Patent Application
20240091513
The present invention relates to devices, assemblies, and systems for gripping and deforming tissue with vacuum pressure, injecting fluid into the tissue, and optionally electroporating the tissue with electrodes.
Plasmid DNA delivery via electroporation (EP) is a clinically proven vaccination platform with potential safety, immunogenicity, and cost advantages compared to traditional vectors. EP is the temporary permeabilization of cell membranes due to electric fields, which greatly enhances DNA uptake within the electroporated tissue. The design of an EP delivery system can directly impact the magnitude and kinetics of immune responses. Clinical EP devices typically use needle electrodes inserted into skin or muscle tissue at the site of DNA injection, and due to the reliability of this method, needle-based EP has become the standard for in vivo DNA delivery.
As an alternative to intramuscular EP (IM-EP), the skin is an attractive target for EP not only due to its accessibility, which permits shallower EP fields, smaller needles, and increased tolerability, but also due to its rich population of immune cells which may permit fractional dosing. Intradermal EP (ID-EP) using a minimally invasive needle array is more pain-tolerable than IM-EP, and is also capable of generating potent immune responses at a fraction of the IM dose.
Although needle electrodes are most commonly used in the clinic because they generate reliable electric fields throughout a predetermined tissue depth, the advent of ID-EP has brought new focus to minimally- or non-invasive EP alternatives. Most non-invasive EP devices function by contacting the skin surface with non-penetrating electrodes and conducting current through the highly resistive stratum corneum and the underlying viable epidermis, generating a shallow electric field at the site of the ID plasmid injection. A challenge associated with non-invasive ID-EP is the risk of the electrodes losing electrical contact during the procedure, which can result in tissue damage, incomplete treatment, and electrical arcing. To overcome this issue, most noninvasive electrode arrays require constant downward pressure, clamping mechanisms, and/or adhesives to prevent electrodes from dislodging or moving during EP. These requirements can be logistical barriers to adoption or can contribute to inconsistency and/or operator error that can compromise successful vaccine delivery. Furthermore, when the electrodes of noninvasive EP devices are placed flat against the skin, the resulting electric field is quite shallow due to the large voltage drop across highly resistive stratum corneum and the superficial path of current flow, resulting in primarily keratinocytes, Langerhans cells, and other superficial cell types being transfected. In contrast, needle-based EP devices additionally transfect deeper tissue layers with a more diverse cellular population, which can include dendritic cells, adipocytes, fibroblasts, and myocytes.
The foregoing summary, as well as the following detailed description of illustrative embodiments of the present application, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the features of the present application, there is shown in the drawings illustrative embodiments. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown. In the drawings:
The present disclosure can be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the scope of the present disclosure. Also, as used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.
The term “plurality,” as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.
The terms “approximately,” “substantially,” and “about,” as used herein with respect to dimensions, angles, and other geometries, takes into account manufacturing tolerances. Further, the terms “approximately,” “substantially,” and “about” can include 10% greater than or less than the stated dimension or angle. Further, the terms “approximately,” “substantially,” and “about” can equally apply to the specific value stated.
The term “agent,” as used herein, means a polypeptide, a polynucleotide, a small molecule, or any combination thereof. The agent may be a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The agent may be formulated in water or a buffer, such as saline-sodium citrate (SSC) or phosphate-buffered saline (PBS), by way of non-limiting examples.
The term “injectate,” as used herein, means an agent configured for injection in tissue.
The term “intradermal” (ID), as used herein, means within the layer of skin that includes the epidermis (i.e., the epidermal layer, from the stratum corneum to the stratum basale) and the dermis (i.e., the dermal layer).
The term “intramuscular” (IM), as used herein, means within muscle tissue.
The term “adipose,” as used herein, means the layer containing adipocytes (i.e., fat cells) that reside in the subcutaneous layer.
The term “electroporation,” as used herein, means employing an electrical field within tissue that temporarily and reversibly increases the permeability and/or porosity of the cell membranes of cells in the tissue, thereby allowing an agent, for example, to be introduced into the cells.
The term “electroporation field,” as used herein, means an electric field capable of electroporating cells. In instances where an electric field includes a portion that is capable of electroporating cells and another portion that is incapable of electroporating cells, the “electroporation field” refers specifically to that portion of the electric field that is capable of electroporating cells. Thus, an electroporation field can be a subset of an electric field.
The term “zone,” as used herein, means a volume of space, such as a volume of space within tissue.
The term “transfection zone,” as used herein, means a volume of tissue in which transfection occurs, and can be used synonymously with the term “transfection volume.”
The embodiments described below pertain to systems and devices that perform vacuum-assisted delivery of injectates to tissue, particularly a targeted layer of tissue, such as intradermal tissue and/or adipose tissue. These embodiments subject a targeted volume of the tissue (or “tissue volume”) to vacuum pressure (i.e., negative pressure) to deform the tissue in a manner favorable for redistribution of fluid, such as migration of an injected agent, within a target zone in the tissue layer. In particular, an open end of a vacuum device, such as a vacuum cup, is placed in contact with an outer surface of the tissue (e.g., the “skin”) overlying the tissue volume, and vacuum pressure is applied to an interior of the cup, thereby drawing the tissue volume into the vacuum cup, which positionally secures the tissue volume to the cup. This can allow, for example, electrodes positioned within the vacuum cup to generate a predictable, substantially uniform electroporation field within the tissue volume, thereby resulting in a predictable, substantially uniform transfection zone within the tissue volume. The vacuum pressure provided by the embodiments described below has also been observed to provide favorable redistribution of fluid within the tissue volume, including favorable in vivo dispersion of an injectate within the tissue volume, and also favorable in vivo ingress and egress of fluid into and out of the target zone. Stated differently, the vacuum pressure enhances the dispersion of the injectate throughout the tissue to enlarge the transfection zone and also draws more in vivo fluids into the target zone, increasing the amount of cells that are exposed to transfected cells. The inventors have observed that the vacuum-assisted electroporation treatments described throughout this disclosure have resulted in subjects' increased responses to injectates.
The inventors have also observed that application of vacuum pressure can cause transfection within the tissue volume even without electroporation, as more fully described in the '941 Reference.
Furthermore, the embodiments described below can also be adaptable between uses and/or during use without mechanical reconfiguration. For example, during and/or between uses, electrical parameters of the electrodes can be adjusted as needed to manipulate the electroporation field in the tissue volume to achieve favorable treatment results. Additionally or alternatively, the vacuum pressure can be adjusted as needed during and/or between uses to physically manipulate the tissue volume to achieve favorable treatment results. For example, a higher vacuum pressure can be applied to draw a larger tissue volume into vacuum cup and a lower vacuum pressure can be applied to draw a lesser tissue volume into the vacuum cup. In this manner, the same vacuum cup can be employed to target different tissue layers for electroporative treatment by selectively exposing different tissue layers to the electroporation field. Additionally, the vacuum pressure can be pulsed during use to manipulate the mechanical behavior of the targeted tissue, such as to enhance fluid redistribution within the tissue.
The embodiments described below can also be mechanically adaptable between uses and/or during use. For example, the vacuum cup can include an outer housing and an internal insert that defines the cup's internal volume for receiving tissue. The internal insert defines an internal cup diameter, and can be interchangeable with various other internal inserts having different internal diameters. In this manner, the outer housing can be coupled with different internal inserts that provide different internal volume characteristics for adapting the vacuum treatment, such between treatments and/or during a single treatment.
Referring now to
In the illustrated examples, the device 2 includes a plurality of electrodes 8. It should be appreciated, however, that in other embodiments, the device 2 can include a single electrode 8. Thus, although the following description of the illustrated embodiments refers to plural electrodes 8, such disclosure also encompasses embodiments with a single electrode 8, unless otherwise stated. In embodiments with plural electrodes 8, the electrodes 8 are arranged into an array 9 of electrodes 8, which can also be referred to as an electrode array 9.
The vacuum cup 2 includes one or more couplings, such as ports, for connection to one or more external components. For example, the vacuum cup 2 has a first port 12 for providing fluid communication between the vacuum chamber 6 and a vacuum source 106, such as a vacuum pump. The vacuum cup 2 can also have a second port 14 for providing access to circuitry 108 providing electrical communication between the electrodes 8 and an energy source 110, such as a power generator. The vacuum cup 2 can further include an optional third port 16, such as for providing an external tool, such as an injection device 18 carrying an injectate with access to the vacuum chamber 6. As shown, the injection device 18 can be a hypodermic needle, although the vacuum cup 2 can be adapted for use with other types of injection devices 18, including jet injection devices, as described in more detail below. It should also be appreciated that the vacuum cup 2 can optionally be employed after the agent is injected into the tissue 102. In such embodiments, the vacuum cup 2 need not include the third port 16.
The energy source 110 can be in electrical communication with a signal generator 112, such as a waveform generator, for generating and transmitting an electric signal in the form of one or more electrical pulses having particular electrical parameters to the electrodes 8 for electroporating cells within the tissue 102 in the vacuum chamber 6. Such electrical parameters include electrical potential (voltage), electric current type (alternating current (AC) or direct current (DC)), electric current magnitude (amperage), pulse duration, pulse quantity (i.e., the number of pulses delivered), and time interval or “delay” between pulses (in multi-pulse deliveries). The signal generator 112 can include a waveform logger for recording the electrical parameters of the pulse(s) delivered. The signal generator 112 can be in electrical communication with a control unit 114 (also referred to herein as a “controller”), which can include a processor 116 configured to control operation of the electroporation system 100, including operation of the signal generator 112. The processor 116 can be in electronic communication with computer memory 118, and can be configured to execute software and/or firmware including one or more algorithms for controlling operation of the system 100. The processor 116 can be in electrical communication with a user interface 120, which can include a display 122 for presenting information relating to operation of the system 100 and a keypad 124 allowing an operator, such as physician, to input information, such as commands, relating to operation of the system 100. It should be appreciated that the display 122 can be a touchscreen display allowing the operator to input information directly at the display 122. It should also be appreciated that the interface 120 can be computer interface, such as a table-top computer or laptop computer, or a hand-held electronic device, such as a smart-phone or the like.
Referring now to
At least one and up to all of the electrodes 8 extend alongside the interior surface 22. As shown, the electrodes 8 can extend alongside the interior surface 22 between the distal end 10 and the proximal end 24 with respect to a longitudinal direction L oriented along a central axis X of the housing 4. The electrodes 8 of the present embodiment are preferably substantially rigid, although in other embodiments the electrodes 8 can have a measure of flexibility. The electrodes 8 can comprise thin layers of conductive material coupled to (e.g., via coating, deposition, bonding, and/or adhesion) associated substantially rigid, non-conductive support bodies, which can be constructed of plastics or other suitable non-conductive materials. The electrodes 8 can have surface geometries that are substantially conformal with the interior surface 22. The electrodes 8 can be elongate along a direction having a directional component along the longitudinal direction L. The electrodes 8 can also extend alongside the interior surface 22 along a circumferential direction C about the central axis X. As shown in
As shown in
The portion of the internal electrode surface 8z within the contact portion 8e can be referred to as a “contact surface” 8z of the electrode. The contact surface 8z can extend arcuately and concentrically (i.e., can share the same centerpoint) with the interior surface 22 of the housing 4 in a reference plane orthogonal to the central axis X. The contact surface 8z can also have a curvilinear contour that is substantially conformal with the interior surface 22 in a direction along the central electrode axis 8x. The contact surfaces 8z can be smooth, as shown, although in other embodiments the contact surfaces 8z can be textured to enhance grip against the tissue 102, such as with protrusions, dimples, knurls, microneedles, and/or a roughened surface, by way of non-limiting examples. In additional embodiments, a coating or adhesive can be applied to the contact surfaces 8z to improve the grip and/or conductivity between the electrode 8 and the tissue 102. The electrodes 8 can also define a secondary portion 8f that extends from the contact portion 8e to the first end 8a and can be configured to connect with a respective lead of the circuitry 108 for transmitting the electroporation pulse(s) to the electrodes 8.
Referring now to
The housing body 26 defines the ports 12, 14, 16. As shown, each of the first, second, and third ports 12, 14, 16 can be adjacent the proximal end 28 of the housing body 26 and remote from the distal end 10. Stated differently, the ports 12, 14, 16 can be located closer to the proximal end 28 than to the distal end 10 of the housing 4. The first port 12, which can also be referred to as a “vacuum port,” extends from the vacuum chamber 6, through the housing body 26, and to a port coupling 34 for connection with a fitting member 36 that interconnects the vacuum port 12 with the vacuum source 106. The port coupling 34 can include a seat 38 and a tubular extension 40 that extends outwardly from the seat 38 and defines a receptacle, such that the seat 38 defines an inner end of the receptacle. The fitting member 36 can include a fitting member coupling 42 and a cannulated stem 44 extending therefrom. The fitting member coupling 42 can be a tubular extension that interconnects with the tubular extension 40 of the port coupling 34, such as by extending within the receptacle defined by the tubular extension 40 in mating fashion. A one-way valve member 46 can be positioned on the seat 38 (which can be referred to as a “valve seat”). The valve member 46 can extend from the valve seat 38 and within an interior space of the fitting member coupling 42, thereby being interposed in the fluid pathway between the vacuum port 12 and the cannulated stem 44 of the fitting member 36. The valve member 46 can be a duckbill valve, as shown, although in other embodiments the valve 46 can be ball valve or an umbrella valve, by way of non-limiting examples.
The second port 14 can extend opposite the first port 12 and can be configured to allow passage for the circuitry 108, such as wires, through the housing 4 and into contact with the electrodes 8 in the vacuum chamber 6. The second port 14 can also be configured to allow passage for one or more additional components, such as one or more tools and/or one or more sensors, through the housing 4 and into the vacuum chamber 6. While positioned inside the vacuum chamber 6, such tools and/or sensors can be positionally secured with respect to the tissue via vacuum pressure supplied to the chamber 6. The third port 16 can extend from the proximal end 24 of the chamber 6 and along the central axis X. The housing 4 can define a mounting formation 48 at an external end of the third port 16. The mounting formation 48 can be configured to mount a cap 50, such as a puncture stopper, over the third port 16. The mounting formation 48 and the puncture stopper 50 can have complimentary, mating geometries that provide an air-tight seal between the puncture stopper 50 and the third port 16. The puncture stopper 50 can be formed of a material that can be pierced by the hypodermic needle 18 allowing the needle 18 to inject the agent into the tissue 102 drawn into the vacuum chamber 6.
With continued reference to
The vacuum chamber 6 defines a chamber volume V, which is defined between the proximal end 24 of the chamber 6 to the opening 10 along the longitudinal direction L, and can also be at least partially defined by the interior surface 22 of the housing body 26, such as along a direction substantially perpendicular to the longitudinal direction L. In the illustrated example, the direction perpendicular to the longitudinal direction L is a radial direction R that intersects the central axis X. The chamber volume V can also be at least partially defined by the sleeve 52, such as along the radial direction R. The chamber 6 can have a depth L2 measured from the proximal end 24 of the chamber 6 to a reference plane circumscribing the distal end 10 of the housing 4. The chamber depth L2 can be in a range from about 1.0 mm to about 50.0 mm, more particularly in a range of about 3 mm to about 20 mm, and more particularly in a range from about 5 mm to about 17 mm. The chamber 6 also has a base width, such as a base chamber diameter D1, which can be measured along the radial direction R adjacent the distal end 56 of the sleeve 52. The chamber diameter D1 can be in a range from about 1.0 mm to about 50.0 mm, particularly in a range of about 3.0 mm to about 20.0 mm, and more particularly in a range from about 6.0 mm to about 17.0 mm. In the present embodiment, the chamber diameter D1 can be measured between opposed portions of an interior surface 55 of the sleeve 52 near the distal end 56 thereof. In other embodiments, the sleeve 52 can be omitted and the electrodes 8 can be attached directly to the interior surface 22 of the housing 4, for example, by being embedded or at least partially embedded within the housing wall 30. In such embodiments, the vacuum chamber 6, and thus the chamber volume V, can be at least partially defined by the interior surface 22 of the housing 4 and the interior surfaces 8z of the electrodes 8. Accordingly, in such embodiments, the chamber diameter D1 can be measured between opposed portions of the interior surface 22 of the housing 4.
Referring now to
Tests
The inventors conducted numerous tests to evaluate the efficacy of vacuum-assisted treatments, including vacuum-assisted treatments with electroporation and without electroporation. As discussed in the '941 Reference and as discussed below in supplementary fashion, the vacuum cups 2 can isolate a controlled volume of skin tissue and perform noninvasive EP to induce transfection and immunogenicity following intradermal injection of a DNA vaccine. Compared to other noninvasive EP methods, vacuum-assisted intradermal EP (ID-VEP) delivery is uniquely advantageous because suction can maintain intimate electrode-tissue contact for the duration of the procedure while isolating a large tissue volume, and the deformation of skin allows electric fields to propagate directly through the injection site and multiple tissue layers rather than following a shallow, lateral path along the skin surface. The impact of vacuum pressure, independent of EP, was also studied to isolate the impact of the regions of localized increased pressure within skin tissue on both transfection and immunogenicity. The tests herein used computational modeling to predict the impact of key ID-VEP design parameters on electric field distribution, and ID-VEP prototypes were built to evaluate in vivo gene expression and immunogenicity in guinea pigs. Immune studies were performed in guinea pigs using a DNA vaccine targeting MERS-CoV as a model system to demonstrate feasibility of this novel, needle-free electroporation system. Test data and results will now be described.
Device Design and Finite Volume Analysis (FVA)
Referring now to
To solve the model, skin was assigned an electrical conductivity of 0.17 S/m, and subcutaneous fat was assigned an electrical conductivity of 0.05 S/m. These properties were based on a publicly available database that aggregates experimental measurements of various tissue properties, as well as historical in-house readouts. Maxwell's equations were solved and a residual error of 10−7 was reached to achieve convergence. The potential difference between the active electrodes (200V for most models) was defined as a constant boundary condition. To model electric field-dependent increases in tissue conductivity, conductivities were progressively transformed based on electric field magnitude at each iteration using a sigmoid equation for a maximum increase of fourfold as electric field magnitude approached 600 V/cm, as shown in
Animals
All in vivo studies were performed using female Hartley guinea pigs 12-16 weeks in age and weighing approximately 600 g. For the duration of all procedures, guinea pigs were maintained under anesthesia by inhaled isoflurane. Treatment sites were shaved immediately prior to each procedure. For terminal studies, euthanasia was performed by intracardiac injection of pentobarbital. All animal studies were performed under a protocol approved by an Institutional Animal Care and Use Committee.
Plasmid DNA Delivery and Electroporation
Intradermal plasmid delivery for all studies consisted of 100 μL ID injection of plasmid DNA onto the flanks of guinea pigs, using a 0.5 mL insulin syringe equipped with a 29-gauge needle. For ID-EP procedures, the CELLECTRA® ID Array—a minimally-invasive array containing three 3 mm needle electrodes—was immediately inserted into the injection site and EP was performed, delivering two sets of two pulses with an intensity of 0.2 A and a pulse duration of 52 msec. For ID-VEP, the vacuum chamber was placed on top of the injection site, the vacuum pump was powered on, and EP was performed once the pressure reached the set-point, delivering two sets of two pulses with an intensity of 200V and a pulse duration of 100 msec. After EP, the vacuum pump was turned off to release the tissue from the treatment chamber. The duration of the entire ID-VEP procedure, following intradermal DNA injection, was approximately 5-10 seconds, which is comparable to the duration of an ID-EP procedure. A GEMINI™ vacuum pump equipped with analog pressure control knob (VWR, Radnor, PA, USA) was used as a vacuum source for all studies. A custom pulse generator was used to deliver the EP pulses.
Gene Expression
Gene expression studies were conducted in guinea pigs to evaluate the breadth and magnitude of plasmid transfection following ID-VEP with different vacuum and electrical limits. In guinea pigs, ID-VEP delivery of plasmid encoding Green Fluorescent Protein (pGFP) at a concentration of 0.5 mg/mL was performed, using voltage limits of 50, 100, 100, or 200V at both low (−40 kPa) and high (−70 kPa) vacuum strengths. As a control, pGFP was delivered using injection alone (without vacuum), as well as via injection followed by −70 kPa vacuum (without EP). Skin was excised for analysis three days after plasmid delivery, and fluorescence was quantified using a FluorChem R imaging system (ProteinSimple, San Jose, CA, USA). Table 1 summarizes the experimental conditions and number of replicates. Injection sites were also photographed prior to tissue harvest to visually compare signs of superficial tissue damage.
Table 1. Gene expression studies. Guinea pigs received 100 μL intradermal injections of 0.5 mg/mL plasmid encoding GFP. Vacuum strength and voltage were varied. Injection alone, or injection with −70 kPa vacuum but no EP, were used as controls. Parameters of these gene expression studies are shown below in Table 1.
Immunogenicity Studies
FOUR INDEPENDENT IMMUNOGENICITY STUDIES were performed in guinea pigs, each comparing ID-VEP to different delivery systems or different ID-VEP parameters. Vaccinations used 100 μL intradermal injection of 50 μg plasmid encoding MERS-CoV spike protein. ID-EP was performed using the CELLECTRA® 3P, a minimally invasive device that uses three needle electrodes, while ID-VEP was performed using VEP prototypes with parameters described in Table 2. Vaccinations were performed at week 0 and week 2 of each study, and an additional vaccination was performed at week 4 for two of the four studies.
ELISA
ELISA was performed as described previously in P. D. Fisher et al., “Adipose tissue: a new target for electroporation-enhanced DNA vaccines,” Gene Therapy, vol. 24, no. 12, Art. no. 12, December 2017, doi: 10.1038/gt.2017.96. Briefly, 96-well plates (Thermo Fisher) were coated overnight with MERS spike protein antigen (Sino Biological, 40069-V08B) in PBS. Plates were then blocked with 3% bovine serum albumin (BSA) for 2 hours at room temperature (RT) and washed. Serum in PBS-t containing 1% BSA (Millipore Sigma) was added for 2 hours at RT and then washed, and then HRP-conjugated goat anti-guinea pig IgG (Millipore Sigma, A7289) was incubated in wells for 1 hour at RT. Tetramethylbenzidine substrate solution (VWR) was used to develop color for 6 minutes, then methylbenzidine stop reagent solution (VWR) was added to halt the reaction. A Synergy HTX plate reader (BioTek Instruments) was used to measure absorbance at 450 nm.
Endpoint binding titers were defined as the intersection point between a 5-parameter logistic curve for each biological replicate at each time point and a single reference curve fitted to the upper limit of the 99% prediction interval calculated at each dilution for all week 0 samples.
ELISPOT
ELISPOT was performed as described previously in Fisher et al., “Adipose tissue: a new target for electroporation-enhanced DNA vaccines” (supra); K. Schultheis et al., “Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model After Vaccination,” Journal of Visualized Experiments, January 2018, doi: 10.3791/58595; and K. Schultheis et al., “Characterization of guinea pig T cell responses elicited after EP-assisted delivery of DNA vaccines to the skin,” Vaccine, vol. 35, no. 1, pp. 61-70, January 2017, doi: 10.1016/j.vaccine.2016.11.052. At 2, 4, and/or 6 weeks following initial vaccination, 3 mL of peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were isolated and plated at a density of 1×105 cells per well on X-coated 96-well Millipore IP plates. Plated PBMCs were stimulated with 5 MERS peptide pools (GenScript) and IFN-γ production was measured using biotinylated mouse monoclonal anti-guinea pig IFN-γ antibody and BCIP/NBT detection reagent substrate. A CTL-Immunospot S6 ELISPOT plate reader and its included software was used to count spot-forming units (SFU). Spot counts were normalized to baseline by subtracting SFU of wells that did not receive any peptide.
Statistical Analysis
Prior to analysis, gene expression data were converted from raw fluorescence values with arbitrary units into ratios relative to a reference group. All immunogenicity data—binding titers for antibody responses and SFU for cellular responses—were log-transformed prior to analysis. For direct comparison of two study groups, a t-test with Welch correction was performed. Otherwise, simple linear regression or multiple linear regression were performed at each time point of each study. Since this work is exploratory in nature and the objective of these studies was to generate new hypotheses for future work, multiplicity corrections which account for Type I error but may increase Type II error were not performed.
Results
By way of summary, the results described below demonstrate, among other things, that DNA vaccines delivered via ID-VEP are highly immunogenic—comparable to needle-based ID-EP—and the delivery procedure itself is noninvasive, repeatable, and requires only a few seconds to perform. Design parameters of the vacuum cup 2, such as chamber size, pulse intensity, and vacuum strength, were shown to influence DNA vaccine immunogenicity and gene expression, and the use of FVA modeling supports the hypothesis that the breadth and magnitude of the electric field can predict and explain performance of ID-VEP systems. Negative pressure was shown to independently enhance transfection and synergized with EP delivery by immobilizing a fixed volume of skin securely against the electrodes lining the EP chamber. This technique provides a reliable, repeatable platform to perform noninvasive EP, and the impact of negative pressure on skin tissue and its interaction with electroporation merits continued research. More specific results are described below.
FVA and Prototype Development
With reference to
In each of
Referring now to
These results demonstrate that, after normalizing the FVA predictions to the mean in vivo current of a reference group for each study, the FVA was capable of accurately predicting the impact of ID-VEP chamber diameter D1, pulse voltage, and vacuum strength on delivered current. As shown in
These results also demonstrate that FVA can be used to prospectively screen the performance of ID-VEP prototype cups 2 and devices and can serve to both inform future design decisions and explain experimental readouts. Reducing pulse voltage from 200V to 100V resulted in electric field magnitudes which were generally less than 100 V/cm, which is below the threshold typically associated with successful transfection, although modeling noninvasive electroporation of skin has been historically difficult due to the less-understood contributions of the stratum corneum. Smaller ID-VEP diameters showed dramatically stronger field strengths for a given voltage, suggesting that the impact of reduced electrode surface area is outweighed by the reduction in inter-electrode spacing. Despite the complex geometry and simplifying assumptions used to build these models, it is promising that the electrical impact of various delivery parameters predicted by FVA closely matches the electrical trends observed in vivo, and these results suggest that FVA may have predictive value when evaluating ID-VEP designs. Furthermore, immune responses (discussed below) also correlated with the observations from simulated electric fields: stronger responses were associated with more intense electric fields.
The FVA performed here assumed that each tissue layer is homogeneous, with electrical conductivity that increases in a sigmoid fashion with electric field strength. Additionally, the stratum corneum was excluded from the model because very thin features can introduce artifacts in finite volume models, and skin was assumed to be a single layer rather than a multilayered composite material. Lastly, thermal effects were not considered in this model. Because these models consistently overestimated total electric current compared to in vivo, it is possible that further optimization of the conductivity transformation and inclusion of the stratum corneum may yield more accurate results. Once normalized to the baseline in vivo readouts, the models were quite accurate and provide evidence that electric field distributions predicted by these simulations can model ID-VEP device performance.
Gene Expression
Studies evaluating in vivo GFP expression of ID-VEP in guinea pigs indicated that stronger vacuum pressure enhances gene expression regardless of EP voltage. For example, with reference to FIG. 36B of the '941 Reference, a combination of 100 V ID-VEP voltage and −70 kPa vacuum strength produced the strongest overall GFP expression on the skin surface in that test. With continued reference to FIG. 36B of the '941 Reference, across injection sites receiving electroporation, increasing vacuum pressure from −40 kPa to −70 kPa doubled GFP expression. Additionally, increasing EP voltage from 50 V to 100 V resulted in a 1.4-fold increase in expression, while further increasing from 100 V to 200 V reduced visible expression by nearly 70%. In the absence of electroporation, briefly applying −70 kPa vacuum pressure to the injection site resulted in more than threefold higher GFP expression on the skin surface than intradermal injection alone (Welch t-test: Fluorescence ratio (95% CI)=3.39 (1.16, 5.62), p=0.038).
With reference to FIG. 36A of the '941 Reference, in addition to quantification of GFP fluorescence, expression patterns at treatment sites receiving high vacuum strength were photographed and visually compared. ID injection of pGFP caused mild erythema even in the absence of vacuum or EP, and resulting GFP expression was concentrated into a small, centralized region. The addition of −70 kPa vacuum strength alone created a broader, circular region of both erythema and transfection, and the further addition of ID-VEP voltages up to 100V increased the intensity, but not the breadth, of fluorescent signal. At 200V, the regions with the darkest erythema corresponded with a complete absence of fluorescence signal.
Parameters involved with the tests shown in FIGS. 36A-36B of the '941 Reference are further identified in Table 3 below. In Table 3, multiple linear regression of superficial GFP expression following ID delivery with combinations of ID-VEP vacuum strengths and voltages, using vacuum pressure and voltage as predictors of fluorescence. Backward difference contrast coding was used for the Voltage variable to provide progressive comparisons between increasing voltage groups.
Referring now to
The gene expression analysis herein revealed potential synergy between vacuum and EP effects on local protein production. Even in the absence of EP, application of vacuum pressure transfected a large region of superficial skin cells. This change in expression breadth suggests that the skin deformation caused is physically disrupting cell membranes and transfecting cells, and possibly redistributing plasmid laterally through the skin to physically contact more cells. The addition of EP showed a voltage-dependent increase in GFP fluorescence intensity, but not breadth, suggesting that the two techniques may be complementary: vacuum can increase the area of transfection, while EP delivers more plasmid copies over the same area. While superficial expression is primarily due to epidermal skin cells, imaging the underside of skin sections highlights expression in the deeper dermal layers, the subcutaneous adipose tissue, and the panniculus carnosus which is present in rodent skin. Although expression in the underlying layers was not detectable in all samples, it is notable that expression in deeper tissue layers was only detectable at treatment sites that had received electroporation. This finding suggests that noninvasive ID-VEP is capable of transfecting superficial as well as deeper layers within skin tissue—similar to ID-EP devices that use needle electrodes—whereas intradermal injection alone, with or without vacuum, is limited mainly to the superficial epidermis. Future ID-VEP studies should investigate these differences in protein expression patterns at the histological level in order to more fully understand the spatial distribution of transfection and the cell populations involved.
Interestingly, when voltage was increased from 100V to 200V, superficial GFP expression in skin sharply declined. This decrease can be attributed to more prominent erythema associated with skin irritation due to higher pulse energy, which effectively quenches visible GFP signal. Therefore, superficial GFP expression readouts are probably unreliable when higher pulse energies are employed, since redness of the tissue may be masking positive signal. It was encouraging to note that none of the ID-VEP treatment sites exhibited any visible signs of irritation beyond transient erythema at the injection site, even at high voltages. These promising initial results suggest that ID-VEP is well-tolerated and causes minimal tissue damage, although future studies should be conducted to more thoroughly characterize the tissue-level and cell-level responses to the ID-VEP procedure.
Immunogenicity
Study 1
Referring now to
All animals (15/15) in this study seroconverted after the second vaccination, while after a single vaccination the seroconversion rates were 4/5 for −70 kPa/200V, 2/5 for −40 kPa/200V, and 3/5 for −70 kPa/100V (
Study 2
With reference to FIGS. 37A-37B of the '941 Reference, the results of a head-to-head immunogenicity study are shown between ID-EP using intradermal needle electrodes and needle-free ID-VEP. Guinea pigs received pMERS DNA vaccine delivered using ID-EP or ID-VEP. A total of three 50 μg vaccinations were performed, one every two weeks beginning at week 0. In FIG. 37A of the '941 Reference, humoral response kinetics of ID-EP and ID-VEP delivery methods are shown, with arrows indicating treatments (although this Figure shows midpoint binding titers, endpoint binding titers were also measured and charted). In FIG. 37B of the '941 Reference, cellular responses are shown, measured at week 2 and week 4 following initial vaccination. IFN-γ spot-forming units (SFU) are presented as the sum of five peptide pools' individual responses.
As demonstrated in this study, ID-VEP appeared to generate a more rapid humoral response than ID-EP (FIG. 37A of the '941 Reference). By 2 weeks following the first vaccination, all animals in both groups had seroconverted and ID-VEP was associated with significantly higher titers than ID-EP. At subsequent time points following additional vaccinations, there was no significant difference between ID-VEP and ID-EP. Cellular responses, measured at week 2 and week 4 of the study (FIG. 37B of the '941 Reference), increased substantially after the second vaccination for each delivery method, and spot counts were comparable for ID-EP and ID-VEP at each time point.
Study 3
With reference to FIGS. 39A-39B of the '941 Reference, a separate study was conducted to evaluate the isolate the impact of vacuum pressure alone (ID-Vacuum) on immunogenicity. Guinea pigs received a pMERS vaccine delivered using three different modes: (1) using an ID-VEP cup 2, (2) ID delivery followed by vacuum fixation using a cup 2 (but without EP); and (3) ID delivery without vacuum or EP. Two 50 ug vaccinations were performed at study weeks 0 and 2. In FIG. 39A of the '941 Reference, humoral responses are shown, with arrows indicating treatments (although this Figure shows midpoint binding titers, endpoint binding titers were also measured and charted). In FIG. 39B of the '941 Reference, cellular responses are shown, measured at week 4. IFN-γ spot-forming units (SFU) are presented as the sum of five peptide pools' individual responses.
As demonstrated in this study, ID-VEP was associated with faster and stronger humoral immune responses compared to ID-Vacuum or ID delivery alone. After one vaccination, all ID-VEP animals (5/5) had seroconverted, compared 1/5 for ID-Vacuum and 0/5 for ID delivery without vacuum or EP. By week 4—two weeks after a second vaccination—each of the ID-Vacuum and ID groups showed seroconversion in 3/5 animals. The presence of electroporation was significantly associated with higher titers at week 2, although this effect was not significant at week 4 due to the highly variable, partial responses in the ID and ID-Vacuum groups. In contrast, the use of vacuum alone did not generate any measurable difference in endpoint titers compared to ID delivery alone at either time point, although it is notable that the two overall strongest responses at week 4 both received ID-Vacuum delivery. Cellular responses were measured at week 4 (FIG. 39B of the '941 Reference), and the use of electroporation was significantly predictive of higher spot counts while the use of vacuum alone was not.
Study 4
With reference to FIGS. 35A-35B of the '941 Reference, a separate study was conducted to evaluate the impact of ID-VEP chamber diameter on immunogenicity. Guinea pigs received a pMERS DNA vaccine delivered using ID-VEP cups 2 with chamber diameters D1 of 8 mm, 10 mm, 12 mm, and 15 mm. A total of three vaccinations were performed, beginning at week 0. In FIG. 35A of the '941 Reference, humoral responses are shown, measured every two weeks for 6 weeks, with arrows indicating ID-VEP treatments (although this Figure shows midpoint binding titers, endpoint binding titers were also measured and charted). In FIG. 35B of the '941 Reference, cellular responses are shown, measured at week 4 and week 6 following initial vaccination. Spot-forming units (SFU) are presented as the sum of five peptide pools' individual responses
As demonstrated in this study, ID-VEP chamber diameter D1 was inversely associated with humoral immune responses, with the smallest (8 mm) diameter generally providing the strongest and most rapid responses. The relationship between ID-VEP diameter and log-transformed binding titers was significant at each time point, and each millimeter of decreasing ID-VEP diameter was associated with a geometric mean endpoint titer increase of approximately 0.2 at week 2, 0.1 at week 4, and 0.09 at week 6. Two weeks after the initial vaccination, the response appeared to be plateauing for the 8 mm and 10 mm diameters. Cellular responses, collected by IFN-ELISpot two weeks after the second and third vaccinations, were generally the strongest for ID-VEP chamber diameters ranging from 8 mm to 12 mm, and increasing the diameter to 15 mm appeared to be associated with lower, more variable spot counts, although the impact of diameter on spot counts was not significantly predictive (see FIG. 35B of the '941 Reference).
A summary of test parameters and result parameters of the foregoing Studies 1-4 is provided below in Table 4. Linear regression models were used for statistical analysis of each immunogenicity study. At each week of each study, a linear regression model was fitted to ELISA and ELISpot readouts, and in cases where multiple independent variables are listed, multiple linear regression was performed. Significant results (p<0.05) are bolded.
In these immunogenicity studies, ID-VEP was shown to be highly immunogenic, and the magnitude of gene expression and immune responses was directly influenced by the device geometry, the EP pulse parameters, and the vacuum pressure. ID-VEP chamber diameters D1 of from 8-10 mm are preferable based on these results, and only a few additional millimeters of size dramatically reduced immune responses.
Recently, a method of suction-mediated transfection to skin tissue was shown to increase localized transfection and immunogenicity of DNA vaccines in rats, as published in E. O. Lallow et al., “Novel suction-based in vivo cutaneous DNA transfection platform,” Science Advances, vol. 7, no. 45, p. eabj0611, doi: 10.1126/sciadv.abj0611. Interestingly, while the results disclosed above agree with the Lallow et al. findings that localized transfection is enhanced through negative pressure alone, the tests described in the present disclosure could not reproduce the finding that negative pressure alone also increases immunogenicity of DNA vaccines. Rather, the results herein show in guinea pigs that there was no meaningful enhancement in immunogenicity through suction alone, and only the addition of EP led to a dramatic improvement in humoral and cellular immune responses. This may be due to the choice of species for these two different studies; rats have substantially thinner stratum corneum, viable epidermis, and dermis thickness than human skin, whereas guinea pigs have skin more structurally similar to humans, with similarly thick epidermal and dermal layers. These divergent findings highlight the importance of multiple animal models to understand the complex phenomena involved in vacuum-mediated DNA delivery, and also suggest that the addition of electroporation may be necessary when delivering DNA vaccines to thicker, more human-like skin.
Human skin thickness (epidermis+dermis) ranges from 0.5 mm to 2 mm, which explains the design of existing ID-EP systems that target the first few millimeters of skin tissue. However, several studies have shown that subdermal cells can be transfected during ID-EP, and prior work from this group showed that subcutaneous fat contributes strongly to DNA vaccine immunogenicity even in the absence of epidermal or dermal transfection. For these reasons, ID-VEP is a rational next step in EP device design, because it can easily immobilize a controlled volume of skin and subcutaneous fat and perform electroporation across that entire volume noninvasively.
In the four immunogenicity studies herein, ID-EP parameters of 200 V pulse intensity and −70 kPa vacuum strength generated the strongest humoral and cellular responses compared to lower vacuum or voltage settings, which agrees with FVA predictions and contrasts with the gene expression findings as explained above. These results raise the possibility that even higher voltages than 200 V may continue to enhance immunogenicity, though it should be considered that increased pulse intensities are also associated with discomfort, thermal damage, and irreversible electroporation. Because the ID-VEP techniques described herein are intended to be highly-tolerable procedures causing no lasting tissue damage, 200 V was selected as a rational upper limit for these studies, though higher voltages merit further investigation since ID-VEP even at 200 V caused only transient irritation. In addition to pulse intensity alone, future work should also consider other combinations of pulse widths and inter-pulse delays in order to provide a more complete picture of how each electrical parameter impacts immunogenicity and superficial tissue damage.
In a head-to-head comparison between ID-VEP and needle-based ID-EP, ID-VEP generated equivalent cellular responses and superior humoral response kinetics and magnitude through 6 weeks of observation. This improvement may be attributable to the broader, more homogeneous electric field that is generated in ID-VEP compared to needle ID-EP. The reason for such effective electric field coverage is the vacuum component of ID-VEP, which positions skin tissue such that electric fields penetrate through all skin layers along two perpendicular axes. These results suggest that ID-VEP overcomes some of the challenges associated with noninvasive EP devices—namely, shallow and inconsistent electric field generation—which have generally led to lower immunogenicity compared to needle-based ID-EP.
Although ID-Vacuum (without EP) delivery generated substantially higher GFP expression than ID delivery (without vacuum or EP), both methods resulted in comparably poor immunogenicity, with virtually no measurable humoral response after 1 vaccine and partial seroconversion after a second vaccination. In these studies, only the combination of vacuum pressure and electroporation was capable of inducing consistent, high-magnitude cellular and humoral responses. This result suggests that transfection alone may not sufficiently predict immune responses to DNA vaccines. Based on these findings, it is proposed that the combination of transfection breadth (due to vacuum pressure) and transfection efficiency (due to EP) act synergistically to increase overall gene expression, while EP is the dominant factor driving immunogenicity. Previous studies have shown that EP can be synergistically or additively combined with other delivery methods such as sonoporation, particle delivery, and jet injection. In addition to its well-characterized enhancement of gene expression, which was corroborated in this study, EP has also been theorized to act as a physical adjuvant since electrical energy produces localized tissue damage which can prompt the localized recruitment of innate and adaptive immune cell types, including macrophages and T-cells. This adjuvant effect may further explain the superiority of ID-VEP compared to ID-Vacuum delivery despite their similar gene expression profiles, and it may also explain why smaller ID-VEP diameters (which had stronger electric fields and more electrical “hot spots”) were associated with stronger immune responses.
The ID-VEP cup 2 prototypes described herein were designed and built with the objective of encompassing sufficient skin and subcutaneous tissue to transfect an entire 100 μL injection site, but not gathering so much tissue that excess tissue is electroporated needlessly. Therefore, most studies were performed using a 10 mm diameter ID-VEP cup 2 having a chamber diameter D1 of 10 mm, which is similar to the diameter of a standard 100 μL intradermal fluid bleb. The smallest ID-VEP chamber diameter D1 tested was 8 mm, and smaller sizes were not tested because the entire injection volume would be unable to fit inside the EP chamber. It should be appreciated, however, that larger or smaller ID-VEP devices may be appropriate for different injection volumes or desired depths of transfection.
The work described here used a MERS-CoV DNA vaccine as a model plasmid to develop the ID-VEP system; this research is now of particular relevance due to the ongoing COVID-19 pandemic caused by another coronavirus, SARS-CoV-2. It is encouraging, therefore, that ID-VEP appears to induce rapid and potent humoral responses, which have shown to correlate with protection from infection, hospitalization, and death, as well as strong cellular responses, which we hypothesize will be critical to provide long-lasting and adaptive protection against current and future mutations. A well-rounded immune response, generated with a noninvasive, easy-to-use, and well-tolerated ID-VEP device, is an attractive product profile in these pandemic situations where widespread vaccination is required.
Although the disclosure has been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. For example, features of the various embodiments described herein can be incorporated into one or more and up to all of the other embodiments described herein. Moreover, the scope of the present disclosure is not intended to be limited to the particular embodiments described in the specification. As one of ordinary skill in the art will readily appreciate from that processes, machines, manufacture, composition of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure.
This application claims the benefit of U.S. Provisional Application No. 63/375,979, filed Sep. 16, 2022, the entire contents of which are incorporated herein by this reference. The present application is related to U.S. Patent Publication No. 2021/0290941 A1, published Sep. 23, 2021, in the name of Fisher, et al. (“the '941 Reference”), the entire contents of which are incorporated by this reference.
| Number | Date | Country | |
|---|---|---|---|
| 63375979 | Sep 2022 | US |
