Attenuation of Semliki Forest Virus Neurovirulence by MicroRNA-Mediated Detargeting

Abstract

cArtificial target sequences for tissue-specific miRNAs have recently been introduced as a new means for altering the tissue tropism of viral replication. This approach can be used to improve the safety of oncolytic viruses for cancer virotherapy by restricting their replication in unwanted tissues, such as the liver. Semliki Forest virus (SFV) is a positive-strand RNA virus and, similar to the related alphaviruses, like Sindbis virus, has potential as a gene therapy vector and an oncolytic virotherapy agent, but this potential is limited by the neurovirulence of these alphaviruses. Here, we have generated a replicative SFV4 carrying six tandem targets for the neuron-specific miR124 between the viral nonstructural protein 3 and 4 (nsp3 and nsp4) genes. When administered intraperitoneally into adult BALB/c mice, SFV4-miRT124 displayed an attenuated spread into the central nervous system (CNS) and greatly increased survival. Peripheral replication was not affected, indicating neuron-specific attenuation. Moreover, a strong protective SFV immunity was elicited in these animals. Intracranial infection of adult mice with SFV4-miRT124 showed greatly reduced infection of neurons in the brain but led to the infection of oligodendrocytes in the corpus callosum. Taken together, our data show that miR124-mediated attenuation of neurovirulence is a feasible and promising strategy for generating safer oncolytic alphavirus virotherapy agents. M icroRNAs (miRNAs) are small noncoding RNA molecules that have important regulatory roles in gene expression by targeting mRNAs for cleavage or translational repression. Incomplete sequence complementarity between the miRNA and the target mRNA can induce translational repression without mRNA degradation, whereas a higher degree of sequence complementarity leads to catalytic degradation of the target mRNA (1). The human genome encodes more than 1,000 miRNAs, and over 50% of cellular mRNAs have been estimated to be under miRNA regulation (2). miRNAs have important regulatory roles in development, cell proliferation, differentiation, apoptosis, and stress responses, explaining why dysregulation of miRNA expression is associated with many diseases, most notably cancer (3). miRNA targeting has recently been used to modify replicative tropism of both RNA and DNA viruses (4-10). By tissue-specific targeting the expression of viral genes that account for viral replication/ pathogenicity, it is possible to generate rationally attenuated live vaccine viruses and safer oncolytic viruses for the treatment of cancer. For example, Edge et al. developed a tumor-specific vesicular stomatitis virus (VSV) by inserting 3 copies of Let7 miRNA target elements in the 3= untranslated region of the VSV genomic RNA (5). Likewise, by inserting four copies of muscle-specific miRNA target elements into the coxsackievirus A21 genome, Kelly et al. could prevent lethal myositis developing in mice infected with the miRNA-targeted coxsackievirus A21 without compromising the tumor cell-killing potency of this virus (7)....

Safe and Effective Treatment of Experimental Neuroblastoma and Glioblastoma Using Systemically Delivered Triple MicroRNA-Detargeted Oncolytic Semliki Forest Virus

“…2B, G-J). This finding is in accordance with previous reports, where miRT124 attenuated SFV4 was used (20) and confirms that IFNb secretion depends on the efficiency of virus replication in the brain (38).…”

“…Previously, miRT124-tagged SFV4 was shown to possess significantly reduced neurovirulence (16,20). But, a few mice still developed severe neurological symptoms, with virus replication detected in the oligodendrocytes of corpus callosum and spine (16,20). The triple miRT de-targeted SFV4 virus (SFV4miRT) presented herein had attenuated replication in mouse brain cells specifically neurons, and oligodendrocytes (Fig.…”

“…Most HGCC lines also had low expression of these miRNAs, which makes these miRNAs suitable targets to be used to attenuate virus replication in normal CNS cells however, expression profiles in healthy human brain cells need to be confirmed before clinical translation. Previously, miRT124-tagged SFV4 was shown to possess significantly reduced neurovirulence (16,20). But, a few mice still developed severe neurological symptoms, with virus replication detected in the oligodendrocytes of corpus callosum and spine (16,20).…”

“…miRNA expression is celltype restricted, which has been taken advantage of when designing oncolytic virus to prevent their replication in healthy tissues (17)(18)(19)(20). This is achieved by introduction of miRNA target sequences (miRT) in the viral genome, leading to that viral messenger RNA (mRNA; in the case of positive stand RNA viruses it is also genomic RNA) degradation in cells where the matching miRNA are expressed.…”

MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma

“…However, the neurotoxicity of virulent alphaviruses precludes use of them as oncolytic agents. In this regard, we previously demonstrated that the replication of virulent SFV4 in neurons can be inhibited by inserting into the viral nonstructural genome multiple target sequences for the micro-RNA (miRNA) miR-124 (SFV4-miRT124) (17). On the other hand, the miR-124 target sites do not interfere with the expression of viral genes in cells lacking miR-T124 expression (17), notably gliomas (18).…”