence of VPA, there is also a significant increase in oxidative stress that is associated with the accumulation of intracellular reactive oxygen species and DNA damage [24].The combined use of H-1 together with the apoptosis inducer ABT-737 also leads to an increase in the oncolytic activity of H-1 in experiments on xenografted rats [10].The same effect is achieved when H-1 is used together with the anti-angiogenic antibody bevacizumab and the PD-1 checkpoint blocker nivolumab [22].A study of the effect of the drug ParvOryx02 on ductal adenocarcinoma of the pancreas (PDAC) conducted at the DKFZ showed that protoparvovirus H-1 was able to induce a virus-specific cellular immune response that led to tumour infiltration by cytotoxic T lymphocytes [14].These facts indicate a significant impact of the oncolytic drug on the creation of an immunogenic intratumoral environment.
To enhance the oncolytic activity of protoparvoviruses (H-1, MVM, etc.), methods of creating point mutations in genes and right-handed palindromes encoding both structural and non-structural proteins have been successfully used [25].An increase in oncolytic activity was also observed after multiple passages of H-1 in semi-permissive conditions, as well as after the introduction of the CpG dinucleotide into the non-translating region of the viral genome that usually enhances the immunogenic properties of viruses.After the introduction of the CpG segment into the H-1 parvovirus genome, a significant enhancement of the immunomodulatory effect was noted, which led to the suppression of most metastases in rats [26].
The use of protoparvovirus H-1 together with gamma interferon led to a significant increase (almost twofold) in the life span of rats with pancreatic cancer, which may indicate the induction of a more pronounced antitumor immunity [8,26].
Since no more than 250 nucleotides can be inserted into the small genome of protoparvoviruses to enhance their oncolytic activity without compromising their reproduction [16], it was proposed to use genetic engineering to create a chimeric virus based on adenovirus-associated virus 2 (AAV2) that contains the P38 promoter and the NS1 gene of the H-1 protoparvovirus.This made it possible to expand the tropism of the chimeric virus to various lines of cancer cells and equip it with immunostimulating agents [8,27].
Pioneers in the design of protoparvovirus vectors are the Maxwell couple from the University of Denver (USA), who together with the laboratory of Prof. Carlson (Fort Collins) created several transgenic vectors based on protoparvovirus Lu III [28].They demonstrated the packaging of a recombinant LuIII genome encoding a firefly luciferase or lacZ transgene into the LuIII protoparvovirus capsid.This vector mediated reliable gene expression in a variety of human cell lines, including HeLa and HepG2 [28].To identify a region in LuIII that gives it the ability to efficiently kill transformed human fibroblasts that are refractory to MVM, MVM/LuIII chimeras were constructed [28].These experiments showed that the region encompassing vp2 of LuIII was sufficient to promote productive cell infection.

Oncolytic potential of erythroparvoviruses
The genus Erythroparvovirus includes several viruses isolated from humans, cows, monkeys, macaques, seals, and chipmunks [7].This is a small group of parvoviruses with a unique tropism for erythrocyte precursors [29].The most studied is parvovirus B-19, which is the etiological agent of the so-called «fifth disease» [30].In Ukraine, parvovirus B-19 was detected for the first time in the blood sera of patients with chronic hemolytic anemia [31] and in nocturnal paroxysmal hemoglobinuria [32].There is little research on the oncolytic properties of erythroparvoviruses.There are reports from Indian clinicians who showed that 35 children with chronic lymphocytic leukemia (CLL) or acute lymphoblastic leukemia (ALL) whose sera contained antibodies to parvovirus B-19 had prolonged remission, and all of them survived [33].The mechanism of oncolytic effect of parvovirus B- 19 has not yet been thoroughly studied.It is known that the non-structural proteins (NS1) of human parvovirus B19, which are highly conserved among parvoviruses, promote apoptosis in erythroid cells through the activation of caspase-3 and caspase-10 [34].A small structural protein of parvovirus B19 with a molecular weight of 11 kDa also plays a significant role in the ability of erythroparvoviruses to affect apoptosis of erythrocyte progenitor cells.Unlike the NS1 protein, which is located in the nucleus of the infected cell, this protein is localized mainly in the cytoplasm.Its expression is almost 100 times more active than NS1 protein [35].
nant B19 virus has proven to be much more effective than other AAV-based recombinant vectors in transducing primary erythroid progenitor cells in mice and humans [37].Further improvement of this vector system based on erythroparvovirus B-19 may be useful in the application of cancer gene therapy to erythroid cell lines in the human hematopoietic system.
Numerous studies continue to be directed at studying the mechanisms associated with the oncolytic properties of autonomous parvoviruses.Oncolytic drugs ParvOryx01 and ParvOryx02 were developed on the basis of rodent protoparvovirus H-1 and are currently undergoing clinical trials against various forms of cancer.The use of recombinant parvoviruses allows their oncolytic potential to be enhanced.