EHV-1 as an Oncolytic Vector Flashcards
What is GBM?
Glioblastoma Multiforme
- Very invasive, lethal, brain cancer
Highly resistant to:
- chemotherapy - radiotherapy - surgical resection
What are the Advantages of oncolytic virotherapy?
1) Viruses can be engineered to be selective for cancer cells (targeted therapy)
2) Some oncolytic viruses, including the herpesviruses, have large genomes
that can be bio-engineered to augment the antitumor effect of the virus and
increase safety
3) Some engineered viruses are selectively replication competent and thus can
efficiently spread throughout the tumor mass without destroying healthy tissue
4) Virotherapy can be combined with other therapeutic approaches such as:
- chemotherapy
- radiotherapy
- surgery
What is the overall goal of oncolytic virotherapy?
Overall idea is to kill the cancer cells
while sparing the healthy tissue
Why do they think EHV-1 has potential to kill cancer cells?
1) Not a human pathogen
- no pre-existing antibodies to the virus
2) Large DNA genome
- genome sequenced
- can easily insert therapeutic genes or delete virulence genes as
needed to engineer a safe and effective oncolytic vector
3) Virus life cycle very well characterized
4) EHV-1 has broad tropism in tissue culture (able to infect cell types from many species in vitro)
5) Antiviral drugs available
- can stop infection once tumor is eradicated
- if vector mutates then you have option to treat it
What did Dr. Frampton do to initially test EHV-1 as an oncolytic vector?
Ordered 6 different cancer cells lines to ATCC (american tissue cell culture)
Used one cell line as positive control for infection (known they are readily infected by virus)
Used mouse skins cancer cells that are highly resistant to virus as negative control
Modified EHV-1 w/ Blue reporter virus: expressed lacZ so you can add substrate and if virus present then you see Blue
MOI: multiplicity of infection
how many virus particles you’re adding per cell
MOI of 1 = 1 infectious particle per cell
MOI 3 = 3 virus particles per cell
Can see positive and negative controls worked from MOI and color
Result: Variation that occurs with the amount of EHV you add to these cell lines. The more you add the more infection is occurring on some of the cell lines. There were 2 cell lines highly resistant to EHV-1.
What questions were raised by Dr. Frampton’s initial test of EHV-1 as an oncolytic vector on the 6 cell lines?
Why are there two cell lines so resistant to EHV?
hypotheses:
these cells might not express the right entry receptor for EHV
MHC I is entry receptor for EHV, so how much MHC I expression is there on these cells and can a human MHC I serve as an entry receptor for EHV-1?
Since MHC I is the entry receptor used by EHV-1, how did Dr. Frampton see if the resistant cell lines expressed MHC-1?
Reverse transcriptase
- isolate RNA from each cancer cell line
- RNA to cDNA
- figure out how much MHC-I is being expressed on these cancer cells
Noticed susceptible cell lines expressed MHC-I early (20 cycles)
HS683 didn’t express any MHC I at all (many cancer cells down regulate MHC I expression)
result: most likey reason the cells were resistant to EHV was b/c they didn’t express MHC-I
How did Dr. Frampton prove that the susceptible cell lines were being infected by EHV-1 because they were expressing MHC-1?
Anti-human MHC-I antibody blocks EHV-1 infection on glioma cells
Infect cells w/ EHV and then add anti-MHC I antibody
shows % infection decreases immediately when MHC-I antibody added
Use HSV-2 (uses nectin 1 as entry receptor) as control to show that EHV isn’t using nectin 1 as entry receptor and only using human MHC I to get in
What did Dr. Frampton prove with his work on EHV-1 and the 6 cell lines and proving they were using MHC I as an entry receptor?
EHV-1 can successfully infect and kill human glioma cells
- however there is variability in cell killing between
different GBM lines
EHV-1 uses human MHC-I as an entry receptor on glioma cells
Positive correlation between the levels of MHC-I expression and EHV-1 infection MOI
How did Dr. Frampton hypothesize he could increase the oncolytic potential of EHV-1? Attack those resistant cell lines?
Histone deacetylase inhibitors - Novel class of anti-cancer agents
Prevent histone proteins on chromosomes from being deacetylated
- Modulates expression of viral and cellular genes
Acetylase opens heterochromatin to Euchromatin and allows for more gene expression
heterochromatin to tightly compacted for RNA polymerase to come in and express genes
Histone deacetylase inhibitors keep the chromatin in relaxed form
Valproic acid (VPA) has been shown both in vitro and in vivo to synergize with oncolytic viruses
What was Dr. Frampton’s experiment design to determine if VPA synergize with EHV-1 and lead to increased oncolysis?
- Treat glioma cells with or without VPA for 24 hours
- Mock-infect or infect cells with EHV-1 at various MOIs
- Assess cell viability 48 hours p.i.
Two groups of cells 1 group gets VPA 1 group doesn’t get VPA Infect both groups Assess % survival 48 hrs later
What were the results of the VPA & EHV-1 experiment?
Mock: control to show that if you add VPA alone and not add EHV then the cells still survive
At MOI 10 you see dramatic increase cell death when you add VPA
Drug is somehow sensitizing the cells to infection (removing some kind of barrier to infection)
can increase amount of cells killed w/ EHV by using VPA
VPA increases EHV-I yield in GBM cells (more virions in GBM cells = more lysis)
What hypothesis did Dr. Frampton propose for how VPA is enhancing EHV-1 oncolysis of GBM cells? What did he do to further investigate?
VPA treatment modulates innate and/or adaptive immune response genes in GBM cells which enables increased EHV-1 infection and cell death.
Treat glioma cells with or without VPA for 24 hours
Isolate total RNA
- convert RNA to cDNA
- PCR amplification and data analysis
Which genes were upregulated after treatment w/ VPA? What experiments can be run to test if this is true?
CD40
create knockout CD40 cell line
treat w/ VPA
infect w/ virus
should decrease infection
Create cell line that overexpress CD40
should increase infection
How do we make EHV-1 selective for GBM cells?
- May already be somewhat selective due to use of MHC-1?
- Engineer gD to recognize tumor-specific receptor?
- Generate selectively replication competent oncolytic vector that contains a tumor-specific promoter driving expression of the immediate early gene (IE). This virus should only replicate inside of GBM cells.
- Replace normal promoter w/ tumor specific promotor
not get activation of oncolytic vector in non-tumor cells
