While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 μs–36 ms and PNPs spanning 0.5–2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 μs–22 ms and PNPs spanning 0.5–2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.
Ultrasound (US)-mediated gene delivery (UMGD) of nonviral vectors was demonstrated in this study to be an effective method to transfer genes into the livers of large animals via a minimally invasive approach. We developed a transhepatic venous nonviral gene delivery protocol in combination with transcutaneous, therapeutic US (tUS) to facilitate significant gene transfer in pig livers. A balloon catheter was inserted into the pig hepatic veins of the target liver lobes via jugular vein access under fluoroscopic guidance. tUS exposure was continuously applied to the lobe with simultaneous infusion of pGL4 plasmid (encoding a luciferase reporter gene) and microbubbles. tUS was delivered via an unfocused, two-element disc transducer (H105) or a novel focused, single-element transducer (H114). We found applying transcutaneous US using H114 and H105 with longer pulses and reduced acoustic pressures resulted in an over 100-fold increase in luciferase activity relative to untreated lobes. We also showed effective UMGD by achieving focal regions of >10 5 relative light units (RLUs)/mg protein with minimal tissue damage, demonstrating the feasibility for clinical translation of this technique to treat patients with genetic diseases.
Physical Methods of delivery and cheMical/Molecular conjugates of the oxygen saturation of the Hb in the blood of the injected lobe from 30 to 0 unit upon the injection and recovered smoothly after the injection. Serum analysis showed a transient, 10-to 20-fold increase in hepatobiliary enzymes, including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase after hydrodynamic injections, which recovered within 4 days. We also explored for the first time, the levels of the cytokines following the hydrodynamic injections to the large animals. There was no increase in the systemic inflammatory cytokines of IFN-γ, IL-8, IL-18, and IL-4, although an increase in serum levels of TNF-α, IL-10, MCP-1, canine KC, and IL-6, which were related to vascular stretching representing the sinusoidal expansion was observed. No impacts on their respiratory, cardiovascular conditions, or long-term body weight changes were observed throughout the study. These results of preclinical assessments support the clinical applications of image-guided, livertargeted hydrodynamic gene delivery.
A saline-based DNA solution corresponding to 5% of body weight (NS) instead of 9%, which is used in a canonical HGD, was injected from the mouse tail vein with pCMV-Luc at 10 mg/ml in 5 sec. The activities of 6 kinds of solutes were evaluated to enhance gene delivery efficiency. Four solutes were contrast media (CM1-4) at 60 mg/ml of iodine concentration, which had various kinds of ionicity and osmolality. The others were mannitol (MAN) and glycerin (GLY), which were solubilized as the same concentration as that of a major component in CM1. Liver samples were collected 24 hours after the injections, and luciferase assay and immunohistochemistry (IHC) were conducted. The median values of luciferase activities after the delivery with CM1-4, MAN, GLY, and NS, and HGD were 3.2x10 9 , 9.2x10 8 , 3.5x10 9 , 2.5x10 8 , 1.4x10 9 , 7.3x10 7 , 1.7x10 6 , and 1.6x10 10 RLU/mg of protein, respectively. In IHC, the distributions of luciferase-positive cells after the injections with the solutes were similar to that of HGD. The luciferase activity of CM1 was significantly higher than that of NS (n=5, 5, p<0.01, Mann-Whitney U test). The levels of luciferase activities were not significantly different among CM1-4 (n=5, 3, 3, 3, p=0.16, Kruskal-Wallis test). The luciferase activities between CM1 and MAN were not significantly different from each other (n=5, 3, p=0.57, Mann-Whitney U test). These results suggested that the ionicity, osmolality, and iodine were not a primary factor of gene delivery efficiency in HGD. Therefore, we focused on another property. Elasticity is one of rheological properties of liquids to return to their original shape after removing stress. When fluids pass though a micro-orifice, the passed liquids attract a flow though the orifice. In general, the attracting force gets higher as the temperature becomes lower. The luciferase activity of GLY, which had the lower elasticity than CM1, was not significantly higher than that of NS (n=3, 5, p=0.14, Mann-Whitney U test). Furthermore, median values of luciferase activities of CM1 at 10, 20, 30, and 37°C were 5.0x10 9 , 1.3x10 9 , 9.7x10 8 , and 1.2x10 8 , respectively (p<0.05, Kruskal-Wallis test), while the elasticity of CM1 got higher as the temperature was lower. Based on these results, we speculated that the solution elasticity was a promising candidate to sustain gene delivery efficiency with a limited injection volume in HGD. The next step is to explore a substance providing higher elasticity to a DNA solution, and to evaluate its safety.
Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.
more uniform heat distribution within the tissue and to potentially elevate temperatures in specific areas of tissue in order to target gene expression. GET is a powerful tool that allows for manipulation of expression levels and kinetics. Adjusting the delivery parameters enables maximum control of the expression profile enhancing the potential for a successful therapeutic outcome. The addition of the thermal component allows further control of delivery and reduces the potential for cellular damage. 492.
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