Bioengineering of G47∆ HSV-1 Combined with Stem Cell Delivery as
an Alternative Virotherapy against Colon Cancer
Kexin Chen
1,†
, Qingqing Huang
2,†
, Mengzhuo Liu
3,*,†
, Ellah Thomas
4,†
, Krystal Wang
5
and Zihan Zhang
6†
1
Shenzhen College of International Education, Shenzhen, 518043, China
2
Department of Biological Science,National University of Singapore, 119077, Singapore
3
Kalamazoo College, Kalamazoo, 49006, U.S.A.
4
United World College of South East Asia, 1207 Dover Road, 139654, Singapore
5
Northfield Mount Hermon, Gill, 01354, U.S.A.
6
Wuhan Britain-China School, Wuhan, 430035, China
4 5 6
†These authors contributed equally this work and should be considered co-first authors
Keywords: Colon Cancer, Oncolytic Virus and Herpes Simplex Virus (HSV).
Abstract: Oncolytic virus (OV) therapy is a recently developed strategy in cancer treatment, especially towards patients
who are unresponsive to conventional therapies. Numerous viruses have been identified efficiently lysing
tumor cells both in vitro and in vivo and eliciting anti-tumor immunity, including but not limited to Herpes
Simplex Virus (HSV), adenovirus, and Newcastle Disease Virus (NDV). However, there are some caveats
with present OV therapies. The delivery of the virus to the human body and its replication before immune
response are essential to the effectiveness of the therapy. Therefore, to maximize the efficacy of existing OV
therapies, we propose a hypothetical herpes simplex virus (HSV)-based OV which combines several
engineered traits to tackle colon cancer. The re-designed virus conceivably limits HSV neutralization by pre-
existing antibodies. It is also conceivable that the engineered oncolytic virus can secrete chimeric molecules
that specifically bind to colon cancer cells. Also, we aim to activate neoantigen-specific T cell responses
through the synergy of PD-L1 inhibition, GM-CSF activation, and viral immunogenic oncolysis. We
hypothesize that a combination of OV therapies with the usage of mesenchymal stem cells (MSCs) as carriers
could enhance the overall efficacy in tumor cell targeting and systemic immune response stimulation. MSC
delivery is likely to aid in the migration of the engineered OV to tumor sites. In summary, the modifications
of HSV enable more effective injection of oncolytic viruses and more accurate binding with tumors,
improving therapeutic outcomes of existing HSV-based immunotherapies.
1 INTRODUCTION
1.1 Colon Cancer
Colorectal cancer develops in the colon or rectum. It
was estimated that 5 to 10 percent of colon cancers
are hereditary (UT Southwestern Medical Center.
2020). The risk of colorectal cancer increases
drastically due to aging and certain lifestyles; almost
90% of colorectal cancer cases result from dietary
contributions. More than 70% of colon cancers can be
prevented by simple dietary and lifestyle
modifications. Sigmoidoscopy and colonoscopy can
be used for the diagnosis of colon cancer, preventing
colon cancer mortality. There are multiple treatments
for colorectal cancer, including surgery of full
excision, chemotherapy, radiation therapy,
immunotherapy with checkpoint inhibitors, and
palliative care, etc (Cancer Net 2021). It is found that
there is recurrent deregulation of STING signalling
and loss of p-53 function in colorectal carcinoma.
1.2 Chemotherapy for Colon Cancer
Chemotherapy is treatment with anti-cancer drugs
like cytotoxins which travel through the bloodstream
and reach most parts of the body (American Cancer
Society 2020). It is often used to treat colorectal
1084
Chen, K., Huang, Q., Liu, M., Thomas, E., Wang, K. and Zhang, Z.
Bioengineering of G47 HSV-1 Combined with Stem Cell Delivery as an Alternative Virotherapy against Colon Cancer.
DOI: 10.5220/0011377800003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1084-1094
ISBN: 978-989-758-595-1
Copyright
c
 2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved

cancer. Chemotherapy will be used at different times
during treatment for colon cancer. For example,
neoadjuvant and adjuvant chemo are given to the
patients before and after surgery, respectively.
However, these drugs have strong side effects since
regular normal cells could also be damaged, resulting
in hair loss, sores, and infection (Cancer Net 2021)
1.3 Oncolytic Virus
Oncolytic viruses (OVs) are viruses engineered to
replicate in and selectively destroy cancer cells
(Chiocca, Rabkin 2014). The common principle of
OV therapy is to reduce or eliminate viral virulence
factors to prevent the viruses from replicating in
normal tissues while retaining the capacity to
reproduce within cancer cells and kill them.
There are many viruses have been studied to
become promising agents for cancer therapy, such as
type 1 herpes simplex virus (HSV), measles virus
(MV), oncolytic adenoviruses, Newcastle disease
virus (NDV), Zika virus and vesicular stomatitis virus
(VSV) (Zheng, Huang, Tong, Yang 2019). Oncolytic
HSV (oHSV) is widely used in clinical trials. For
example, the U.S. Food and Drug Administration
(FDA) approve the use of T-VEC in biological cancer
therapy, which is an HSV-based oncolytic virus
completed phase Ш clinical trial (Mondal, Guo, He,
Zhoub 2020).
Limitations exist in these current oncolytic
therapies. One limitation is the pre-existing
neutralization of the HSV-based oncolytic virus. Of
the more than 100 known herpesviruses, 8 routinely
infect only humans (Whitley 1996). Thus, the
oncolytic effect of this therapy is compromised. The
pre-existing antibodies neutralize the oHSV upon its
administration. The oncolysis is compromised and
the therapeutic effect is reduced.
In our study, we plan to employ G47∆, a third-
generation oncolytic herpes simplex virus type 1
(Sugawara, Iwai, Yajima, Tanaka, Yanagihara, Seto,
Todo 2020). As HSV has a large, double-stranded
DNA genome in its core (Whitley, 1996), it is suitable
for multiple gene insertions. G47∆ has three
mutations in the γ34. 5, ICP6 and α47 genes which
make the virus replicate only in dividing cells, such
as tumor cells (Cancer Treatments - from Research to
Application. 2019).
1.4 Hypothesis
To address the limitations in the current therapies, we
propose a new model of oncolytic virus design based
on some primary research articles. We plan to employ
the genome of G47∆ oncolytic virus. Modifications
will be made to the virus genome, including point
mutations on the genes that encode for antibody-
binding surface proteins of G47∆, insertion of gene
segments of specific chimeric molecules, PD-L1
inhibitor, and granulocyte-macrophage colony-
stimulating factor (GM-CSF).
We will use mesenchymal stem cells as a carrier
to deliver our oncolytic virus. Additionally, this
oncolytic virotherapy will be especially effective in
treating advanced cancer with suppressed STING
expression.
We hypothesize that using point mutations on
surface proteins, triple gene insertion, and
mesenchymal stem cell carriers will increase the
general accuracy of tumor targeting and oncolysis
efficacy for colon cancer.
In our research paper, we draw insights from six
primary research articles on the existing oncolytic
viral therapies. Following the summary of those
primary research articles, we will present our study
design of the proposed new OV model, methods and
anticipated results.
2 PRIMARY RESEARCH
2.1 Redirecting Innate Immunity to
Target Tumor Cells by Arming
HSV-based Oncolytic Viruses
A major obstacle of cancer immunotherapy is the
patients’ innate immunity. This problem has been
resolved by redirection of innate immune cells like
macrophages or NK (natural killer) cells (Fu, Tao,
Wu, Zhang 2020). This is achieved by arming the
HSV with secreted chimeric molecules. These
chimeric molecules consist of a tumor-associated
antigen (TAA) binding moiety at their N terminus
and protein L (PL) that binds to immunoglobulins
(Igs) at their C terminus. The binding ability of Igs to
PL exists among a variety of classes, such as IgM,
IgG, IgA, IgE, and IgD (Bjorck 1988). The binding
of Igs to the Ig-binding domains exposes Fc to the Fc
receptors on the surface of innate immune cells,
triggering them to attach to the tumor cells.
Oncolytic viruses, which were based on HSV-1
and HSV-2 were built in this experiment. They
inserted EGF-PL chimeric gene cassette into the
genome of Synco-2D, an oncolytic virus based on
HSV-1. FusOn-H2, an HSV-2-based oncolytic virus
was engineered with an insertion of the affibody-PL
chimeric gene cassette. Synco-4 and FusOn-PL were
Bioengineering of G47 HSV-1 Combined with Stem Cell Delivery as an Alternative Virotherapy against Colon Cancer
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developed from Synco-2D and FusOn-H2,
respectively. The results show that CT26-EGFR cells
(a kind of colon cancer cells) was tightly bind with
the supernatant containing EGF-PL. In addition, it
was found that SKOV3 (ovarian cancer) and MCF7
(breast cancer) cell lines expressed high and medium
levels of the human epidermal growth factor receptor
type 2 affibody (HER2), respectively. A murine colon
tumor model was used on mice, Synco-4 and FusOn-
PL treatments demonstrated a more efficient
therapeutic effect. This suggests that these chimeric
molecules successfully redirect the innate immunity
in attacking the specific tumor cells.
Moreover, this research also showed that
neoantigen-specific antitumor immunity can be
enhanced by the combination effect of the innate
immune responses and oncolytic virus, rather than the
virotherapy alone.
2.2 Third-Generation Oncolytic
Herpes Virus G47∆ Used in Human
Gastric Cancer
A research paper goes over how certain types of
cancer such as Scirrhous gastric cancer are resistant
to common treatments and how G47∆ could be a
potential solution (Sugawara, Iwai, Yajima, Tanaka,
Yanagihara, Seto, Todo 2020). G47∆ was developed
through a deletion mutation to the genome of HSV-1,
G207. This oncolytic virus has had success with
being inoculated into the human brain suggesting it is
a safe course of treatment.
Many clinical trials have been conducted
indicating that G47∆ has a higher success rate in
comparison to common courses of treatment for other
types of cancer such as chemotherapy radiation.
The G47∆ treatment can successfully modify
immunosuppressive molecules of the tumor
including regulatory T cells and macrophages which
reduces function. Through clinical trials, it has been
discovered that NK cells are significant in the earlier
phases as they are able to support healthy cells.
There is not enough data at the moment to support
the hypothesis that the G47∆ third generation mutated
oncolytic virus is able to become a successful cure to
Scirrhous gastric cancer however further testing of
high and low doses of treatment on tumors of
different sizes are being conducted on mice and
scientists are hopeful for more data soon.
2.3 STING Signaling in Cancer Cells
Stimulator of interferon genes (STING) is an adaptor
protein that mediates type I INF activation responsive
to cytosolic DNA ligands. During infection, STING
in infected cells senses the nucleic acids of
intracellular pathogens, thereby stimulating the
production of multiple interferons. It is observed that
STING expression is down-regulated more
frequently than up-regulated in advanced diseases
(Sokolowska, Nowis 2018), leading to poor
prognosis of cancer and less effective
immunotherapy (Kol et al 2021).
The oncolytic virus Talimogene laherparepvec
(T-VEC), which was approved for melanoma
treatment, has shown great efficacy in treating murine
tumors with high levels of STING expression.
However, in advanced melanoma, STING expression
is usually suppressed, rendering PD-1 blockade
therapy ineffective. Under these situations, a negative
correlation between melanoma cell STING
expression and sensitivity to T-VEC was found,
suggesting that T-VEC oncolytic treatment would be
particularly effective when treating advanced cancer
with lower STING expression and is stubborn to PD-
1 blockade therapy. It is shown that T-VEC can
recruit CD8+ T cells and induce a pro-inflammatory
gene expression, generating a systemic anti-tumor
immune response. T-VEC infection also promotes the
release of damage-associated molecular patterns;
since immunogenic cell death is signified by the
release of DAMP, which suggests that T-VEC
infection would induce ICD. In summary, T-VEC
could induce cytokine production, immunogenic cell
death, and induction of inflammatory gene expression
both in vitro and in vivo. Besides, a combinatorial
treatment of anti-PD1 and T-VEC has a great
potential to control systemic diseases with a higher
overall response rate and complete response rate (Sun
et al 2018).
Future studies can target other signalling
pathways such as Toll-like receptor (TLR) signalling
and investigate ways of optimizing intratumoral
delivery (Sokolowska, Nowis 2018).
2.4 Induction of Antitumor Innate and
Adaptive Immune Response by
Oncolytic Newcastle Disease Virus
A challenge most Oncolytic Virus (OV) therapies
face is to replicate in the human circulation for a
sufficient amount of time to achieve efficacy.
Usually, there are pre-existing immunities, causing
most OVs constricted to intratumoral delivery. A
promising solution to overcome this obstacle is the
utilization of the Newcastle disease virus (NDV).
Naturally, NDV does not infect humans, implying no
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pre-existing immunity, ensuring the engineered NDV
remains in the human circulation long enough.
By inserting granulocyte-macrophage colony-
stimulating factor (GM-CSF), MEDI5395, a
recombinant NDV, presents potent oncolytic and
immunostimulatory activities. In a previous study
(Burke et al 2020), MEDI5395 was shown to exhibit
characteristics of preferential virus uptake and non-
productive infection in myeloid cells, inducing
upregulations of cell surface activation markers and
releases of proinflammatory cytokines.
Consequently, DCs with NDV infection stimulate
higher levels of allogeneic T cell proliferation in
comparison with non-infected DCs. Furthermore,
infected myeloid cells in a co-culture system are
demonstrated to be virus vectors, transferring the
viruses to enter and reproduce in tumor cells, causing
cell death. Following cell lysis, the antigens are
released and cross-presented by the DCs, activating
specific autologous T cells.
MEDI5395’s capability to promote immune
responses suggests the potentials to tackle therapy
and anti-viral immunity resistance. However,
application of MEDI5395 will cause the upregulation
of the protein PD-L1. To add on, these experiments
are only tested in vitro, indicating the necessity of
further investigations, as well as a lack of
standardized protocol for the application of
MEDI5395 to patients (Schirrmacher, van Gool,
Stuecker 2019).
2.5 Tumor Neoantigen-Specific T Cell
Responses Activated by PD-L1
Inhibition
Tumor cells can only express a small proportion of
nonsynonymous mutations, which enables
neoantigen-specific T cell responses stimulation.
Besides, PD-1/PD-L1, as T cell checkpoint molecules
should be responsible for the immunosuppressive
tumor microenvironment in tumor cells, which
inhibits anti-tumor T cell responses. There was a
study designing an oncolytic vaccinia virus, co-
expressing a PD-L1 inhibitor and a GM-CSF, for
investigating the ability of engineered oncolytic
viruses in activating tumor neoantigen-specific T cell
responses (Wang et al 2020).
In the engineered oncolytic virus, IgG1 Fc was
fused with a murine soluble PD-1 extracellular
domain as a PD-L1 inhibitor (iPDL1). iPDL1 co-
expressed with murine GM-CSF, with the backbone
of a tumor-selective double-deleted oncolytic
vaccinia virus, with the deletion of thymidine kinase
and viral growth factor genes. Two recombinant
controls were generated: only with GM-CSF or
marker RFP expression. MC38 cell lines carried
oncolytic viruses inoculated into the C57BL/6 mice,
with PBS (negative control), anti-PD-L1 antibody
(positive control).
It was found that the newly engineered oncolytic
virus can produce PD-L1 inhibitors and bind to PD-
L1+ tumor cells and immune cells (Wang et al 2020).
In addition, the intratumoral injection of the (VV)-
iPDL1/GM promotes maturation of the dendritic cells
and presentation of neoantigen on tumor cells via PD-
1/PD-L1 inhibition, leading to active neoantigen-
specific T cell responses. Therefore, this study
indicates that the synergy of PD-L1 inhibitors, GM-
CSF, and viral replication activates neoantigen-
specific T cell responses in tumor cells, resulting in
effective tumor-specific oncolytic immunotherapy
(Wang et al 2020).
2.6 Delivery of Mesenchymal Stem Cell
by Oncolytic Virus and Prodrug
Activation in Colorectal Cancer
Therapy
Although oncolytic virus therapy has been used in
various clinical approaches, many factors have been
proven to prevent them from reaching the tumor sites.
A new strategy of OV therapy improved efficacy by
using the mesenchymal stem cells (MSCs) to deliver
the oncolytic viruses (Ho, Wu, Chen, Lin, Yen, Hung
2021). This ability of MSCs to target tumor cells
makes them suitable carriers for oncolytic viruses
(OVs). The MSCs can further undergo numerous
modifications to carry the viruses, improving tumor
homing, especially for p-53 pathway deficient cancer.
The MSCs are primed with trichostatin A (TSA)
under hypoxia conditions to maintain their properties,
then loaded with oncolytic viruses to encode an
enzyme called E. coli nitroreductase (NTR) for tumor
cell targeting (Ho, Wu, Chen, Lin, Yen, Hung 2021).
The primed MSCs can increase tumor tropism, which
makes tumor cells more susceptible for viral
infections and protects NTR from neutralisation of
the immune system. The priming also leads to
upregulation of CXCR4--a chemokine receptor,
further contributing to targeting accuracy since
CXCR4 is involved in tumor tropism for cancer
homing.
Furthermore, the gene-directed enzymes-prodrug
therapy (GDEPT), also known as suicide gene
therapy, was used together with the stem cell
delivered OV therapy. The mechanism included a
combination of the prodrug-activating enzyme gene,
here NTR, with a non-toxic prodrug (NTR+CB1954)
Bioengineering of G47 HSV-1 Combined with Stem Cell Delivery as an Alternative Virotherapy against Colon Cancer
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that induces apoptosis in tumor cells. The prodrug is
converted into cytotoxic metabolites to trigger
oncolysis and inhibit tumor growth without damaging
other vital tissues and organs.
2.7 Limitations
One limitation in the primary research is the
neutralization of HSV-based oncolytic viruses by the
pre-existing antibodies. HSV infection is pretty
common, as it was estimated that 67% people under
age 50 have HSV-1 infection globally (World Health
Organization 2020). This would result in high anti-
HSV seroprevalence in the general population. The
human immune system can respond in a short time.
Thus, the killing effect of the virus will be
compromised.
One major setback of using MSCs is that some of
their previously proved tumor-promoting properties
could lead to the opposite results when used with
OVs. Another limitation is that MSCs from different
tissues can produce varying results, thus it is difficult
to predict the effectiveness of this therapy on
different patients.
We decided to design a new hypothetical model
to minimize setbacks and optimize the
immunotherapy effects in oncolytic virus treatment.
3 STUDY DESIGN
3.1 Overview
As shown in Figure 1, the proposed oncolytic
virotherapy consists of three sections, engineering of
glycoprotein, triple gene insertions, and stem cell
carrier. In our design, we plan to use the genome of
G47∆, an HSV-based OV, and target specifically
colon cancer.
Figure 1: The schematic structure of the new model of
G47∆ oncolytic virus.
The first section of approaches is the engineering
of the glycoprotein of the G47∆. We plan to perform
point mutations on the gene encoding the binding site
of the glycoprotein of HSV. Thus, the pre-existing
neutralization by the immune system can be avoided.
Our proposal of triple gene insertion includes
insertion of genes encoding for special chimeric
molecules, PD-L1 inhibitors and GM-CSF, aiming to
enhance the killing effect of G47∆ and direct innate
immune cells. The last method in our proposal is the
stem cell carrier. Stem cell delivery can elevate the
accuracy of OV migration to tumor sites and reduce
the off-target issue.
The overall purpose is to improve the efficacy of
the existing oncolytic virotherapies. We want to
achieve this goal by combining the effective methods
of each and maximally reduce the limitations.
3.2 Approaches
3.2.1 Engineering of Glycoproteins to Avoid
the Pre-Existing Neutralization
One problem we aim to resolve is the pre-existing
immunity against HSV in human populations, which
poses a difficulty for the HSV to remain long enough
in the human body to achieve efficacy. According to
the National Health and Nutrition Examination
Survey conducted in 2015-2016, HSV-1 infects
47.8% of the U.S. population, and HSV-2 infects
11.9% of the U.S. population (McQuillan, Kruszon-
Moran, Flagg, Paulose-Ram 2018). To prevent the
virus from the swift elimination by the immune
system in these populations, we first proposed the
solution of transferring the genes encoding the
protein coating of the NDV onto HSV. As humans are
not natural hosts of the NDV, if the engineered HSV
expresses NDV surface proteins, it will endure longer
in the human body to pass down genetic information.
However, this plan has considerable flaws. Firstly,
the execution of this process will be challenging.
Similar efforts have been made without great success
(Davidsson et al 2019). As changing the entire
genetic makeup of the protein coating is a difficult
and inefficient task. Secondly, excessive gene
modifications will drop overall efficacy of the virus,
impacting other genes that we want to implement on
this virus. Thirdly, glycoproteins are encoded by the
essential genes of HSV (Nishiyama 2004), and a
complete altercation of its protein coat could affect
the virus’s ability to survive and replicate (Synthego
| Full Stack Genome Engineering 2020).
Therefore, we abandoned the concept of
combining the two viruses, and decided to do point
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mutation on the glycoproteins of the HSV. A recent
research proved that Human adenovirus-C5 (HAdv-
C5) with mutated hypervariable region 1 (HVR1) of
the capsid are more resistant to complement-
mediated inactivation (Atasheva, Emerson, Yao,
Young, Stewart, Shayakhmetov 2020), indicating the
possibility of modifying the glycoproteins on HSV to
elude the immune system. To achieve this,
identification and artificial mutation of the principal
antigenic determinant (epitope) of HSV is required,
while ensuring no impacts on the entry of human
cells. Using targeted mutagenesis and the screening
process, determining modifiable amino acid
sequences, the ideal point mutation might be found
through trial and error. Other modifications on the
HSV will continue after the most suitable
glycoprotein mutation is discovered.
3.2.2 Gene Insertions to Enhance Binding
and Oncolytic Effect
In our model, we choose to use EGF-PL chimeric
molecules. The genes encoding this specific chimeric
molecule will be inserted into the genome of G47∆,
an HSV-based OV. The key features of the EGF-PL
molecules are its single protein (sp), human
epidermal growth factor (EGF), and protein L (PL).
A soluble form of the chimeric molecules can be
secreted with the help with single protein - EGF,
which is critical to trigger the intermolecular reaction
(Fu, Tao, Wu, Zhang 2020). EGF is a TAA-binding
moiety that specifically targets colon cancer cells. PL
can bind to the Igs, including IgG, IgM, IgA, IgE, and
IgD (Bjorck 1988). The Fc region of the binded Igs is
available to bind to the Fc receptors on the surface of
natural killer cells or macrophages. In this
conformation, the EGF-PL molecule can redirect the
innate immune cells to attach to colon cancer cells,
instead of the viruses.
T cell responses to tumor cells are usually
inhibited in immunosuppressive tumor environment,
due to the presence of T cell checkpoint molecules
(Gray, Gong, Hatch, Nguyen, Hughes, Hutchins,
Freimark 2016). The first discovered checkpoint
molecule is cytotoxic T-lymphocyte-associated
protein-4 (CTLA-4), a T cell receptor with high
homology to CD28 binding with B7 molecules to
impede CD28 co-stimulation and downregulate T cell
responses (Sharma, Allison 2015). Unlike CTLA-4,
programmed cell death protein 1 (PD-1), as another
checkpoint molecule, interacts with its ligands (PD-
L1) blocking inflammatory reactions of T cells by
mediating signaling pathways (Grabie, Lichtman,
Padera 2019). For dealing with T cell checkpoint
inhibition caused by PD-1/PD-L1, relative blockade
therapy has been issued, during which there appears
some challenges like the failure of tumor cells to
provoke spontaneous T cell responses to mutant
tumor neoantigens (Wang et al 2020). However, it
was proved that synergy of PDL1 inhibition, GM-
CSF stimulation, and viral replication can activate
neoantigen-specific T cell responses (Wang et al
2020). Therefore, another modification is an insertion
of PD-L1 inhibitor, GM-CSF, and a marker RFP to
explore the synergistic functions.
PDL1 inhibitor can be formed with the fusion of
a murine soluble PD-1 extracellular domain and IgG1
Fc. The extracellular domain functions as a PD-L1
binding site, and the IgG1 Fc is a tracing signal for
iPDL1 to be tested by using anti-IgG Fc. iPDL1
impedes PD-L1 to partially reverse the
immunosuppressive tumor microenvironment,
indirectly activating specific T cell responses for
neoantigen (Karabajakian et al 2020). Besides, GM-
CSF will act as a stimulator to promote intratumoral
T cell infiltration and the anti-PD-1/CTLA
immunotherapy (Puzanov 2016).
3.2.3 Stem Cell Carrier to Improve
Targeting
The purpose of using Mesenchymal Stem Cells
(MSCs) delivery is to solve the problem of possible
failures of oncolytic viral migrations to tumor sites
(Ho, Wu, Chen, Lin, Yen, Hung 2021), using their
inherent tumor tropism, through numerous aspects.
First, MSCs can provide shielding protection of the
internalized OVs by avoiding immune recognition
and further neutralization with their low
immunogenicity (Hadryś, Sochanik, McFadden,
Jazowiecka-Rakus 2020). In addition, MSCs can
migrate directionally to specific tissues that release an
“unusual signal”, triggered by a signalling
mechanism, to improve the general accuracy of
targeting. Moreover, MSCs are suited for gene
transduction, thus can be transduced with therapeutic
genes for viral vectors (Amara, Touati, Beaune, de
Waziers 2014), they are treated with TSA before
being administered to the OVs for priming (Ho, Wu,
Chen, Lin, Yen, Hung 2021). Not only an up-
regulation of CXCR4 level and improved migration
ability towards targeted regions are shown after
priming but also results in increased susceptibility of
OV infections and enhanced capacity of CRAdNTR
loading. Afterward, the primed MSCs with OVs
expressed will be intravenously injected into tumors.
Although several pieces of research have proven
that MSCs have undesired tumor-promoting
Bioengineering of G47 HSV-1 Combined with Stem Cell Delivery as an Alternative Virotherapy against Colon Cancer
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characteristics that can be presented via many
mechanisms (Lin, Huang, Li, Fang, Li, Chen, Xu
2019,
Mahasa, de, Rachid, Amina, Maini, A-Rum,
Chae-Ok, 2020), the preclinical studies have shown
promising safety and efficacy of MSCs carriers for
OV therapies since they have also revealed abilities
that contributed to tumor growth inhibitions. In the
primary research, the prodrug activation is also used
to assist the OV:MSC combination. However, we
choose not to use active prodrugs in our design since
another insertion of genes encoding for a prodrug-
activating enzyme will be risky for the overall
presentation of the engineered model due to the
increased possibility of flaws occurring. However,
without the prodrug being used, a pathway for
oncolysis of the tumor cells might not be efficiently
presented, and the inhibition of metastasis may not be
carried to an ideal extent.
3.3 Experimental Design
3.3.1 Experiments in Vitro
We plan to use CT26-EGFR, a derivative of CT 26
(murine cell line) for our in vitro experiment. Parental
CT26 cells with the human EGFR gene will be
transduced to established our cell line: CT26-EGFR
(Fu, Tao, Wu, Zhang 2020). We will test for oncolytic
virus count, the number of antibodies and
complement proteins found on the virion surface, the
expression and the binding effect of chimeric
molecules, iPDL1 and GM-CSF with the help of
RFP.
To test the performance of the mutated virus,
wild-type HSV, mock HSV, and the mutated HSV
will be incubated individually in human serums
containing antibodies and complement proteins, and
CT26-EGFR, the tumor cells to provide comparisons.
Assays, ELISA, will be conducted for the number of
antibodies and complement proteins deposition found
on the virion surfaces (Atasheva, Emerson, Yao,
Young, Stewart, Shayakhmetov 2020). We use
western blotting to confirm the transgene expression
from the engineered G47∆ virus. We will use flow
cytometry analysis to measure the selective binding
efficacy of the EGF-PL chimeric molecules to CT26-
EGFR. Virus counts will be registered over time for
survival and replication rates, and the number of
tumor cells will also be recorded.
Furthermore, in order to explore the synergistic
function of iPDL1, GM-CSF, and viral replication, it
is necessary to test the expression of those molecules,
as well as the binding efficacy of iPD-L1. iPDL1
expression can be tested using anti-IgG Fc and anti-
PD-1 in western blot (Wang et al 2020). Serum
purification can re-confirm the expression of iPDL1
and test the presence of GM-CSF at the same time.
Flow cytometric analysis will be conducted to
investigate the target binding of iPDL1 to tumor cells
with PD-L1 expression, with IgG-Fc at the x-axis and
iPDL1 at the y axis. CT26-EGFR with PD-L1-knock
down acts as the control.
3.3.2 Experiments in Vivo
To evaluate the therapeutic effect of our new model,
we plan to use the chemical approach to induce a
colon tumor in mice models. We will inject 1,2-
dimethylhydrazine (DMH), a carcinogen to the mice.
We will inject each mouse subcutaneously (s.c.) with
15 µg of DMH per gram of body weight (Gurley,
Moser, Kempn2015). We will perform each injection
once weekly for 12 weeks. When tumors reach the
size of about 5mm in diameter, grouped mice will be
rejected with the new oncolytic virus inside an MSC
or PBS as a mock control (Fu, Tao, Wu, Zhang 2020).
We will also vaccinate mice with recombinant HSV
glycoproteins to induce anti-HSV antibodies, thereby
imitating pre-existing immunity against the HSV
virus as seen in patients.
We will test whether an intratumoral injection of
engineered OV functions in enhancing T cell
responses against neoantigen epitopes. We will use
non-engineered OV as a control group. Four groups
of mice with tumors are required, which will be
treated with PBS, anti-PD-L1, non-engineered OV,
and engineered OV. After ten days of the last
injection of them, tumor-infiltrating T cells from the
tumor-bearing mice should be isolated and cultured
with a mixture of 11 neoepitope peptides (Yadav et al
2014), covering the majority of MHC-I restricted
neoepitopes in MC38 cells (a colon cancer cell line of
mice). Eighty hours incubation later, supernatants
will be collected for IFN-gamma ELISA, which
reveals the concentration of IFN-gamma, a
chemokine released with splenic T cells activation
(Wang et al 2020). Also, [3H] thymidine
incorporation will be measured to figure out the T cell
proliferation. If anti-PD-L1 cannot induce specific T
cell responses to neoantigen as efficiently as iPD-L1,
the potency of iPD-L1/GM-CSF will be confirmed in
stimulating neoantigen-specific T cell responses.
In addition, by carrying out a combination
cytotoxicity assay, we can test if MSCs are
successfully infected by our engineered virus.
Tumor size will be recorded, and mice treated
with mutated variations are expected to have a
decrease in tumor size faster than the wild-type HSV
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
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and the mock infected mice. The population count for
the virus will be registered.
3.4 Anticipated Results
3.4.1 In Vitro Characterization of Our
Engineered Oncolytic Virus
We hypothesize that the virus with the desired
mutations will be resistant against neutralizing
antibodies and complement proteins while remaining
effective in killing cancer cells. We anticipate that the
chimeric molecules, iPD-L1 and GM-CSF will
function properly and are effective against cancer
cells.
A previous study on adenovirus with engineered
capsid protein hexon resulted in reduced immune
response (Atasheva, Emerson, Yao, Young, Stewart,
Shayakhmetov 2020). A beneficial mutation in the
glycoproteins HSV will hinder recognition by the
immune system, while not compromising the OV’s
abilities to enter tumor cells and replication to cause
cell death. Accordingly, the results should display
relatively lower levels of antibodies and complement
proteins detected, while the tumor cell counts are
similar or lower than the wild-type and mock HSV.
Other studies might support our prediction for
chimeric molecules, iPD-L1, and GM-CSF’s
efficacy. Evidence from the primary research paper
showed that the EGF-PL molecules was found in the
supernatants from cells infected with OVs, indicating
that EGF-PL can be produced and released into the
mulieu (Fu, Tao, Wu, Zhang 2020). Therefore, it can
be hypothesized that EGF-PL molecules will be
successfully produced during virus infection in our
experiment.
From similar experiments, iPD-L1 and GM-CSF
mount with engineered OV effectively co-express
those genes (Wang et al 2020). Thus, it can be
estimated that iPD-L1 and GM-CSF might function
in colon cancer cells with the engineered OV
infection.
3.4.2 In Vivo Characterization of the New
Oncolytic Virus
We hypothesize that iPD-L1 and GM-CSF will
induce T cell responses with higher levels of
chemokine released, while MSC-delivery should
show effectiveness in helping the engineered virus to
target cancer cells. The gene mutation should also
improve the virus’s survival in the mice. It can be
inferred that iPD-L1/GM-CSF infection is likely to
provoke neoantigen-specific T cell responses with
elevated proliferation and chemokine (IFN-gamma)
secretion (Wang et al 2020).
It has been proven that the MSC-delivered OV
therapy shows an impressive improvement in
successful migration and targeting to the tumor sites
without being attacked by cellular and humoral
immune systems. Therefore, we predict that the
conduction of our new engineered oncolytic virus
will give out similar results. The result should show
that the administration of our new virus contained in
MSC slows down the growth of tumors and increases
the survival rate of mice in the comparison with the
control. By the end of our experiment, we should
achieve tumor-free in the mice group injected with
the new virus.
The data should present a higher amount of
mutated HSV in the mice relative to the other two
conditions, indicating higher survival and replication
rates. ELISA measurement of antibodies and
complement proteins binding to the viruses will be
conducted. With the right mutation, the antibody and
complement protein counts should show a lower
figure in the mice injected with mutated HSV.
4 DISCUSSION
In our study design, we addressed the limitations in
current oncolytic virotherapies. We addressed the
issue of pre-existing neutralizing antibodies by
editing the glycoproteins on the surface of G47∆
oncolytic virus. Point mutation in gene encoding for
glycoproteins results in the conformational change in
glycoprotein. Thus, the hypothetical oncolytic virus
tends to evade the immune system and survive for a
longer duration. The OVs can successfully replicate
their genetic materials and secrete chimeric
molecules and iPD-L1/GM-CSF. Moreover, the
chimeric molecules direct the immune system to
facilitate virotherapy. This helps the hypothetical
OVs further evade the attack of the immune system.
The chimeric molecules can also recruit the innate
immune cells, attack the tumor cells and increasing
the efficacy of our proposed therapy.
The modifications of the virus can have some
downsides. Point mutations and testing can be highly
time-consuming without producing desired results.
The mice model may not well resemble human pre-
existing immunity, as mice are not natural hosts for
HSV, and antibodies do not create a perfect imitation.
Reversion of the engineered OVs to wildtype by
spontaneous mutation is also possible (Paquet et al
2011). Furthermore, the editing of surface protein
might affect the virus entry into stem cells and tumor
Bioengineering of G47 HSV-1 Combined with Stem Cell Delivery as an Alternative Virotherapy against Colon Cancer
1091

cells. Viral replication might be compromised. Thus,
the oncolytic effect of the engineered OV is reduced.
Additionally, some previous studies have shown the
proliferative effects of MSCs on tumor cells after
being recruited into tumor sites; however, the precise
mechanism of MSCs’ cancer-promoting properties
remains unknown.
Our study design suggests several possible
improvement strategies in the future. One of them is
to replace the EGF with other TAA-binding
molecules. In this way, we can target a wide range of
tumors, broadening the scope of our virotherapy. For
example, we can replace the EGF by the human
epidermal growth factor receptor (EGFR) type 2
(HER2) affibody. Evidence shows that SKOV3 and
MCF7 were found to express HER2 (Fu, Tao, Wu,
Zhang 2020), indicating that affibody-PL chimeric
molecules can bind to ovarian and breast cancer cells.
A thorough investigation of the genetic makeup of
HSV is required for further mastery of gene editing,
improving the initial gene mutation to a greater
extent. Further investigations can also target other
cancer types, with high STING signalling or normal
p-53 function.
5 CONCLUSION
We raised a new HSV-based therapy model in this
paper, combining cell surface protein point
mutations, multiple gene insertions including
chimeric molecules, PD-L1 inhibitors, and GM-CSF,
and delivered through an MSC carrier. We believe
our design has a promising future, but there may still
be some possible factors limiting the efficacy due to
the lack of practical experiments being carried out.
More experiments are required to examine
feasibility of the point mutations of the glycoprotein-
encoding gene, the successful expression of PD-L1
inhibitor and chimeric molecules after gene
insertions, and the comparison of effectiveness
between primed MSCs and unprimed MSCs. Further
in-vivo studies should be conducted to test the
practicality of this new model, even on other cancer
types. Additional experimentations can also focus on
using prodrug with the MSC delivery. Since the
precise mechanism of MSCs’ cancer-promoting
properties remains unknown, the results of further
experiments might reveal their pathway. This might
lead to the promotion of developing a new treatment
based on the inhibition of tumor promotion on MSC
carriers, contributing to more therapeutic
improvements based on the manipulation of immune
systems.
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