Tissue-Specific Attenuation of Oncolytic Sindbis Virus Without Compromised Genetic Stability

Abstract

Wild-type Sindbis virus (SV) shows promise as an oncolytic agent, although potential off-target replication is a safety concern. To remove possible pathology reflecting virus replication in liver, muscle, and/or hematopoietic cells, microRNA (miR)-response elements (MREs) to liver-specific miR122, muscle-specific miR133a and miR206, or hematopoietic-specific miR142-3p were inserted into the Sindbis viral genome. We compared the effectiveness of MREs in two distinct genomic locations and found better tissue-specific attenuation when they were inserted into the structural polyprotein coding region (up to 6000-fold selectivity with miR142-3p) rather than into the 3' untranslated region (up to 850-fold with miR142-3p). While this degree of tissue-specific attenuation may be effective for relieving pathology in vivo, genetic instability of RNA viruses raises concerns over the mutation or loss of MREs conferring safety. Genetically modified SVs containing a reporter transgene, used as a surrogate for virus replication, mutated quickly in vitro, losing 50% transgene sequence within 6.2 passages. Using a shorter insert containing MREs but no transgene, complete genetic stability was observed over at least 10 passages. We conclude that SV may be genetically modified to improve clinical properties, but attention must be paid to ensure that genetic stability is sufficient for intended applications.

CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo

“…CD62L expression characterises populations of T cells that possess lymphoid homing capability and is routinely used to identify naïve and effector memory T cells (T EM ). However, CD62L expression on leukocytes is highly susceptible to freeze-thaw damage [ 30 ] and there is the suggestion that CD62L expression on CD4 + T cells is associated with a T h 2 cytokine profile rather than preferred T h 1 profile for cancer immunotherapy [ 31 ].…”

MicroRNA-based Regulation of Picornavirus Tropism

“…Optimizing these will require trial and error. However, rational design based on RNA structural analysis and previous studies of viral replication and microRNA signatures aids in the implementation of this technique with minimal optimization 10,11,12,13,38 .…”

MicroRNAs and oncolytic viruses

“…Targeting of a single gene may not be sufficient and control of various genes simultaneously may be warranted. Location within the 3 0 UTR may not be optimal for target insertion and enhanced control may be achieved by incorporating the miRNA target within the open reading frame of the gene as was demonstrated for Sindbis virus [59]. Finally, the miRNAs chosen may not be optimal.…”

“…Finally, the insertion site is a major factor in dictating suppressive activity. While most MRE elements are found within the 3 0 UTR of mRNAs, relocation to the 5 0 UTR or the open reading frame of a gene or genome can be more efficacious [59]. Insertion sites need to allow high accessibility of the miRNA target, which can be hindered by secondary structures normally formed by the virus or additional structures introduced by the incorporated target sequences.…”

“…The first is the potential evolution of escape mutants, which has been demonstrated with miRNA-targeted CVA21, poliovirus, and Dengue virus [36,45,62]. Using perfectly complementary target sequences in tandem and at various stable locations throughout the genome decreases the potential for the evolution of escape mutants [59,61]. Additionally, MREs are differentially effective and susceptible to different selective pressures to generate escape mutations, which may be target and/or tissue-specific [63].…”