Symmetric Wave and Applicator
Optimization for DNA Vaccine
Transfection Protocol
By James Ingram
Introduction
Clinical application of DNA vaccines is an
emerging field with prospects for engineering a
targeted immunotherapy response within the
body. With the arrival of SARS-CoV-2
(COVID-19), vaccine synthesis and application
have a renewed focus from multiple disciplines
in the scientific community. It is my goal, under
the supervision of Dr. Mike Sano, to assist in
developing the optimal protocol for non-viral
immunotherapy using electroporation within the
derma to transfect cells with DNA vaccines.
Background Information
DNA vaccines are solutions composed of
bacterial plasmids that contain an antigen
expression along with promoter and encoding
sequences [​1​]. This plasmid vector contains a
protein of interest to be produced by the cells
and presented on the surface to induce CD8+
cytotoxic T cell (CTL) response. CTLs then
prevent virus replication through mechanisms
such as cytolysis and cytokine production [​2​].
Electroporation is the process in which cells
become permeable through inducing an electric
field across the cell’s surface [​3​]. By creating
pores, DNA and other molecules can enter the
cell when normally impossible. Controlling the
level of permeability, from surface
destabilization to cellular death (ablation),
depends on factors not limited to waveform
(symmetrical), dosage (sum of pulse delivered,
i.e. time), and geometry (electrode) [​3​]. This
concept is the main focus of the project.
Research Plans
Dr. Sano’s lab is focused on creating the
successful protocol for derma transfection using
electroporation. The task that I have been
assigned is to model and test different forms of
symmetric wave propagation to maximize
transfection and minimize ablation. In addition
to optimizing transfection, I will be assigned to
create a new electrode for this process. The
electrode will need to administer the vaccine
subdermally and apply the electric field. By
preventing a two-step system vaccine uptake
will be increased (less mechanical trauma).
Fig. 1.​ A two component electrode designed in
SolidWorks​.
Currently, a base model for the electrode exists
that I have created previously (Fig. 1). This
model can be easily adapted to fit the needs of a
two-step process by implementing the wiring
into the applicator and exchanging the
depression system for vaccine administration.
Internal pressure similar to a syringe can be used
for dispensing, with numerous possibilities of
loading the vaccine into the system. A luer
locking system to exchange needles after
treatment is a simple internal adjustment. The
ring structure of the system can be easily altered
as well to fit clinical injection angle (45​°​).
A second set of safety measures is
implementable, while the pulse generator has its
own, ideally the applicator will be able to house
a microprocessor (​Arduino Nano​) to receive and
send information on the device. This, and
implementation of a servo for vaccine
administration, depends on interference caused
by electromagnetic radiation during the process
of electroporation.
Fig. 2.​ Conceptual tissue model of electroporation.
Cylindrical region represents tissue while the grey
regions are electrode parts. Gradients represent the
project’s desired goal.
Symmetric waveforms are the project's second
component which will have a timeline and
testing process. By implementing the proper
symmetric waveform, ablation can be
significantly reduced while vaccine uptake is
increased. Fig. 2 is a visual representation of the
overall process by model. The goal is to
decrease the red zone (ablated) while
maintaining the radius of the green (transfected).
Prior research shows that asymmetric waves
lower the lethal threshold, making ablation
easier at low dosage [​3​]. Symmetric waves have
a smaller, more predictable ablative region when
used at high frequencies compared to
asymmetric waves at the same frequency [​3​]. By
using symmetric waves to increase the
threshold and using high frequency pulses,
ablation will become more difficult and more
cells can sustain transfection. As there is little
research showing optimal parameters, due to
geometric differences, a binary search algorithm
will be employed starting from scratch.
Methods
Electrode design will work in conjunction with
waveform optimization. As idyllic parameters
are defined, geometry and design will adjust to
further the project’s end goal. Parameters to be
kept constant during the experiment will begin
with voltage, temperature, and cell model. The
voltage will be kept constant at 500 V and the
temperature will be at room (20 ​°C). The cell
model consists of a 6 well plate: glioblastomas
will adhere to the base under fixative collagen.
Each well will contain approximately 250,000
cells. The parameters to be varied are dosage,
pulse duration, and molecule size. The
molecules then increase in size as tests become
more successful (Propidium Iodide, FITC
Dextran, Enhanced GFP) . This will determine
the size of the pores that can form on the surface
of the cell without reaching the lethal threshold
or decreasing the model’s viability. Success will
be measured by a percentage of cells alive,
expressing, and ablated. Low percentage of
ablation and high percentage of viable cells
transfected. Viability and expression studies will
be performed through referencing control
ablations during microscopy—without the
molecule—and image processing in MATLAB.
Cell model values will be defined when
experimentation begins.
 Fig. 3.​ A flowchart showing the timeline/iterative process for optimizing a DNA Vaccine protocol.

binary search process begins until success is

determined (Fig. 4). The next molecule
undergoes the previous successful parameters
and either succeeds or the binary search is
implemented. During this process, data will be
collected and compared to an endothelial model
which better represents ​in vivo.​ Once target
dosage is found for all molecules, the process
begins again with changing pulse duration to
optimize further. Lastly, waveforms will be
investigated similarly, by varying forms of
symmetry.

Fig. 4.​ A binary search algorithm of determining Future Research

successful dosage (green) compared to ablation When optimal parameters are defined, electrode
dosage (red). Success was earlier defined in ​Methods.​
geometry will be adjusted further to ease the
procedure towards ​in vivo​ testing (if not during
Fig. 3 conceptually represents the approach that
the ​in vitro​ process). Modeling and simulation of
will be taken. Lethal precedence has already
the electrode will be performed in SolidWorks
been established with the parameters of 100 µs
and COMSOL. Versions will be 3D printed to
pulse, 0.02 s dosage at 500 V for asymmetric
prevent design creep and ensure functionality. ​In
positive waves [​3​]. The same parameters will
vivo​ will not be performed during this project.
need to be tested for symmetric waves. Next the
References

[1] Koprowski, H, and D.B. Weiner. 1998. DNA Vaccination/ Genetic Vaccination.
Spriner-Verlag, Heidelberg, 198p.

[2] Schirmbeck, R. and Jorg Reimann. April 2001. Revealing the Potential of DNA-based
Vaccination: Lessons Learned from the Hepatitis B Virus Surface Antigen. Biol. Chem.,
382:543-552.

[3] M. B. Sano, R. E. Fan, and L. Xing, “Asymmetric Waveforms Decrease Lethal Thresholds in
High Frequency Irreversible Electroporation Therapies,” Scientific Reports, vol. 7, no. 1, Art. no.
1, Jan. 2017, doi: 10.1038/srep40747.