SignaGen Laboratories (SignaGen) is a
small business focusing on developing and manufacturing gene delivery tools for
bio-medical research community. Based on our unique technology platforms
(patent pending, US PTO # 61135606 & 072308),
SignaGen has successfully developed three categories of DNA/RNA transfection
reagents, which were validated to beat the leading products easily with
extremely high efficiency & low cytotoxicity. What's more important is we are supplying these transfection reagents
with logical prices.
1. Biodegradable Polymer (BDP) Synthesis:
This BDP technology is our patent pending (US
PTO # 61135606 & 072308)
propriety technique which
allows us to synthesize a biodegradable polycationic polymer. Unlike other
available cationic polymers such as poly(L-lysine) and poly(ethylenimine), and
cationic liposome, this novel polymer has an unique backbone which can be
degraded after the gene vector unloads DNA/RNA inside of mammalian cells (Figure
1),
leading to extremely low cytotoxicity. In addition, this novel polymer was
conformed to possess excellent nucleic acid binding affinity and high buffering
capacity, meeting the fundamental design criteria of good gene carriers.
The in vitro DNA transfection experiments showed that this novel biodegradable
polymer was 2 to 10 times more efficient than poly(ethylenimine) in delivering DNA to HEK293T cells.
Figure 1. A Cartoon
Showing DNA Transfection with A Biodegradable Polymer
2. Virus Proteins Simulating (VIPs)
Technology:
Gene therapy is advanced technology in the
treatment of human genetic and acquired diseases through the delivery and
expression of therapeutic gene-based drugs. The use of safe, efficient, and
controllable gene carriers is one prerequisite for the success of clinical gene
therapy. While viral vectors are very efficient in gene delivery, their
potential safety and immunogenicity concerns raise their risk in clinical
applications. As an alternative to viral vectors, we developed an unique VIPs
technology to simulate virus nuclear targeting peptide sequences. Based on this
VIPs platform, series of simulated virus (e.g., lentivirus) nuclear targeting peptide sequences are
created, synthesized and screened. It was confirmed that combined with a
liposome DNA transfection reagent, series of these simulated peptides were able
to penetrate cell nuclear pore, thus delivering DNA/RNA directly to cell nuclear
(Figure 2).
The initial results showed that several simulated peptides can significantly (up to
100 times)
enhance liposome based gene delivery efficiency on variety of mammalian cells.
Interestingly the synergy effect was observed on hard-to-transfect non-dividing
cells, providing a potential non-viral nucleic acids vector for
hard-to-transfect cells.
Figure 2. A Cartoon
Showing Synergy Effect of VIPs Simulated Peptide with a Liposome Transfection
Reagent
3. Shaved Cell Transfection (SCT)
Technology:
Cell membrane architecture is
that of a lipid bilayer. The lipids are amphipathic in that they have
hydrophilic polar heads pointing out and the hydrophobic portion forming the
core.
However cell membrane surface is uneven (Figure 3-I).
Lots of peripheral membrane
molecules including
glycolipids,
glycoproteins, carbohydrate of glycolipids and transmembrane proteins, ion
channels are
also
constituents of membrane.
In addition,
most
cells are decorated with other several types of proteins that allow their
binding to other cells or to the extracellular matrix. They are known as cell
adhesion molecules (CAMs). All these
peripheral membrane molecules
are termed
as cell's hairs (Figure 3-I), which hinder transfection complex (Figure
3-II & III) accessing and penetrating the cell, compromising transfection complex
endocytosis. This is why regular transfection procedure usually leads to
lower transfection efficiency especially on the hard-to-transfect cells.
To circumvent barrier of cell's hair in preventing transfection complex
endocytosis, we developed unique transfection reagents with proprietary
transfection protocols. With our proprietary transfection procedures called "Shaved Cell Transfection (SCT)",
cell's hairs are temporarily shaved before conducting transfection followed by
re-plating the shaved cells (Figure 3-IV & V). SCT technology allows transfection complex easier and more efficient in crossing and penetrating
the cell membrane, leading
to 3~15 times better efficiency than that on unshaved cells (Figure 3-V & VI).
The SCT technology is extremely useful for transfecting the hard-to-transfection
cells.
Figure 3. A Cartoon
Showing How Shaved Cell Transfection Technology Works
4. Transfection Mediated Cytotoxicity Removal (TMCR) Technology:
Researches often encounter the
following dilemma during DNA/siRNA transfection operations: transfection reagent with high efficiency usually gives
strong cytotoxicity. Sometimes the toxicity is so strong that large amount
of cells are killed after transfection. Therefore researchers have to sacrifice
transfection efficiency to trade for relative healthy cells. Now we
provide a solution to achieve maximum transfection efficiency while keeping
relative good cell viability by removing transfection mediated cytotoxicity
which otherwise kills the cells. After years of research, we
identified that it is the transfection complex sticking to the edges that leads to cytotoxicity
(Figure 4, upper panel). Due to the nature of transfection
complex, it is impossible to wash the complex out with just medium change post transfection. We develop an unique product in which several
chemicals are mixed to make a cocktail (Patent pending,
US PTO # 62375686) which was confirmed to effectively remove all the transfection
complex bound to cell edges with 5 minutes incubation at room temperature
(Figure 4, lower panel), thus removing the transfection mediated cytotoxicity.
Figure 4. A Cartoon
Showing How Transfection Mediated Cytotoxcity Removal (TMCR) Technology Works
5. pH
Dependent Conformational Change (PDCC) Technology for Efficient siRNA Delivery:
While
liposome or polymer reagents often give very good DNA delivery efficacy, they failed to
efficiently drive siRNA into mammalian cells. The poor performance of liposome
or polymer based siRNA transfection reagents could be linked to the length of the siRNA
anionic segment that is too short to maintain electrostatic cohesion with the
cationic lipids or polycationic polymer. Consequently, siRNA lipoplex or polyplex breaks apart too easily upon
touching polyanionic cell surface. We modified the liposome and polymer with
specific
hydrophobic groups which confers the pH dependent conformational changes (PDCC)
at physiological pH condition and greatly stabilizes siRNA lipolplex or polyplex. The equal important property
resulting from PDCC technology is that siRNA lipoplex or polyplex nanoparticles can be
controlled to be virus-like size by adding specific hydrophobic groups, leading to much better siRNA transfection
efficiency.
Figure 5. A Cartoon
Showing How
pH
Dependent Conformational Change (PDCC) Technology
Works
6.
Ad.MAX™ Technology for Maximum Adenovirus Production:
Ad.MAX™ technology was developed by genetically modifying virus packaging
cell, HEK293 cell and
adenoviral shuttle vector or adenoviral genome for maximum adenovirus production. The core of
this proprietary technology lies in the genetically engineered HEK293 in which adenoviral
replication is kept intact while viral protein expression during adenovirus
packaging process is
significantly suppressed. In combination with a trans-element in adenovirus
genome which recognizes the suppressor cassette of HEK293 cell, Ad.MAX™
system allows maximum adenovirus production with minimum protein expression
during viral packaging.
This system is extremely useful for packaging adenoviral vectors with toxic
genes of interests (GOI),
which will otherwise kill HEK293 during adenovirus replication and production
(Figure 6), leading to dramatic reduction in adenovirus yield. Ad.MAX™ technology eventually allows researchers to construct
all genes (<7.5 kb) to adenovirus.
Figure 6. A Cartoon Showing
How Ad.MAX™ Technology
Works
7. Multi Approaches to Produce
Super Infectious rAAV Particles at Super High Titer:
Not like adenovirus, it is relatively harder to achieve high yield of rAAV
with regular rAAV production procedures. By adopting the following
multi strategies, we have managed to produce super infectious (~30 times
better infectious capability than conventional rAAV) rAAV particles at super high titer (up to 1E+15 GC).
- AAV·HT™ Packaging Cell:
We have screened different HEK293 cell types and developed a highly productive
strain------AAV·HT™ packaging cell which per our validation data produces ~10
times more rAAV particles than regular HEK293.
- Modify rAAV cis Vector: Customer has an option to choose rAAV cis
vector with a truncated WPRE cassette downstream of transgene. Inclusion
of WPRE cassette in rAAV cis vector allows to produce ~8 times more rAAV
particles than rAAV cis vector without WPRE.
- Modify rAAV Capsid: rAAV capsid mutations at several specific sites
significantly (~30 folds better in vitro & in vivo) enhanced rAAV transduction efficiency.
Customers have an option to choose packaging rAAV with mutant capsids to produce
more infectious rAAV vector.
- Package Double-stranded AAV (dsAAV): Double-stranded AAV (dsAAV) also
known as self-complementary AAV (scAAV)
is confirmed to deliver up to 50 times better
transduction efficiency in both vitro and vivo. Custom service is
available to generate and package gene of interests to a scAAV vector*.
- Advanced Double CsCl Ultra-centrifugation Protocol: The main feature of
the advanced protocol is treatment with a special formulated detergent after
3xfreeze/thaw cycle and pre-precipitation step before loading to
ultra-centrifugation. The advanced protocol produces super purified
(clinical trial grade) and super infectious rAAV vector at high yield. See
Figure 8 for details. The differences with conventional
protocol are being framed in red.
Figure 7.
Multi Approaches
to Produce Super Infectious rAAV Particles at Super High Titer
Figure 8. Outline and comparison of advanced protocol and conventional
protocol. The differences in advanced protocol are being framed in red.
8. LentiMAX™ Lentivirus Packaging
System for Maximum Yield:
After co-transfection of lentivector and lentivirus packaging mix, large amount
of double-stranded RNA (dsRNA) will be generated inside of packaging cells. The
accumulation of dsRNA in the cytoplasm will then activate interferon-induced
antiviral defense pathways. Specifically upon binding to dsRNA, the
interferon-inducible protein kinase (PKR) becomes activated. Activated PKR
phosphorylates the eukaryotic initiation factor-2α (eIF-2α), resulting in a
block in protein synthesis initiation. This leads to the inhibition of viral RNA
packaging and translation, which consequently affects the stability and
processing of lentivirus-specific proteins (Figure 9 upper panel). The PKR
inhibitor was confirmed to significantly increase the lentivirus titer.
Based on this principle, we have successfully developed LentiMAX™ lentivirus
packaging system by including a shRNA construct targeting a specific variant of
PKR under U6 promoter. With inhibition of PKR by introduction of PKR shRNA
during lentivirus packaging, we obtained lentivirus at up to 10^7 TU/mL from
supernatant of packaging cells, 10 times higher than conventional packaging
approach (Figure 9 lower panel).
Figure 9. A carton shows how the LentiMAX™ packaging system works.
* Some terms may apply for custom scAAV production and packaging services.