Oncolytic Herpes Simplex Virus

Oncolytic herpes simplex virus (oHSV) is one of the oncolytic viruses being studied in cancer therapeutic research and several oHSVs have been investigated in clinical trials. With years of experience in immunology and oncology, Creative Biolabs has established a comprehensive platform called OncoVirapy™ for providing oncolytic virus development service. By virtue of OncoVirapy™, we are able to design, engineer and generate the most efficacious oncolytic herpes viral vector and viral particles.

Brief Introduction of Oncolytic HSV

HSV is a double-stranded DNA virus belonging to the Herpesviridae family. There're two existing variants of HSV, including HSV type 1 (HSV-1) and HSV type 2 (HSV-2). Among these two subtypes, HSV-1 has been widely investigated in cancer oncolytic viral therapy. HSV-1 contains a double-stranded DNA (dsDNA) genome of about 152 kb, which is arranged by two unique sequences: Unique Long (UL) and Unique Short (US), an icosadeltahedral capsid, an amorphous tegument, and an outer lipid bilayer envelope with glycoprotein spikes for viral entry. Some characteristics including large foreign genes capacity (estimated to be about 30 kb), neural sensitivity tropism and high titers of manufacture enable HSV-1 to be used as a viral-delivery candidate for cancer treatment. Although it is best known as a neurotropic virus due to its latency in neurons, HSV-1 actually can infect a wide variety of cell types and thus tumor types. Talimogene laherparepvec (T-Vec) was an oncolytic HSV that was first approved for the melanoma treatment by the Food and Drug Administration (FDA) in October 2015, and then successively approved in Europe, Australia, Switzerland, and Israel.

HSV-1 Gene Mutations in Oncolytic Virotherapy

• Safety

HSV-1 can be genetically-engineered or significantly attenuated to confer safety by deleting or mutating some virulence genes.

• Tumor targeting specificity

HSV-1 genome has many genes that can be deleted, replaced or mutated to confer tumor-targeting specificity through tumor-selective receptor binding and transcriptional- or post-transcriptional miRNA-targeting, respectively.

• “Armed” with transgenes

HSV can be “armed” with transgenes that are: reporters, to track virus replication and spread; cytotoxic, to kill uninfected tumor cells; immune modulatory, to stimulate antitumor immunity; or tumor microenvironment altering, to enhance virus spread or to inhibit tumor growth.

Fig.1 Map of HSV-1 genome and schematic representation of essential genes and non-essential genes. (Artusi, 2018)

Cases of oHSV Genetic Modifications

Oncolytic virotherapy with mutants derived from HSV-1 exhibits significant antitumor effects in preclinical models. Several mutants have now been tested in clinical trials for a variety of cancer types, such as colorectal cancer, melanoma and osteosarcoma. Excitingly, some mutants have been found to be safe and effective. Table.1 listed several common genetic alterations of oHSVs.

Table.1 Common genetic modifications of oncolytic HSV.

• Thymidine kinase/UL23 (E)

HSV-1 thymidine kinase (TK) is a multifunctional enzyme that catalyzes the creation of deoxythymidine 5’-phosphate (dTMP) from deoxythymidine, as well as phosphorylating deoxycytidine and nucleoside analogs, creating precursors for viral DNA synthesis. Loss of TK activity halts HSV-1 replication in nondividing cells, making TK mutants safer than wild type HSV-1. Dlsptk, an HSV-1 lacking TK, was the first genetically-engineered oncolytic virus devised to kill tumor cells while sparing normal cells.

• ICP6/UL39 (E)

ICP6 is the large subunit of the viral ribonucleotide reductase, which transforms ribonucleotides into deoxyribonucleotides (dNMP) by reducing the 2’-COH group. ICP6 is essential for HSV replication in noncycling cells, making ICP6-null oHSVs selective for dividing cells.

• γ34.5 (ICP34.5)/RL1 (L)

γ34.5, one of the most frequently mutated genes for creating oHSVs, has many functions. HSV-1 is diploid for the γ34.5 gene, and mature virions contain the protein in their tegument in order to deliver it upon entry. Attenuation of both copies of this gene leads to a reduction of neurovirulence associated with HSV-1 in vitro and in vivo. Despite the reduced replication in normal cells, HSV-1 virions deleted for ICP34.5 efficiently replicate in and are cytotoxic to a majority of glioma cell lines and primary tumor-derived cells.

• ICP47

ICP47 deletion enhances anti-tumor efficacy while retaining safety characteristics. G47Δ showed efficacy in all solid tumor models tested in vivo, such as hepatocellular carcinoma, schwannoma, prostate cancer, nasopharyngeal carcinoma, glioma, thyroid carcinoma, colorectal cancer, breast cancer and malignant peripheral nerve sheath tumor.

• ICP0 (IE)

ICP0 encodes a RING finger ubiquitin E3 ligase that targets cellular proteins. ICP0-null HSV-1 is extremely sensitive to IFN and PML-mediated disruption of the viral lifecycle. ICP0-null HSV-1 is extremely sensitive to IFN and PML-mediated disruption of the viral lifecycle.

• UL56 (L)

UL56 is another gene associated with pathogenicity and neuroinvasiveness. UL56 is a good candidate for mutation when creating a safer oHSV. In addition, it may contribute to tumor selectivity.

Featured Services and Construction Workflow

We provide a comprehensive range of HSV services based on our advanced OncoVirapy™ platform.

• GOI cloning (e.g., antibody, bispecific antibody, cytokine, chemokine, shRNA or gRNA) and confirmation through restriction mapping and sequencing verification
• Functional validation of transgenes
• Expression cassette design with the most appropriate promoter to guarantee the high-level expression
• Viral genes engineering (single gene or different genes combination)
• Different construction strategies (homologous recombination, BAC system, etc.)
• HSV amplification, purification, titering services
• HSV whole genome sequencing (WGS) service

With years of experience and continuous optimization, Creative Biolabs has developed a systematic workflow for oncolytic HSV construction as follows. In addition to the standard construction workflow, we also developed bacterial artificial chromosome (BAC) cloning technology, which significantly advances the capacity of herpesvirus researchers to manipulate virus genomes.

Fig.2 Workflow of oncolytic HSV construction at Creative Biolabs.

Oncolytic HSV has been widely used in pre-clinical and clinical cases; moreover, there’s hope that its curative effect will be further enhanced through the combination of oHSV with both traditional and emerging therapeutics. With the development of modern genetic engineering techniques, more and more strategies have been discovered to optimize the construction of oncolytic HSV, to reduce toxicity, as well as increase the specificity and efficacy. Creative Biolabs offers a broad range of custom oncolytic HSV cloning and construction services at a reasonable cost and with quick turnaround time. Besides, we also provide in vitro and in vivo validation services for engineered oncolytic HSV to facilitate your whole project. 

Reference

1. Artusi, S.; et al. Herpes simplex virus vectors for gene transfer to the central nervous system. Diseases. 2018, 6(3): 74.

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