Team:Bielefeld-CeBiTec/Background
Background
Summary
To realize our aim to selectively and efficiently transform eukaryotic cells of interest, we take advantage of the high specificity of bacteriophages. A well-characterized bacteriophage is M13, a filamentous, non-lytic phage that infects Escherichia coli.
About 10 years ago, the approach to use a M13-derived gene delivery system as an alternative to virus-based gene therapy was developed. Fusing target cell specific ligands to the coat proteins induces endocytosis, which allows the hybrid phage to enter the cell and deliver its gene of interest.
Inspired by the described approach we decided to design an easily adaptable platform system, called Troygenics, to enable transformation of eukaryotic cells that are currently difficult to transform. The uptake of our Troygenics relies on Endocytosis, a very conserved process among all eukaryotes. It mediates the internalization of extracellular material by the cell.
As a possible application in fighting eukaryotic pathogens, we designed a highly specific CRISPR/Cas13a-based Cell Death Inducing System (CeDIS) as gene of interest which is delivered into the target cell.
To realize our aim to selectively and efficiently transform eukaryotic cells of interest, we take advantage of the high specificity of bacteriophages. A well-characterized bacteriophage is M13, a filamentous, non-lytic phage that infects Escherichia coli.
About 10 years ago, the approach to use a M13-derived gene delivery system as an alternative to virus-based gene therapy was developed. Fusing target cell specific ligands to the coat proteins induces endocytosis, which allows the hybrid phage to enter the cell and deliver its gene of interest.
Inspired by the described approach we decided to design an easily adaptable platform system, called Troygenics, to enable transformation of eukaryotic cells that are currently difficult to transform. The uptake of our Troygenics relies on Endocytosis, a very conserved process among all eukaryotes. It mediates the internalization of extracellular material by the cell.
As a possible application in fighting eukaryotic pathogens, we designed a highly specific CRISPR/Cas13a-based Cell Death Inducing System (CeDIS) as gene of interest which is delivered into the target cell.
M13 Bacteriophage
The basic of our Troygenics is the filamentous bacteriophage M13, an isolate of the F-pilus specific phage. M13 is non-lytic
and infects E. coli. Replication is possible exclusively in E. coli (Smeal et al. 2017). The M13 phage is about 6 nm wide in
diameter and 800-2000 nm long (Marvin 1998).
The genome of the M13 phage consists of single stranded DNA. In the genome there are eleven genes, promoters, ribosome binding
sites and terminators which compose a complex control system (Smeal et al. 2017). Because of this, the independent manipulation and controlled
regulation of one gene is very complicated.
The genome is separated into two separate transcriptional units, one is more frequently transcribed and codes for pII, pV, pVII, pVIII, pIX and pX. The other one is less
frequently transcribed and contains pI, pIII, pIV, pVI and pXI. The coat proteins pIII, pVI, pVII and pIX are replicated five times, just the major coat protein pVIII is
copied up to
2700 times. pI, pIV and pXI, together with an inner and an outer membrane spanning
protein and an inner membrane anchored periplasmatic protein, build the membrane spanning phage assembly complex. pII,
pX and pV
are control proteins in the M13 phage, enabling DNA synthesis and mediating the fate of the newly-synthesized DNA
(Smeal et al. 2017).
The life cycle of M13 consists of 10 steps.
- Passage of the phage genome into the host cell mediated by coat protein pIII
- Conversion of the ssDNA to double stranded form by E. coli
- Initiation of mRNA transcription with E. coli polymerase
- Synthesizing single stranded copies of M13 genome through rolling circle replication using the dsDNA as template
- if the concentration of phage proteins is low: ssDNA copies converted into additional dsDNA
- if the concentration of phage proteins (especially pV) is high: pV binds to ssDNA copies preventing conversion, packing ssDNA into new phages
- Recognition of pV-sequestered ssDNA by the membrane spanning phage assembly complex
- Attachment of the minor coat proteins pVII and pIX to the ssDNA by the assembly complex
- Pass of the pV-sequestered ssDNA trough the cell membranes by the assembly complex
- Removal of pV and assembly of pVIII around the ssDNA genome
- Addition of the other minor coat proteins pIII and pIV
- Release of the particle by the cell, conformational change within pIII
M13K07
M13K07 is an M13 phage with a kanamycin resistance originated from the transposon Tn-903 and an origin of replication
from p15A that was inserted into the M13 ori. With this ori M13K07 is able to produce single-stranded DNA, and therefore can
be used for
mutagenesis or sequencing. The new origin of replication causes the original phage ori to lose its efficiency. Althrough, M13K07
is able to replicate in absence of phagemid DNA (Vieira and Messing 1989).
Hybrid Phage
The delivery of genes into specific target cells is still very challenging. Virus based gene delivery is a commonly
used method for gene therapy and other applications. However, it bares several disadvantages (Yoo 2016). Viruses
often do not reach their destined targets and many of them have a limited range of host cells. Thus, there is a need
for a novel bio material which can specifically and efficiently deliver genes of interest into cells. To engineer a
product like this, the properties of two types of viruses can be utilized; the gene
integration ability of viruses infecting eukaryotic hosts, as well as the high specificity of viruses for prokaryotes,
known as phages.
The aim of previous projects using M13 as shuttle for genetic information, was to use this delivery system as a gene therapy against cancer. To achieve this, the hybrid-phage needed different properties, one of them being able to recognize the eukaryotic tumor cells. Therefore, they fused the pIII minor coat protein with an RGD-peptide as ligand, which binds to αv integrins, cell-surface receptors that are overexpressed on tumor cells. The occupation of the receptor leads to the internalization of the phage (Pranjol 2015). Another feature of the vector is a cassette located in the inter genomic region of the phage. The gene of interest for gene therapy, which will be expressed in the host cell, is flanked by inverted terminal repeats (ITR) from Adeno Associated Virus (AAV). Those ITRs allow the genomic integration of the gene of interest.
The aim of previous projects using M13 as shuttle for genetic information, was to use this delivery system as a gene therapy against cancer. To achieve this, the hybrid-phage needed different properties, one of them being able to recognize the eukaryotic tumor cells. Therefore, they fused the pIII minor coat protein with an RGD-peptide as ligand, which binds to αv integrins, cell-surface receptors that are overexpressed on tumor cells. The occupation of the receptor leads to the internalization of the phage (Pranjol 2015). Another feature of the vector is a cassette located in the inter genomic region of the phage. The gene of interest for gene therapy, which will be expressed in the host cell, is flanked by inverted terminal repeats (ITR) from Adeno Associated Virus (AAV). Those ITRs allow the genomic integration of the gene of interest.
Some years later, scientists were able to enhance the uptake of the hybrid phage into HeLa cells by
presenting the RGD-motif on the major coat protein pVIII of the M13 phage (Yoo, 2016). By using this hybrid
phage approach, HeLa cells could be transformed with the gene for GFP with an efficiency of 40 % (Yoo, 2016).
It has been shown that a hybrid phage like this was able to target a tumor in mice in vivo. One systemic dose inhibited tumor growth significantly (Hajitou, 2006).
It has been shown that a hybrid phage like this was able to target a tumor in mice in vivo. One systemic dose inhibited tumor growth significantly (Hajitou, 2006).
pIII Minor Coat Protein
One of the proteins responsible for building the M13 protein coat is called pIII minor coat protein and can be found at the
top of the phage. In total, only five copies of the protein are being incorporated into the phage’s coat.
pIII consists of three domains, namely N1, N2 and a C-terminal domain which are connected through flexible glycine-rich
linkers. The N2 domain recognizes and binds the tip of the F pilus of E. coli. As soon as M13 is bound to the
F pilus, an unknown mechanism causes its retraction so the N1 domain can interact with the C-terminal domain of the
bacterial TolA protein. This interaction enables the phage to insert its DNA into the bacterial cytoplasm through an
unknown mechanism (Lubkowski et al., 1999). The C-terminal domain of pIII is required for releasing the newly assembled
phages from
the bacterial membrane, and for higher stability (Armstrong et al., 1981; Jakes et al., 1988; Stengele et al., ).
pVIII Major Coat Protein
pVIII is the major coat protein of the M13 bacteriophage. It is replicated about 2700 times inside one phage
(Smeal et al. 2017). Thousands of α-helical pVIII proteins are arranged in a helical structure around the
ssDNA core. One helical protein is about 1 - 7 nm small. The N-terminal end of pVIII has a hollow
cup shape while the C-terminal end has a pointed arrowhead shape. The α-helical axis is in small angle to the
virion axis (Marvin 1998).
Endocytosis
Proteins bind to receptors on the cell surface and are actively taken up into the cell via endocytosis.
Every organism is in close contact with its environment. To sense and communicate with its environment, almost every
eukaryote performs endocytosis (Doherty and McMahon 2009). Endocytosis is the process of internalization of extracellular
material within an invagination in the plasmalemma (Besterman and Low 1983). The conserved process for all eucaryotic cells
is required for diverse cellular functions. It is for example involved in the turnover and degradation of transmembrane
proteins and receptors, the transduction and dispersal of signals in and between cells, in cell-cell-communication and
nutrient uptake (Besterman and Low 1983; Samaj et al. 2004). Additionally, endocytosis is the main route through which
viruses and bioparticles enter and leave cells (Gao et al. 2005). Especially in protozoa endocytosis plays an obvious
nutritional role (Gao et al. 2005).
There are different types of endocytosis, called phagocytosis and pinocytosis (Besterman and Low 1983; Samaj et al. 2004). While phagocytosis describes the uptake of large particles like whole cells, pinocytosis deals with smaller proteins and peptides. The mechanism of phago- and pinocytosis are similar to each other. At first the extracellular particle binds to the cell surface. The next step involves extruding of philopodia which surround the extracellular fluid and lead to engulfment of the particles attached to the cell surface (Besterman and Low 1983; Samaj et al. 2004).
Endocytosis can happen in a constitutive, unregulated way when particles bind non-specifically to the cell surface (Doherty and McMahon 2009; Besterman and Low 1983; Samaj et al. 2004) and is internalized due to the plasma membrane dynamics. The plasma membrane is turned over constitutively to retain cell homeostasis (Samaj et al. 2004; Besterman and Low 1983) and regulates the protein and lipid composition of the membrane (Besterman and Low 1983). Furthermore, there is the receptor mediated endocytosis (Goldstein et al. 1979; Besterman and Low 1983; Doherty and McMahon 2009; Gao et al. 2005). This mechanism is highly specific. A substrate, for example a signaling or a nutritional molecule, binds its specific receptor in the plasma membrane. Those ligand receptor complexes cluster in clathrin coated pits in the membrane and transmit the information that they are bound to their substrate. This process triggers a signal cascade which leads to endocytosis of the ligand receptor complexes. Vesicles are forming by invagination of- and budding off from the plasma membrane so they can deliver their cargo to the correct destination (Goldstein et al. 1979; Samaj et al. 2004). Often this destination is the lysosome or the vacuole of the cell where the ligand receptor complex is degraded but sometimes the receptors are recycled back to the plasma membrane (Doherty and McMahon 2009; Goldstein et al. 1985).
There are different types of endocytosis, called phagocytosis and pinocytosis (Besterman and Low 1983; Samaj et al. 2004). While phagocytosis describes the uptake of large particles like whole cells, pinocytosis deals with smaller proteins and peptides. The mechanism of phago- and pinocytosis are similar to each other. At first the extracellular particle binds to the cell surface. The next step involves extruding of philopodia which surround the extracellular fluid and lead to engulfment of the particles attached to the cell surface (Besterman and Low 1983; Samaj et al. 2004).
Endocytosis can happen in a constitutive, unregulated way when particles bind non-specifically to the cell surface (Doherty and McMahon 2009; Besterman and Low 1983; Samaj et al. 2004) and is internalized due to the plasma membrane dynamics. The plasma membrane is turned over constitutively to retain cell homeostasis (Samaj et al. 2004; Besterman and Low 1983) and regulates the protein and lipid composition of the membrane (Besterman and Low 1983). Furthermore, there is the receptor mediated endocytosis (Goldstein et al. 1979; Besterman and Low 1983; Doherty and McMahon 2009; Gao et al. 2005). This mechanism is highly specific. A substrate, for example a signaling or a nutritional molecule, binds its specific receptor in the plasma membrane. Those ligand receptor complexes cluster in clathrin coated pits in the membrane and transmit the information that they are bound to their substrate. This process triggers a signal cascade which leads to endocytosis of the ligand receptor complexes. Vesicles are forming by invagination of- and budding off from the plasma membrane so they can deliver their cargo to the correct destination (Goldstein et al. 1979; Samaj et al. 2004). Often this destination is the lysosome or the vacuole of the cell where the ligand receptor complex is degraded but sometimes the receptors are recycled back to the plasma membrane (Doherty and McMahon 2009; Goldstein et al. 1985).
CeDIS
For our Cell Death Inducing System (CeDIS) we intended to use a system based on CRISPR/Cas13a – a protein that is
known to cause collateral cleavage of
RNA, causing cell toxicity, in prokaryotic cells (Abudayyeh et al., 2016).
Cas13a consists of a recognition site including crRNA, which binds to complementary ssRNA within the cell. Upon binding,
the nuclease of the Cas13a complex is activated. This results in collateral RNA cleavage, ultimately leading to cell death
(Liu et al., 2017).
Within organisms like Leptotrichia wadei, Leptotrichia buccalis and
Leptotrichia shahii, this system has been assumed to serve as an immune system inducing cell death upon infection with RNA
viruses, or, more probable, upon expression of dormant DNA viruses (Koonin and Zhang, 2017).
It has previously been used to detect certain viruses or bacteria in blood, milk or other suspensions (Gootenberg et al.,
2017; iGEM TU Delft, 2017). It is known to be able to differentiate very similar RNA strains, in some systems even single
nucleotide polymorphisms (Gootenberg et al., 2017). Therefore, Cas13a is considered to be highly specific and can be
used to differentiate between species.
However, Cas13a is not able to induce collateral RNA cleavage in mammalian cells (Cox et al., 2017), disabling it from
triggering their cell death.
To recognize its specific target RNAs Cas13a is equipped with guide RNAs. Those 21 nucleotides short sequences are
complementary to the desired target and flanked by direct repeat sequences (Abudayyeh, 2016).
In the course of our project we aim to induce collateral RNA cleavage specifically inside the target organism resulting in the
pathogens cell death.
References
iGEM TU Delft, T. (2017). Team:TUDelft—2017.igem.org. Retrieved May 14, 2019.
Vieira, JEFFREY, and JOACHIM Messing. 1989. “11 - Production of Single-Stranded Plasmid DNA.” In Recombinant DNA Methodology, edited by Ray Wu, Lawrence Grossman, and Kivie Moldave, 225–33. Selected Methods in Enzymology. San Diego: Academic Press.