Available online at www.sciencedirect.com ScienceDirect Evolution of oncolytic viruses 1 2 Rafael Sanjua´ n and Valery Z Grdzelishvili Owing to their replicative capacity, oncolytic viruses (OVs) can genetically engineered OVs have been created in which evolve under the action of natural selection. Reversion to virulence genes have been deleted; the viral tropism or the virulence and recombination with wild-type strains may ability to escape premature neutralization has been reset by compromise OV safety, therefore requiring evolutionary risk modifying viral envelope proteins; and viruses have been assessment studies. On the other hand, evolution can be armed with tumor suppressor or suicide genes expressed directed in the laboratory to create more potent and safer OVs. only in cancer cells, with immunostimulatory genes, or with Previous work in the experimental evolution field provides a genes that increase susceptibility of infected cells to chemo background for OV directed evolution, and has identified and radiotherapy [1–3]. interesting exploitable features. While genetic engineering has greatly advanced the field of oncolytic virotherapy, this These approaches have undeniably led to important approach is sometimes curtailed by the complexity and advances, but have also met some difficulties. Virus–host diversity of virus–host interactions. Directed evolution provides interactions are extremely complex and often still poorly an alternative approach that may help to obtain new OVs understood, and therefore our ability to manipulate them without prejudice toward the underlying molecular is limited. This is particularly challenging within the mechanisms involved. context of virus–tumor cell interactions, as tumor cells Addresses vary widely depending on the cancer type and between 1 Instituto Cavanilles de Biodiversidad y Biologia Evolutiva and and within patients [4]. As a result, the rational design of Departament de Gene` tica, Universitat de Vale` ncia, Valencia, Spain OVs for each specific tumor becomes a formidable task. 2 Department of Biological Sciences, University of North Carolina at Additionally, extensive manipulation of viruses often Charlotte, Charlotte, NC, USA renders them so attenuated, that, in many cases, it abro- Corresponding author: Sanjua´ n, Rafael ([email protected]) gates replication. Although strong attenuation is desirable for safety, many OVs have proven to be insufficiently potent against tumors. A unique feature of OV, though, is Current Opinion in Virology 2015, 13:1–5 that, unlike other therapeutic agents, they replicate and This review comes from a themed issue on Oncolytic viruses mutate, and therefore have the potential to evolve. This has two important implications. First, OVs are amenable Edited by Grant McFadden and John Bell to optimization by directed evolution. This approach can help improve the efficacy of previously engineered virus- es, and may allow creation of new OVs by acting on still http://dx.doi.org/10.1016/j.coviro.2015.01.014 uncharacterized molecular pathways. Second, evolution 1879-6257/# 2015 Elsevier B.V. All rights reserved. of therapeutic viruses in vivo, once delivered to patients, needs to be seriously assessed and, particularly, the probability of reversion to virulence. Directed evolution to generate more effective OVs Introduction Directed and experimental evolution has been used for Oncolytic virus (OV) therapy uses replication-competent practical applications in various research areas. For in- viruses to selectively target malignant cells, in contrast to stance, pioneer procedures for creating oral polio vaccines virus-based gene therapy which uses replication-deficient included serial passages in non-human hosts [5]. In light viruses. Despite a large number of preclinical studies with of today’s knowledge, this probably favored adaptation to impressive results that have led to several OVs being the alternate hosts at the cost of reduced fitness in currently tested in human clinical trials, some critical humans and promoted the accumulation of deleterious challenges need to be addressed, including insufficient mutations by random genetic drift, thus making these selectivity for tumoral cells, low oncolytic potency, inabili- basic evolutionary processes instrumental in the success ty to penetrate and spread in tumor tissues, premature viral of polio vaccines. Evolutionary studies have also been clearance, and poor stimulation of tumor-specific immuni- used to predict the appearance of drug resistances in HIV- ty. Many efforts have addressed these issues. Due to the 1 [6], or the pathogenesis and pandemic potential of increasing availability of techniques for the genetic ma- influenza viruses [7]. nipulation of animal viruses during the last decades, today the field is dominated by an engineer’s view based on Using modern experimental evolution techniques, it is the rational modification of viral genomes. Hundreds of possible to adapt viruses to cells showing the hallmarks of www.sciencedirect.com Current Opinion in Virology 2015, 13:1–5 2 Oncolytic viruses cancer, to characterize the genetic and molecular basis of cells increased viral fitness [8]. Another example is serial this adaptation, and to subsequently evaluate the useful- passages of wild-type VSV in human glioblastoma cells ness of these OVs in cell cultures and animal models with the aim of promoting selective attachment to these (Figure 1). However, this approach has not been system- cells and replication [11]. Interestingly, the evolved virus atically applied in the field, albeit a few notable excep- was later found to be effective also against other tumor tions [8,9 ,10,11,12 ,13,14 ]. In one study, pools of cell lines [12 ]. Recently, VSV was adapted to MEF different adenovirus serotypes were serially passaged in p53À/À cells by serial passaging and then tested in cultures of human colon cancer cells, favoring among- isogenic p53+/+ cells, as well as in p53-positive and serotype recombination and the selection of the fittest p53-negative tumors in vivo [14 ]. This revealed gene- variants in these cells [9 ]. This led to isolation of a specific adaptation, suggesting that VSV can be selec- recombinant virus (ColoAd1) that outperformed existing tively adapted to a broad cancer feature such as p53 oncolytic adenoviruses and was approved for phase I/II inactivation. clinical trials. A limitation of using directed evolution with double-stranded DNA viruses, though, is their low Relevant factors in directed evolution rates of spontaneous mutations compared to RNA viruses experiments [15] thus making the production of new potentially adap- Owing to their extremely high rates of spontaneous tive variants slower. However, DNA virus variability can mutation, RNA viruses are ideal candidates for directed be enhanced using chemical mutagenesis [13] or by evolution. This, combined with the often high titers engineering viral polymerases with reduced replication achieved under cell culture conditions, increases the fidelity [10]. efficacy of selection and allows for deterministic evolu- tion of fitness-related traits in the laboratory [16]. Addi- There are also a few examples of directed evolution of tionally, the genomes of most RNA viruses are less than RNA viruses in the context of oncolytic virotherapy. In 20 kb, making it easy to identify mutations responsible for one study, a pseudotyped vesicular stomatitis virus (VSV) adaptation. However, with the advent of next-generation was engineered to express a single-chain antibody against sequencing, the genetic analysis of large oncolytic DNA the Her2/neu receptor (ErbB2). Although the engineered viruses such as vaccinia or herpes viruses has been greatly virus initially showed a very low titer in target mammary facilitated, allowing direct identification of the molecular cancer cells expressing ErbB2, serial passages in these basis of adaption in these viruses as well [17]. Using Figure 1 (a) Directed evolution (b) Phenotypic assays ... line 1 founder evolved virus target tumor cells virus line 2 growth toxicity ... (c) Sequencing of replicate evolution lines (d) Oncoselectivity line 1 line 2 tumor ... cells normal cells beneficial (driver) non-beneficial viral fitness mutation (passenger) mutation virus genotype Current Opinion in Virology Directed evolution of oncolytic viruses. (a) Starting from a founder virus, which can be the wild-type or a previously engineered virus which has to be optimized, serial transfers can be performed in target tumor cells under conditions that favor the action of selection (high effective population size, low multiplicity of infection). The duration of this process can vary depending on the type of virus (RNA/DNA) and the strength of the selective pressure, but should typically range 10–50 transfers. The repeatability of evolution can be assessed by establishing several replicate lines. (b) The growth rate, toxicity, or other interesting phenotypic properties can then be evaluated for the evolved virus and compared against the founder. (c) Sequencing of replicate evolution lines can help identify relevant mutations responsible for the observed increases in viral fitness (driver mutations), since these mutations typically appear in more than one line (parallel evolution). (d) In many cases, adaptation to a specific cell type (here a given tumor cell line) is accompanied by a loss of fitness in other cell types (here, normal cells). These fitness tradeoffs should increase the oncolselectivity of the evolved viruses. Current Opinion