Development of circRNA based

treatment for glioblastoma

A project proposal submitted to

Department of Microbiology and Biotechnology Centre

The Maharaja Sayajirao University of Baroda

As a part of

Master’s Degree in Microbiology

(2019-2021)
By

Sandip Bambhaniya
Department of Microbiology and Biotechnology Centre
Faculty of Science
The Maharaja Sayajirao University of Baroda
Vadodara- 390002
Gujarat, India.

1
Contents

Summary 4
Introduction 5
Literature survey 6
Rationale 8
Objectives 9
Workplan and Methodology 11
Schedule 12
Budget 13
References 14

2
SUMMARY:

Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules

in eukaryotes with tissue- specific and cell- specific expression patterns. Some
circRNAs are abundant and evolutionarily conserved, and many circRNAs
exert important biological functions by acting as microRNA or protein
inhibitors (‘sponges’). circRNAs have been implicated in diseases such as
diabetes mellitus, neurological disorders, cardiovascular diseases and cancer.
Glioblastoma multiforme (GBM) is the most common and lethal cancer of the
adult brain, remaining incurable with a median survival time of only
15 months. Despite recent advances in understanding of its pathogenesis,
GBM remains incurable with standard treatment options contributing little to
survival time. Synthesis of modified circRNA (containing binding sites for
target miRNA) with the help of site directed mutagenesis, provides a way to
play with expression level of oncogenes and tumor-supressor genes and
control cell proliferation.

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Introduction
Circular RNAs (circRNAs) comprise a large class of non- coding RNAs that are
produced by a non- canonical splicing event called backsplicing; during
backsplicing, a downstream splice- donor site is covalently linked to an upstream
splice- acceptor site. circRNAs were mainly considered to be ‘junk’ generated by
aberrant splicing events and only the testis- specific circRNA from the sex-
determining region Y (Sry) gene was thought to have a possible function (in
mouse testis) (Xia, S. et al., 2017). In recent years, high- throughput RNA
sequencing (RNA- seq) and circRNA- specific bioinformatics algorithms have
identified thousands of circRNAs in eukaryotes, including in fungi, protists,
plants, worms, fish, insects and mammals, and found them to have tissue- specific
expression patterns (Rybak-Wolf, A. et al., 2015). Despite a lack of
polyadenylation (poly(A)) and capping, circRNAs generally localize to the
cytoplasm. Although backsplicing is generally less efficient than linear splicing,
circRNAs can accumulate in specific cell types in a temporally regulated manner,
owing to their high stability. This high stability is presumably the result of their
covalently closed ring structure protecting these molecules from exonuclease-
mediated degradation. Interestingly, a high number of circRNAs are upregulated
during neurogenesis, and some circRNAs are enriched in synapses. To date,
circRNAs have been implicated in several human diseases, including diabetes
mellitus, neurological disorders, cardiovascular diseases, chronic inflammatory
diseases and cancer, and they accumulate during ageing (Kristensen et al., 2020).
Glioblastoma multiforme (GBM) is the most common and lethal cancer of the
brain, with approximately 10,000 newly diagnosed cases each year in the United
States. Despite recent advances in understanding of its pathogenesis, GBM
remains incurable with standard treatment options contributing little to survival
time. Currently, GBM has a 5‐year survival rate of only 10% and a median
survival time of 15 months following treatment. Contributing to its poor
prognosis are numerous therapeutic challenges including aggressive growth rates,
tumor heterogeneity, drug resistance, and obstacles to drug delivery such as the
blood–brain barrier(Shea et al., 2016).
In an effort to find novel approaches to GBM treatment, recent studies have
focused on molecular phenotyping of GBM subtypes to identify new targets for
biomarkers and therapeutics.

4
Literature survey:

circRNAs are derived from canonical splice sites, and mutational analyses in
circRNA expression vectors as well as treatment of HeLa cells with the splicing
inhibitor isoginkgetin, which blocks spliceosome assembly, showed that
circRNA biogenesis is dependent on the canonical splicing machinery (Starke, S.
et al. 2015). Base pairing between inverted repeat elements (such as Alu elem
ents), which are located in the upstream and downstream introns is important for
circularization of RNA. Additionally, lariat formation during exon skipping, an
event during which alternative exons are spliced out of the final mRNA product
and end up contained within the excised lariat, can lead to the formation of
circRNA when the lariat undergoes internal backsplicing (Kelly, S. et al., 2015).

Since the recent discovery that endogenous circular RNAs (circRNAs) are widely
expressed in all human tissues, there has been an increasing focus on
characterizing their relevance and function in disease. the majority of studies have
focused on the role of circRNAs in solid tumours, in which individual circRNAs
have been described as either oncogenes (for example, circPvt1 in head and neck
squamous cell carcinoma and cirs-7 (CDr1as) in colorectal cancer, oesophageal
squamous cell carcinoma and hepatocellular carcinoma) or tumour suppressors
(for example, circsMarCa5 and circ-sHPrH in glioblastoma). the function of other
circRNAs in solid tumours may be cell type- specific; for example, circHiPK3
has been described as an oncogene in colorectal cancer and a tumour suppressor
in bladder cancer. Moreover, as the expression level of circRNAs often correlates
with clinical and pathological characteristics, these RNAs have the potential to
be diagnostic, prognostic and predictive biomarkers. Furthermore, the high
stability of circRNAs may allow them to be detected non- invasively in bodily
fluids, as exemplified by the detection of circCNOt2 in plasma from patients with
breast cancer and the detection of circ-KLDHC10 in serum exosomes from
patients with colorectal cancer(Kristensen et al., 2020.).

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FIG. miRNA sponging activity of circRNA

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Rationale
• Glioblastoma multiforme (GBM) is the most common and lethal cancer of
the adult brain, remaining incurable with a median survival time of only
15 months.

• In an effort to identify new targets for GBM diagnostics and therapeutics,

recent studies have focused on molecular phenotyping of GBM subtypes.

• circRNAs have important non-coding functions that reflect their high

stability. However, biological functions have only been investigated for a
minor fraction of the circRNAs identified to date, most of which have been
proposed to act as miRNA sponges.

• ciRS-7, which is perhaps the most well- characterized circRNA, contains

more than 70 conserved binding sites for miR-7 and is highly and stably
expressed in many tissues, in particular in the brain.

• Thus, it has the potential to regulate the expression of miR-7 target genes.

• Synthesis of modified circRNA (containing binding sites for target

miRNA) with the help of site directed mutagenesis, provides a way to play
with expression level of oncogenes and tumor-supressor genes and control
cell proliferation.

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 • Objectives
1. Genome wide profiling of circRNA in glioblastoma cells.
2. Effect of deletion and overexpression of circRNA on cell proliferation.
3. Analysis of interaction between circRNA and miRNA and identification of
miRNA which repress tumor suppressor gene.
4. Synthesis of tumor suppressor gene specific miRNA binding site
containing circRNA
5. Transformation of modified circRNA producing gene containing plasmid
into cell and analysis of proliferation.
6. In vivo transfer of pCDNA3 into mouse model.

Outcome
• Based on profiling of circRNA in glioblastoma cells, circRNAs are
identified which are dysregulated in tumor cells.

• Effect of dysregulated circRNAs on cell proliferation and target miRNA is

to be identified.

• Development of novel cancer treatment method based on modified

circRNA.

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Workplan and Methodology

Objective 1: genome wide profiling of circRNA in glioblastoma cells.

• Glioblastoma sample collection.

• The GBM cells were maintained in Dulbecco's modified Eagle's medium
(DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS;
HyClone) and incubated at 37˚C in a 5% CO2 atmosphere.
• With the help of Two independent algorithms (plethora and CIRI2)
genome wide profiling of circRNA to be done.
• Further validation of circRNA detected by algorithms is done by RT-PCR
using backsplice region specific divergent primers.

Objective 2: Effect of deletion and overexpression of circRNA on cell

proliferation.
• Flanking intronic motifs that are important for circRNA biogenesis,
removing these elements from the genome can eliminate or diminish
circRNA expression without altering steady-state RNA levels of the host
gene.
• For overexpressing a certain circRNA cells can be transfected or
transduced with an circRNA producing plasmid.
• Change in cell proliferation to be analysed by MTT assay for both type of
cells.

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Objective 3: Analysis of interaction between circRNA and miRNA and
identification of miRNA which repress tumor suppressor gene.

• Immunoprecipitation or argonaute immunoprecipitation are used to be

prove miRNA sponging activity of circRNA.

• miRNA which are repressing tumor suppressor genes are identified based
on effect of knockout of miRNA producing gene on cell proliferation.

OBJECTIVE 4: Synthesis of tumor suppressor gene specific miRNA

binding site containing circRNA.
• Tumor suppressor gene repressing miRNA specific binding site to be
incorporated into circRNA producing gene by CRISPER-CAS system or by
site directed mutagenesis.

• Cloning of this gene in the mammalian expression vector pCDNA3.

OBJECTIVE 5: Transformation of modified circRNA producing gene

containing plasmid into cell and analysis of proliferation.
• Plasmid pCDNA3 is transfected into glioblastoma cells by calcium
phosphate transfection protocol and cell proliferation is estimated by
MTT ASSAY.

OBJECTIVE 6: In vivo transfer of pCDNA3 into mouse model.

• pCDNA3 containing gene for modified circRNA is transported to
glioblastoma cells in brain by gene transfer technology that uses pegylated
immunoliposomes (PILs).

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Schedule

Objective Expected Duration

Profiling of circRNA 4 months
Effect of circRNA expression on cell 6 months
proliferation

circRNA miRNA interaction study 3 months

Development of modified gene of 4 months

circRNA and transfection into
plasmid pCDNA3
Analysis of modified circRNA on cell 4 months
proliferation
In vivo study in mouse model 3 months

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Budget

Non-Recurring Expenditures
Product Cost
Cell lines ₹ 90000
Media and reagents ₹ 80,00,00.00
Maintenance of cell lines ₹ 100000.00
Glasswares and Plasticwares ₹ 30,000.00
contingency ₹ 10,00,000.00
Incubator (Thermo fisher) ₹ 1,00,939.00
Primers ₹ 50,000.00
RT-PCR ₹ 15,00,000.00
Monoclonal antibody ₹ 10,0000.00
Gene transfer technology ₹ 50,480.00
Mouse model ₹ 7000.00
Total ₹ 3,828,419.00

Recurring Expenditures
Product Quantity Cost
Other requirements and consumables ₹ 5,00,000.00
Junior Research Fellow 1 ₹ 6,20,000.00
Overheads ₹ 2,00,000.00
Total ₹ 2,613,627.00

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References

1. Kristensen, L. S., Andersen, M. S., Stagsted, L. V., Ebbesen, K. K.,

Hansen, T. B., & Kjems, J. (2019). The biogenesis, biology and
characterization of circular RNAs. Nature Reviews Genetics, 20(11), 675-
691.
2. Shea, A., Harish, V., Afzal, Z., Chijioke, J., Kedir, H., Dusmatova, S., &
Kumar, D. (2016). MicroRNAs in glioblastoma multiforme pathogenesis
and therapeutics. Cancer medicine, 5(8), 1917-1946.
3. Memczak, S., Jens, M., Elefsinioti, A., Torti, F., Krueger, J., Rybak, A., &
Rajewsky, N. (2013). Circular RNAs are a large class of animal RNAs with
regulatory potency. Nature, 495(7441), 333-338.
4. Boado, R. J. (2005). RNA interference and nonviral targeted gene therapy
of experimental brain cancer. NeuroRx, 2(1), 139-150.
5. Xia, X., Li, Y., Wang, W., Tang, F., Tan, J., Sun, L., & He, S. (2015).
MicroRNA-1908 functions as a glioblastoma oncogene by suppressing
PTEN tumor suppressor pathway. Molecular cancer, 14(1), 1-14.
6. Jeck, W. R., Sorrentino, J. A., Wang, K., Slevin, M. K., Burd, C. E., Liu,
J., & Sharpless, N. E. (2013). Circular RNAs are abundant, conserved, and
associated with ALU repeats. Rna, 19(2), 141-157.
7. Zeng, K., Chen, X., Xu, M. U., Liu, X., Hu, X., Xu, T., ... & Wang, S.
(2018). CircHIPK3 promotes colorectal cancer growth and metastasis by
sponging miR-7. Cell death & disease, 9(4), 1-15.
8. Starke, S., Jost, I., Rossbach, O., Schneider, T., Schreiner, S., Hung, L. H.,
& Bindereif, A. (2015). Exon circularization requires canonical splice
signals. Cell reports, 10(1), 103-111.

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