Methods and systems for improved nucleic acid delivery via ultrasound

Abstract

Disclosed herein are sonoporogenic methods that allow for repeating sonoporogenic gene therapy treatments in a safe and efficient manner to improve nucleic acid delivery and expression in target cells. The methods include a combination for multiplex application of ultrasound in combination with application of an acoustically active agent and a nucleic acid payload to increase delivery of the nucleic acid payload to target cells in the subject, thereby increasing gene transfection and expression, and in some cases, in some cases, the delivery of the nucleic acid payload to the target cells in the subject. A process to increase the persistence of gene expression following treatment by sonoporosis-based gene therapy. The methods disclosed herein can include providing ultrasound energy to a plurality of locations in a target tissue comprising the target cells, and can apply ultrasound energy during a plurality of repeatable treatment procedures.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 462,515, filed April 27, 2023; U.S. Provisional Patent Application No. 63 / 516,489, filed July 28, 2023; U.S. Provisional Patent Application No. 63 / 537,160, filed September 7, 2023; U.S. Provisional Patent Application No. 63 / 625,277, filed January 25, 2024; U.S. Provisional Patent Application No. 63 / 462,516, filed April 27, 2023; and U.S. Provisional Patent Application No. 63 / 516,491, filed July 28, 2023, each of which is incorporated herein by reference in its entirety for all purposes. Background Technology

[0002] Gene therapy, which involves transfecting functional copies of genes into cells, has been proposed as a potential approach to treating genetic diseases. However, existing gene therapy techniques using ultrasound or sonoporation have significant drawbacks, such as low transfection rates, insufficient gene expression, and short duration of gene expression, hindering their clinical development and commercialization. There remains a need in the field for effective gene therapy technologies that can safely, effectively, and persistently transfect genes into cells in a subject's organs or tissues. Summary of the Invention

[0003] One possible approach to achieving safe and effective gene expression using sonopore-based gene therapy is repetitive gene therapy, as it can potentially be reapplied, unlike viral vector-based gene therapy. However, determining suitable protocols and conditions for safely and effectively repeating sonopore-based gene therapy remains a challenge, as repeating ultrasound-based gene therapy can lead to substantial cell damage, inflammation, and / or death due to the ultrasound treatment and gene transfection process, and such sonopore-based procedures have not been shown to provide safe and effective therapeutic treatment, thus demonstrating the risks of reapplying sonopore-based gene therapy. Improved methods for delivering and expressing enhanced nucleic acids to target cells represent an improvement over existing technologies.

[0004] This document discloses a sonopore-forming method that allows for the safe and efficient repetition of sonopore-based gene therapy treatments to improve nucleic acid delivery and expression in target cells. The method includes a combination of multiple applications of ultrasound with the administration of a sonoactive agent and a nucleic acid payload to increase the delivery of the nucleic acid payload to target cells in a subject, thereby increasing gene transfection and expression, and in some cases, increasing the persistence of gene expression after sonopore-based gene therapy treatment. The disclosed method may include providing ultrasound energy to multiple sites in a target tissue containing target cells, and the ultrasound energy may be applied in multiple treatment sessions that can be repeated without significant cell damage, inflammation, and / or death following the treatment sessions. The disclosed method may include applying or reapplying sonopore-forming treatment processes at different intervals, applying ultrasound to multiple sites in a target organ or tissue, and applying ultrasound energy with different parameters to safely and efficiently deliver nucleic acid payloads. Such ultrasound parameters may include applying with reduced power, applying with a reduced mechanical index, alternating between high and low mechanical indices, applying short flash pulses at a high mechanical index, and / or applying them during a short total treatment period to obtain beneficial results.

[0005] The present invention discloses a method for delivering a nucleic acid payload to target cells of a subject, the method comprising: applying a first treatment process to the subject, the first treatment process comprising: applying an amount of a first treatment composition to the subject, the first treatment composition comprising: i) a nucleic acid payload, and ii) multiple acoustic agents; applying ultrasonic energy to target cells at a first location in a target tissue of the subject; and applying ultrasonic energy to target cells at a second location in a target tissue of the subject, wherein the first location and the second location are different; and applying a second treatment process to the subject after the first treatment process, the second treatment process comprising: applying an amount of a second treatment composition to the subject, the second treatment composition comprising: i) a nucleic acid payload, and ii) multiple acoustic agents; applying ultrasonic energy to target cells at a third location in a target tissue of the subject; and applying ultrasonic energy to target cells at a fourth location in a target tissue of the subject, wherein the third location and the fourth location are different. In some embodiments, the method includes: administering a subsequent treatment to the subject after a second treatment process, the subsequent treatment process including: administering to the subject a quantity of a first treatment composition or a second treatment composition, the composition comprising: i) a nucleic acid payload, and ii) multiple acoustic agents; administering ultrasonic energy to target cells at a fifth location in the subject's target tissue; and administering ultrasonic energy to target cells at a sixth location in the subject's target tissue, wherein the fifth and sixth locations are different. In some embodiments, the method includes, during the first treatment process, administering ultrasonic energy to target cells at a subsequent location in the subject's target tissue after administering ultrasonic energy to a second location. In some embodiments, the method includes, during the second treatment process, administering ultrasonic energy to target cells at a subsequent location in the subject's target tissue after administering ultrasonic energy to a fourth location. In some embodiments, the method includes, during the subsequent treatment process, administering ultrasonic energy to target cells at a subsequent location in the subject's target tissue after administering ultrasonic energy to a sixth location. In some embodiments, the second treatment process occurs more than 6 hours after the first treatment process but within 10 days of the first treatment process. In some embodiments, the second treatment process occurs more than 21 days after the first treatment process. In some embodiments, the first therapeutic composition and / or the second therapeutic composition are administered intravenously via a peripheral vein. In some embodiments, the nucleic acid payload comprises a therapeutic transgene of greater than 4.7 kbp in length.In some embodiments, the therapeutic transgenics include FVIII, COL4A5, or PKD1, GLP-1, INS, Reg3g, MafA, PDX-1, NUEROG3, NGN3, DRYK, DYRK1A, DYRK1B, factor VIII, factor IX, PKD2, COL4A3, COL4A4, Klotho, Smad7, TGF-β, SLC7A1, SLC3A9, UMOD, REN, HNF1B, MUC1, SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, NPHS1, NPHS2, CTNS, or combinations thereof. In some embodiments, administering the first therapeutic composition in the stated amount comprises administering a first dose of the first therapeutic composition and a second dose of the first therapeutic composition to the subject during a first treatment process. In some embodiments, during or after administering the second dose of the first therapeutic composition during the first treatment process, ultrasound energy is administered to a second location in the target tissue of the subject. In some embodiments, administering the amount of the first therapeutic composition during a first treatment includes administering at least a third dose of the first therapeutic composition. In some embodiments, the method includes administering ultrasound energy to a subsequent location on the subject during or after administering the third dose during the first treatment. In some embodiments, administering the amount of the second therapeutic composition during a second treatment includes administering a first dose of the second therapeutic composition and a second dose of the second therapeutic composition to the subject. In some embodiments, during or after administering the second dose of the second therapeutic composition during the second treatment, ultrasound energy is administered to a third location in the subject's target tissue. In some embodiments, administering the amount of the second therapeutic composition during a second treatment includes administering at least a third dose of the second therapeutic composition. In some embodiments, each dose of the first or second therapeutic composition is administered by intravenous injection. In some embodiments, intravenous injection is administered over discrete time intervals. In some embodiments, the discrete time interval does not exceed 60 seconds, 120 seconds, or 180 seconds. In some embodiments, the first location and the third location in the target tissue are the same location. In some embodiments, the first location and the third location in the target tissue are different locations. In some implementations, during the first treatment, the first location and the subsequent location in the target tissue are the same location. In some implementations, during the first treatment, the first location and the subsequent location in the target tissue are different. In some implementations, the second location and the fourth location in the target tissue are the same location. In some implementations, the second location and the fourth location in the target tissue are different.In some embodiments, during the second treatment, the target tissue and the subsequent location within the target tissue are the same location during the second treatment. In some embodiments, during the second treatment, the second location within the target tissue and the subsequent location within the target tissue are different. In some embodiments, the fifth location is the same location as any of the first through fourth locations. In some embodiments, the sixth location is the same location as any of the first through fourth locations. In some embodiments, ultrasound energy is applied at an MI of at least 1.6, 2.1, or 2.3. In some embodiments, the acoustic agent comprises a protein-stabilized shell, a lipid-stabilized shell, a perfluoropropane (perflutran) gas core, an SF6 gas core, or a combination thereof. In some embodiments, ultrasound energy is applied percutaneously. In some embodiments, the target cells and / or target tissue are in the liver. In some embodiments, the first location is the first lobe of the liver, and the second or subsequent location is the second or subsequent lobe of the liver. In some embodiments, the first lobe of the liver is the right lobe, the second lobe is the left lobe, and the subsequent lobe is one or both of the caudate or quadrate lobe. In some embodiments, the nucleic acid payload comprises a therapeutic transgene, wherein the target cells are hepatocytes, and at least 50% of the cells expressing the therapeutic transgene in the liver are hepatocytes. In some embodiments, the target cells and / or target tissue are in the kidney. In some embodiments, the first location is in a first region of the kidney, and the second location is in a second region of the kidney. In some embodiments, expression of the nucleic acid payload is induced in multiple cell types of the kidney. In some embodiments, the transgene encoded by the nucleic acid payload is expressed in cells in all regions of the kidney. In some embodiments, the transgene encoded by the nucleic acid payload is expressed in cells across four non-overlapping spatial regions of the kidney, which define the entire kidney. In some embodiments, the distance between the first location and the second or subsequent location in the target tissue is at least 25% of the maximum distance of the long axis of the organ comprising the target tissue. In some embodiments, the distance between the third location and the fourth or subsequent location in the target tissue is at least 25% of the maximum distance of the long axis of the organ comprising the target tissue. In some embodiments, the distance between the first location and the second or subsequent location in the target tissue is at least 1 cm, 2 cm, or 3 cm. In some embodiments, the distance between the third and fourth or subsequent locations in the target tissue is at least 1 cm, 2 cm, or 3 cm. In some embodiments, applying ultrasound energy at the second or subsequent location in the target tissue increases microvascular perfusion of the target tissue. In some embodiments, the target tissue exhibits cystic lesions. In some embodiments, the delivery and / or expression of nucleic acid payloads to target cells in the target tissue exhibiting cystic lesions is increased.In some embodiments, the first therapeutic composition and the second therapeutic composition are the same therapeutic composition. In some embodiments, the first and second therapeutic compositions contain different doses of nucleic acid payload. In some embodiments, the first and second therapeutic compositions contain different doses of acoustic active agent or different acoustic active agents. In some embodiments, applying ultrasound energy to a first and second location on a subject includes moving an ultrasound probe across the subject's skin surface from the first location to the second location. In some cases, compared to a method that includes applying the first therapeutic process without applying the second therapeutic process, applying the second therapeutic process increases the delivery or expression of the nucleic acid payload by at least 25%, 50%, 75%, 100%, 250%, 300%, 350%, 400%, 450%, or 500%. In some cases, compared to a method that includes applying the first therapeutic process without applying the second therapeutic process, or compared to a method that includes applying the first and second therapeutic processes without applying a subsequent therapeutic process, applying the subsequent therapeutic process increases the delivery or expression of the nucleic acid payload by at least 100%, 250%, 300%, 350%, 400%, 450%, or 500%. In some cases, the first and second locations are adjacent. In some cases, the second position and the subsequent position are adjacent. In some cases, the third position and the fourth position are adjacent. In some cases, the fourth position and the subsequent position are adjacent. In some cases, the fifth position and the sixth position are adjacent. In some cases, the sixth position and the subsequent position are adjacent. In some cases, the fifth or sixth position is adjacent to any of the first through fourth positions.

[0006] The disclosed aspect provides a method for delivering a nucleic acid payload to target cells of a subject, comprising: providing a treatment procedure to the subject, wherein the treatment procedure includes: applying the nucleic acid payload to the subject; applying a plurality of microbubbles to the subject; and applying ultrasound energy near the target cells of the subject; and repeating the treatment procedure at least once more than 6 hours after the start of the first treatment procedure but within 10 days after the start of the first treatment procedure. The disclosed aspect provides a method for delivering a nucleic acid payload to target cells of a subject, comprising: providing a treatment procedure to the subject, wherein the treatment procedure includes: applying the nucleic acid payload to the subject; applying a plurality of microbubbles to the subject; and applying ultrasound energy near the target cells of the subject; and repeating the treatment procedure at least once more than 21 days after the start of the first treatment procedure. The aspects disclosed herein provide a method for delivering a nucleic acid payload to target cells of a subject, comprising: providing the subject with a treatment process, wherein the treatment process includes: administering the nucleic acid payload to the subject; administering a plurality of microbubbles to the subject; and administering ultrasonic energy near the target cells of the subject; and repeating the treatment process, wherein repeating the treatment process increases the expression of a protein encoded by the nucleic acid payload by at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 250%, 500%, 750%, 1000%, or 2000% compared to a single dose. The aspects disclosed herein provide a method for delivering a nucleic acid payload to target cells of a subject, comprising: providing the subject with a treatment process, wherein the treatment process includes: administering two or more doses of the nucleic acid payload to the subject; administering two or more doses of a plurality of microbubbles to the subject; and administering ultrasonic energy near the target cells of the subject; and repeating the treatment process. In some embodiments, the two or more doses of the nucleic acid payload comprise three or more doses of a plurality of microbubbles. In some embodiments, the two or more doses of the nucleic acid payload comprise three or more doses of a plurality of microbubbles. In some embodiments, repeated treatment procedures include reapplying ultrasound energy to target cells at a second or subsequent location on the target organ. In some embodiments, administering a nucleic acid payload to the subject includes administering two or more doses of the nucleic acid payload to the subject during the treatment procedure. In some embodiments, administering a nucleic acid payload to the subject includes administering three or more doses of the nucleic acid payload to the subject during the treatment procedure. In some embodiments, administering multiple microbubbles to the subject includes administering two or more doses of multiple microbubbles to the subject during the treatment procedure. In some embodiments, administering multiple microbubbles to the subject includes administering three or more doses of multiple microbubbles to the subject during the treatment procedure.In some embodiments, the repeated treatment process includes re-administering two or more doses of the nucleic acid payload to the subject during the treatment process. In some embodiments, the repeated treatment process includes re-administering three or more doses of the nucleic acid payload to the subject during the treatment process. In some embodiments, the repeated treatment process includes re-administering two or more doses of multiple microbubbles to the subject during the treatment process. In some embodiments, the repeated treatment process includes re-administering three or more doses of multiple microbubbles to the subject during the treatment process. In some embodiments, compared to a single dose, the repeated treatment process increases the expression of the protein encoded by the nucleic acid payload by at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 250%, 500%, 750%, 1000%, or 2000%. In some embodiments, the repeated treatment process includes re-applying ultrasonic energy near target cells at a second location on the target organ. In some embodiments, the repeated treatment process includes re-applying ultrasonic energy near target cells at the same location on the target organ. In some embodiments, the nucleic acid payload is a nanoparticle. In some embodiments, the nucleic acid payload comprises a therapeutic transgene coupled to a promoter other than the CMV promoter or the ubiquitin C promoter. In some embodiments, the ultrasound energy is sufficient to disrupt microvesicles. In some embodiments, the ultrasound energy is sufficient to allow the nucleic acid payload to enter the target cell. In some embodiments, the ultrasound energy is sufficient to allow the nucleic acid payload to enter the target cell, as demonstrated by the expression of the nucleic acid or amino acid sequence. In some embodiments, the ultrasound energy comprises applying ultrasound energy with a mechanical index ranging from 0.05 to 2.3. In some embodiments, the ultrasound energy comprises applying ultrasound energy with a mechanical index ranging from 0.05 to 2.1. In some embodiments, the ultrasound energy comprises applying ultrasound energy with a mechanical index ranging from 0.05 to 1.6. In some embodiments, the ultrasound energy comprises applying ultrasound energy for up to 30, 60, 90, 120, 150, 180, 240, 300, or 360 seconds. In some embodiments, the ultrasound energy comprises applying ultrasound energy for up to 10, 15, 20, 25, or 30 minutes. In some embodiments, applying ultrasound energy includes continuously applying ultrasound energy for the duration of the treatment process. In some embodiments, applying ultrasound energy includes continuously applying ultrasound energy. In some embodiments, continuously applying ultrasound energy includes maintaining continuous contact between the subject and an ultrasound transducer. In some embodiments, continuously applying ultrasound energy includes the ultrasound transducer transmitting or receiving reflected ultrasound energy for at least 95% of the time the ultrasound transducer is in continuous contact with the subject. In some embodiments, continuously applying ultrasound energy includes continuously applying or reflecting ultrasound energy. In some embodiments, continuously applying ultrasound energy includes the ultrasound transducer continuously emitting or receiving reflected ultrasound energy.In some embodiments, continuous application of ultrasound energy includes applying ultrasound energy at a first mechanical index for a first duration, applying ultrasound energy at a second mechanical index for a second duration, and reapplying ultrasound energy at the first mechanical index without stopping the application of ultrasound energy, for a duration of continuous application of ultrasound energy. In some embodiments, the second mechanical index is higher than the first mechanical index. In some embodiments, continuous application of ultrasound energy includes applying ultrasound energy at a first mechanical index for a first duration, applying ultrasound energy at a second mechanical index for a second duration, and reapplying ultrasound energy at the first mechanical index without stopping the application of ultrasound energy, for a duration of continuous treatment. In some embodiments, the treatment process, as determined by the length of continuous application of ultrasound energy, is as long as 30, 60, 90, 120, 150, 180, 240, 300, or 360 seconds. In some embodiments, the application of ultrasound energy c. includes the application of multiple ultrasound flashes with increasing mechanical index, said multiple ultrasound flashes being applied at intervals of at least 2 seconds between each other. In some embodiments, the application of ultrasound energy c. includes the application of multiple ultrasound flashes with increasing mechanical index, each of said multiple ultrasound flashes being applied at intervals of 1 s to about 10 s between each other. In some embodiments, the application of ultrasound energy c. comprises the application of multiple ultrasound flashes with increasing mechanical index, each of the multiple ultrasound flashes being applied at intervals of 5 s to about 10 s and lasting about 0.5 s. In some embodiments, the application of ultrasound energy c. comprises the application of a total of 10 ultrasound flashes with increasing mechanical index, each of the total 10 ultrasound flashes being applied at intervals of 1 s to about 2 s and lasting about 0.5 s. In some embodiments, an ultrasound flash is a change in the application of ultrasound energy from a first mechanical index to a second mechanical index, and a reapplication of ultrasound energy from the second mechanical index to the first mechanical index within a time period. In some embodiments, the time period is less than 1 s. In some embodiments, the time period is from about 0.7 μs to about 3 μs (microseconds). In some embodiments, the time period is about 0.72, 0.82, 0.72, 0.98, or 2.28 μs. In some embodiments, the application of ultrasound energy c. comprises the application of at least 5 ultrasound flashes with a second mechanical index that increases relative to the first mechanical index, and intervals of less than 10 seconds. In some embodiments, administering the nucleic acid payload or multiple microbubbles includes intravenous administration via a peripheral vein. In some embodiments, the expression of the nucleic acid or amino acid sequence encoded by the nucleic acid payload persists at at least 50% of the peak protein or mRNA expression level for at least one week. In some embodiments, the expression of the nucleic acid or amino acid sequence encoded by the nucleic acid payload persists at at least 50% of the peak protein or mRNA expression level for at least one week.In some embodiments, the treatment process is repeated within 72 hours after the initial first treatment. In some embodiments, the treatment process is repeated within 48 hours after the initial first treatment. In some embodiments, the treatment process is repeated twice within 24 hours after the first treatment. In some embodiments, the treatment process is repeated 30 days after the first treatment. In some embodiments, the treatment process is repeated 60 days after the first treatment. In some embodiments, the treatment process is repeated 90 days after the first treatment. In some embodiments, the treatment process is repeated 120 days after the first treatment. In some embodiments, the repeated treatment does not result in a significant increase in inflammatory biomarkers. In some embodiments, the repeated treatment results in a significant increase in inflammatory biomarkers within 20% of the baseline level of the inflammatory biomarkers. In some embodiments, the biomarkers are transaminases, ALT, AST, IL6, or combinations thereof. In some embodiments, the nanoparticle construct is administered at a concentration of 0.4 mg / kg body weight to 1.5 mg / kg body weight. In some embodiments, a.-c. induces expression of the payload in target cells within 24 hours. In some embodiments, the expression of the nucleic acid payload in target cells is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks after a.-c. In some embodiments, the expression of the nucleic acid payload in target cells is maintained at an elevated level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks after a.-c., compared to baseline expression levels. In some embodiments, the elevated level of the nucleic acid payload is at least 10% higher than baseline expression levels. In some embodiments, the nucleic acid payload of a. is administered at a dose of about 3.5 μg / μl. In some embodiments, the microbubble contains a protein-stabilized shell. In some embodiments, the microbubble contains an albumin-stabilized shell. In some embodiments, the microbubble contains perfluorobromoethane gas. In some embodiments, the microbubble is an Optison microbubble. In some embodiments, the microbubble contains a lipid-stabilized shell. In some embodiments, the concentration of the applied microbubbles is up to about 1.2 × 10¹⁰ microbubbles / ml. In some embodiments, the microbubbles are applied at a concentration of at least 5 × 10⁸ microbubbles / ml. In some embodiments, the concentration of the applied microbubbles is between 5 × 10⁸ and 8 × 10⁸ microbubbles / ml. In some embodiments, ultrasound energy is applied percutaneously. In some embodiments, the target cells include hepatocytes. In some embodiments, the target cells include kidney cells. In some embodiments, the target cells include pancreatic cells. In some embodiments, the target cells include cardiomyocytes or endothelial cells.In some embodiments, the target cells include myocytes, skeletal muscle cells, or smooth muscle cells. In some embodiments, the nucleic acid payload comprises a therapeutic transgene. In some embodiments, the therapeutic transgene includes: GLP-1, INS, Reg3g, MafA, PDX-1, NUEROG3, NGN3, DRYK, DYRK1A, DYRK1B, factor VIII (FVIII), factor IX (FIX), PKD1, PKD2, COL4A3, COL4A4, COL4A5, Klotho, Smad7, TGF-β, SLC7A1, SLC3A9, UMOD, REN, HNF1B, MUC1, SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, NPHS1, NPHS2, CTNS, or combinations thereof. In some embodiments, the nucleic acid payload comprises a therapeutic transgene coupled to a promoter sequence, said promoter sequence including: ApoE promoter, CAG promoter, AAT promoter, or a combination thereof. In some embodiments, the nucleic acid payload and a plurality of microbubbles are administered at a volume ratio of at least 1 part nucleic acid payload solution to 4 parts microbubble solution, wherein there are at least 4 parts microbubble solution to nucleic acid payload solution.

[0007] In some aspects, this document provides a method for increasing the expression of a nucleic acid or amino acid sequence encoded by a payload in a subject's target tissue, comprising administering a first dose of: i) the payload and ii) multiple acoustically active microstructures to the subject via a peripheral vein; percutaneously applying ultrasound energy to the vicinity of target cells at a first location in the subject's target tissue; and percutaneously applying ultrasound energy to the vicinity of target cells at a second location in the subject's target tissue. In some embodiments, the method further comprises administering a second dose of i) the payload and ii) multiple acoustically active microstructures to the subject. In some embodiments, the method further comprises percutaneously applying ultrasound energy to the vicinity of target cells at a third location in the subject's target tissue. In some embodiments, the method further comprises administering a third dose of i) the payload and ii) multiple acoustically active microstructures to the subject via a peripheral vein. In some embodiments, any one of the first dose, the second dose, or optionally the third dose is administered within a first time period. In some embodiments, the first time period is up to 15, 30, 45, or 60 seconds. In some embodiments, the first dose, the second dose, and optionally the third dose are administered within 6 hours of each other. In some implementations, the first dose, the second dose, and optionally the third dose are administered within one hour of each other.

[0008] In some embodiments, compared to a single dose, after 24 hours, the method increases the expression of the nucleic acid or amino acid sequence encoded by the payload by at least 5%, 10%, 20%, 30%, 40%, 50%, or more. In some embodiments, the method includes applying an effective amount of ultrasonic energy. In some embodiments, the effective amount of ultrasonic energy is sufficient to disrupt acoustically active microstructures. In some embodiments, the effective amount of ultrasonic energy is sufficient to allow the nucleic acid payload to enter the target cell. In some embodiments, the effective amount of ultrasonic energy is sufficient to allow the nucleic acid payload to enter the target cell, as demonstrated by the expression of the nucleic acid or amino acid sequence. In some embodiments, the effective amount of ultrasonic energy includes applying ultrasonic energy with a mechanical index ranging from 0.05 to 1.8. In some embodiments, the effective amount of ultrasonic energy includes applying ultrasonic energy for up to 30, 60, 90, 120, 150, 180, 240, 300, or 360 seconds. In some embodiments, applying ultrasonic energy includes the duration of a continuous application period of ultrasonic energy. In some embodiments, continuously applying ultrasonic energy includes applying ultrasonic energy at a first mechanical exponent for a first duration, applying ultrasonic energy at a second mechanical exponent for a second duration, and reapplying ultrasonic energy at the first mechanical exponent without stopping the application of ultrasonic energy, for a duration of continuous application of ultrasonic energy. In some embodiments, applying an effective amount of ultrasonic energy includes applying multiple ultrasonic flashes spaced several seconds apart. In some embodiments, applying an effective amount of ultrasonic energy includes applying multiple ultrasonic flashes spaced several seconds apart. In some embodiments, multiple ultrasonic flashes are applied spaced one millisecond or more milliseconds apart. In some embodiments, multiple ultrasonic flashes are applied spaced one second or more seconds apart. In some embodiments, applying ultrasonic energy includes applying multiple ultrasonic flashes with increasing mechanical exponents, each of the multiple ultrasonic flashes being applied spaced from 0.9 μs to about 2.5 μs apart. In some embodiments, applying ultrasonic energy includes applying multiple ultrasonic flashes with increasing mechanical exponents, each of the multiple ultrasonic flashes being applied spaced from 0.9 μs to about 2.5 μs apart, for a duration of about 0.5 s. In some embodiments, applying ultrasound energy comprises applying 10 ultrasound flashes with an increasing mechanical index, each of the 10 ultrasound flashes being applied at 2.2 μs intervals and lasting approximately 0.5 s. In some embodiments, ultrasound flashes represent a change in ultrasound energy application from a first mechanical index to a second mechanical index, and a reapplication of ultrasound energy from the second mechanical index back to the first mechanical index over a period of time. In some embodiments, applying an effective amount of ultrasound energy comprises applying at least 5 ultrasound flashes at a second mechanical index, increasing relative to the first mechanical index, at intervals of less than 10 seconds. In some embodiments, the expression of the nucleic acid payload in the target tissue is maintained for at least 5 days compared to baseline expression levels.In some embodiments, the expression of the nucleic acid payload in the target tissue is maintained for at least 7 days compared to the baseline expression level. In some embodiments, the expression of the nucleic acid payload in the target tissue is maintained for at least 2 weeks. In some embodiments, the target tissue is an organ. In some embodiments, the organ is the liver. In some embodiments, the first location is the first lobe of the liver, and the second location is the second lobe of the liver. In some embodiments, the first location and the second location are different locations within the same lobe of the liver. In some embodiments, the organ is the kidney. In some embodiments, the first location is in a first region of the kidney, and the second location is in a second region of the kidney. In some embodiments, the first location and the second location are different locations within the same region of the kidney. In some embodiments, the organ is the pancreas. In some embodiments, the first location is in a first region of the pancreas, and the second location is in a second region of the pancreas. In some embodiments, the first location and the second location are different locations within the same region of the pancreas. In some embodiments, the organ is the heart. In some embodiments, the organ is the brain. In some embodiments, the target tissue is skeletal muscle. In some embodiments, the target tissue is skeletal muscle. In some embodiments, the nucleic acid payload is a nanoparticle.

[0009] In some embodiments, the nucleic acid payload is a transgene. In some embodiments, the nucleic acid payload includes a therapeutic transgene. In some embodiments, increasing the expression of the nucleic acid payload encoded by the payload includes inducing the expression of a therapeutic transgene. In some embodiments, the transgene includes a detectable biomarker. In some embodiments, the detectable biomarker includes luciferase. In some embodiments, the therapeutic transgene includes GLP-1, INS, Reg3g, MafA, PDX-1, NUEROG3, NGN3, DRYK, DYRK1A, DYRK1B, Factor VIII, Factor IX, PKD1, PKD2, COL4A3, COL4A4, COL4A5, Klotho, Smad7, TGF-β, SLC7A1, SLC3A9, UMOD, REN, HNF1B, MUC1, SLC12A1, KCNJ1, C LCNKA, CLCNKB, BSND, MAGED2, NPHS1, NPHS2, CTNS, PKD1, PKD2, COL4A3, COL4A4, COL4A5, Klotho, Smad7, TGF-β, SLC7A1, SLC3A9, UMOD, REN, HNF1B, MUC1, SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, NPHS1, NPHS2, CTNS, or combinations thereof. In some embodiments, the payload comprises a therapeutic transgene operatively linked to a promoter sequence. In some embodiments, the promoter sequence is a promoter sequence other than the CMV promoter or the ubiquitin C promoter. In some embodiments, the promoter sequence comprises the ApoE promoter, the CAG promoter, the AAT promoter, or combinations thereof. In some embodiments, the microbubble comprises a protein-stabilized shell. In some embodiments, the protein-stabilized shell comprises albumin. In some embodiments, the microbubble comprises perfluoropropane gas. In some embodiments, the acoustically active microstructures are applied at a concentration of at least 5 × 10^8 acoustically active microstructures / mL. In some embodiments, the acoustically active microstructures are applied at a concentration between about 5 × 10^8 and about 8 × 10^8 acoustically active microstructures / mL. In some embodiments, the plurality of acoustically active microstructures includes Optison acoustically active microstructures. In some embodiments, the acoustically active microstructures include a lipid-stabilized shell. In some embodiments, the concentration of the applied acoustically active microstructures is at most about 1.2 × 10^10 acoustically active microstructures / mL. In some embodiments, the method does not lead to a significant increase in inflammatory biomarkers.In some embodiments, after administering the nucleic acid payload to a subject, the level of the inflammatory biomarker is maintained within 20% of the baseline level of the inflammatory biomarker; multiple microbubbles are administered to the subject; and ultrasonic energy is administered near the target cells of the subject, and optionally, ultrasonic energy is administered near target cells at a second location in the subject's target tissue. In some embodiments, the biomarker is one or more of ALT, AST, and / or IL6. In some embodiments, the nucleic acid payload and multiple acoustically active microstructures are administered at a volume ratio of at least 1 part nucleic acid payload solution to 4 parts microbubble solution. In some aspects, this document provides a method for delivering a payload to one or more target cells, the method comprising: administering a first dose of i) a payload containing a therapeutic transgene, and ii) multiple acoustically active microstructures intravenously to a subject via a peripheral vein; percutaneously administering ultrasonic energy near target cells at a first location in the subject's target tissue; and percutaneously administering ultrasonic energy near target cells at a second location in the subject's target tissue. In some embodiments, the therapeutic transgene contains a sequence encoding FVIII. In some embodiments, the therapeutic transgene contains a sequence encoding COL4A3. In some embodiments, the therapeutic transgene contains a sequence encoding COL4A4. In some embodiments, the therapeutic transgene contains a sequence encoding COL4A5. In some embodiments, the therapeutic transgene contains a sequence encoding PKD1. In some embodiments, the therapeutic transgene includes a sequence encoding PKD2. In some embodiments, applying ultrasound energy near the acoustically active microstructure increases microvascular perfusion of the target tissue. In some embodiments, applying ultrasound energy at a second location within the target tissue increases microvascular perfusion of the target tissue. In some embodiments, repeated sonopore treatment increases microvascular perfusion of the target tissue. In some embodiments, the target cells are located in target tissue exhibiting cystic lesions. In some embodiments, the target cells are located in target tissue exhibiting cystic lesions, wherein delivery of nucleic acids to the target cells in the tissue exhibiting cystic lesions is increased. In some embodiments, the target cells are included in a subject with polycystic kidney disease, wherein the therapeutic transgene includes a sequence encoding PKD1 or PDK2, wherein delivery of the therapeutic transgene to the target cells is increased, thereby treating polycystic kidney disease. Attached Figure Description

[0010] The novel features of this disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of this disclosure, and in the accompanying drawings: Figure 1 The experimental protocol used in the experiment was described, in which the sonoporogenic gene therapy treatment was repeated in subjects; Figure 2 Fluorescence images of experiments collected via IVIS fluorescence imaging are provided, in which repeat acoustic pore-forming gene therapy treatment was performed in subjects. Figure 3A Provided Figure 2 The bar graph shown shows the average fluorescence radiance measurements for each group of subjects; Figure 3B Provided Figure 2 The line graph shown shows the mean fluorescence irradiance measurements for each group of subjects, which demonstrates that repeated sonoporosis gene therapy treatment provided an increase of more than an order of magnitude compared to a single dose; Figure 4 This describes the percentage change in mean fluorescence radiance measured 24 hours after the first dose measurement of repeated acoustic pore-forming gene therapy in subjects; Figure 5 The experimental protocol used in the experiment was described, in which the sonoporogenic gene therapy treatment was repeated in subjects; Figure 6 Fluorescence images of experiments collected via IVIS fluorescence imaging are provided, in which repeat acoustic pore-forming gene therapy treatment was performed in subjects. Figure 7A A bar graph of the mean fluorescence radiance measurements collected by IVIS fluorescence imaging is provided for subjects who underwent repeated sonoporosis gene therapy treatment. Figure 7B Provided Figure 6 The bar graph shown shows the average fluorescence radiance measurements of the subjects; Figure 7C Explanation Figure 6 The mean fluorescence radiance measurements of the subjects shown are given, where repeated sonoporosis gene therapy treatment resulted in an order of magnitude increase in the measured fluorescence radiance. Figure 8 Serum levels of ALT, IL6, and AST measured from subjects 24 hours after sonopore treatment are shown, and it is demonstrated that sonopore treatment did not lead to an increase in serum levels of ALT, IL6, and AST. Figure 9A The implementation plan for sonoporosis treatment at the cellular level was explained; Figure 9B The implementation plan for sonoporosis treatment at the cellular level was explained; Figure 10 A bar graph of mean fluorescence emissivity measurements is provided, showing repeated acoustic pore-forming gene therapy treatments in different target organs of the subjects. Figure 11 The experimental protocol used in the study was described, in which the sonoporogenic gene therapy was repeated in subjects 16 weeks after the initial treatment. Figure 12A Bar graphs of mean fluorescence radiance measurements collected by IVIS fluorescence imaging are provided for subjects who underwent repeat sonoporosis gene therapy treatment 16 weeks after initial treatment. Figure 12B Bar graphs of mean fluorescence radiance measurements collected via IVIS fluorescence imaging are provided for experiments in which sonoporosis gene therapy was repeated 16 weeks after initial treatment in two groups of mice. Mice in one group received a smaller dose of DNA (light gray bars), and mice in the other group received a larger dose of DNA (dark gray bars, also shown in...). Figure 12A middle); Figure 13 The average fluorescence emissivity measurements of experiments collected by IVIS fluorescence imaging are shown, which tested the efficacy of different doses of DNA delivered during sonoporotic therapy of the liver. Figure 14 The average fluorescence emissivity measurements of experiments collected by IVIS fluorescence imaging are shown, which tested the efficacy of different doses of DNA delivered during sonoporosis treatment of the kidney. Figure 15 The mean fluorescence emissivity measurements of the experiment, collected by IVIS fluorescence imaging, are shown. The experiment tested the efficacy of repeated doses of DNA delivered during sonoporotic therapy of the kidney. Figure 16 An exemplary experimental protocol is described in which one, two, or three doses (“pump”) of a DNA construct and an acoustically active microstructure (“acoustically active microstructure”) are delivered to a subject while ultrasound is delivered to one, two, or three sites in the target tissue. Figure 17 Provides support for multi-dose, multi-site experiments (e.g.) Figure 16 Fluorescence images collected by IVIS fluorescence imaging in the experiment described in the paper, and demonstrated that subjects who received two or three doses (“push”) of DNA constructs and acoustically active microstructures exhibited increased gene expression and increased persistence of gene expression within weeks after treatment. Figure 18 Provided data collected by IVIS fluorescence imaging at the indicated time points. Figure 17 The fluorescence emissivity measurements (p / s / cm) for each group of subjects are shown below. 2 The study described how subjects who received two or three doses (“pumps”) of DNA constructs and acoustically active microstructures exhibited increased gene expression and increased persistence of gene expression within weeks following treatment. Figure 19Fluorescence emissivity measurements (p / s / cm) were provided for four groups of subjects in a kidney sonopore transfection study. 2 / sr). Compared with control mice, mice with polycystic kidney disease (Nek8) receiving ultrasound-mediated gene delivery (UMGD) showed significantly better results. jck The highest mean fluorescence emissivity was measured in the mouse model, indicating that Nek8... jck UMGD in mice leads to microvascular perfusion, efficient gene delivery, and robust expression in cystic structures; Figure 20 52-week fluorescence radiometric data (p / s / cm) are provided for subjects who underwent sonoporosis treatment in the liver using the methods described herein. 2 / sr); Figure 21 52-week fluorescence radiometric data (p / s / cm) are provided for subjects who underwent sonotomy in the kidneys using the methods described herein. 2 / sr); Figure 22 26-week fluorescence radiometric data (p / s / cm) of subjects who underwent acoustic pore formation therapy using the methods described herein at various nucleic acid payload doses are provided. 2 / sr); Figure 23A Histological images are provided, showing that the enhanced green fluorescent protein (EGFP) genetic payload is expressed in up to about 70% of the glomeruli in the kidneys of non-human primates (NHPs); Figure 23B This study provides quantification of the expression of enhanced green fluorescent protein (EGFP) genetic payload in NHP kidney glomeruli; Figure 24 Histological images are provided, showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in the kidney cells of NHP, including podocytes, renal tubular epithelial cells, and endothelial cells; Figure 25A Histological images are provided showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in renal cells in the non-glomerular region of NHP kidneys, including podocytes, renal tubular epithelial cells, and endothelial cells; Figure 25B Quantification of enhanced green fluorescent protein (EGFP) genetic payload expression in NHP renal cells in non-glomerular regions, including podocytes, renal tubular epithelial cells (Tubl), and endothelial cells (Endo), was provided. Figure 26AHistological images are provided showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in renal cells of the glomerular region of an NHP kidney, including podocytes, renal tubular epithelial cells, and endothelial cells; Figure 26B Quantification of enhanced green fluorescent protein (EGFP) genetic payload expression in NHP renal cells in the glomerular region is provided, said renal cells including podocytes, renal tubular epithelial cells (Tubl), and endothelial cells (Endo). Figure 27 Histological images are provided showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in mouse kidney cells, including podocytes, tubular epithelial cells (Tub. cells), and endothelial cells (Endo. cells); Figure 28A Histological images are provided, showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in renal cells in the non-glomerular region of a mouse kidney, including podocytes, renal tubular epithelial cells, and endothelial cells; Figure 28B Quantification of enhanced green fluorescent protein genetic payload (EGFP) expression in non-glomerular region mouse kidney cells, including podocytes, tubular epithelial cells (Tubl), and endothelial cells (Endo), was provided. Figure 29A Histological images are provided showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in renal cells of the glomerular region of a mouse kidney, including podocytes, renal tubular epithelial cells, and endothelial cells; Figure 29B Quantification of the expression of enhanced green fluorescent protein (EGFP) genetic payload in mouse kidney cells in the glomerular region is provided, said kidney cells including podocytes, renal tubular epithelial cells (Tubl), and endothelial cells (Endo). Figure 30A The distribution of kidney cell types (podocytes, renal tubular epithelial cells, endothelial cells, and unknown cells) in mouse kidney samples is provided; Figure 30B The percentages of different types of kidney cells (podocytes, renal tubular epithelial cells, endothelial cells, and unknown cells) identified by snRNA-seq (left) and RNAscope (right) in mouse kidney samples are quantified. Figure 31A Histological images are provided showing the expression of enhanced green fluorescent protein (EGFP) genetic payload in cells of mouse liver, including hepatocytes, Kupffer cells / immune cells, and hepatic sinusoidal endothelial cells (LSEC). Figure 31B Quantification of enhanced green fluorescent protein (EGFP) expression in mouse hepatocytes, including hepatocytes, Kupffer cells / immune cells, and hepatic sinusoidal endothelial cells (LSEC), is provided. Figure 32A The distribution of hepatocyte types (hepatocytes, Kupffer cells / immune cells, sinusoidal endothelial cells (LSEC), and unknown cells) in mouse liver samples is provided; Figure 32B The percentage of different types of hepatocytes (hepatocytes, Kupffer cells / immune cells, hepatic sinusoidal endothelial cells (LSEC) and unknown cells) identified by snRNA-seq (left) and RNAscope (right) in mouse liver samples is quantified. Figure 33 The image shows luciferase immunofluorescence staining of acoustically perforated kidney sections; Figure 34A Data illustrating gene delivery and expression in mouse livers using the sonoporosis treatment protocol described herein are presented; and Figure 34B Data illustrating gene delivery and gene expression in mouse livers using the sonoporosis treatment protocol described herein are presented. Detailed Implementation

[0011] One possible approach to achieving safe and effective gene expression using sonopore-based gene therapy is repetitive gene therapy, as it can potentially be reapplied, unlike viral vector-based gene therapy. However, determining suitable protocols and conditions for safely and effectively repeating sonopore-based gene therapy remains a challenge, as repeating ultrasound-based gene therapy can lead to substantial cell damage, inflammation, and / or death due to the ultrasound treatment and gene transfection process, and such sonopore-based procedures have not been shown to provide safe and effective therapeutic treatment, thus demonstrating the risks of reapplying sonopore-based gene therapy. Improved methods for delivering and expressing enhanced nucleic acids to target cells represent an improvement over existing technologies.

[0012] This document discloses a sonopore-forming method that allows for the safe and efficient repetition of sonopore-based gene therapy treatments to improve nucleic acid delivery and expression in target cells. The method includes a combination of multiple applications of ultrasound with the administration of a sonoactive agent and a nucleic acid payload to increase the delivery of the nucleic acid payload to target cells in a subject, thereby increasing gene transfection and expression, and in some cases, increasing the persistence of gene expression after sonopore-based gene therapy treatment. The disclosed method may include providing ultrasound energy to multiple sites in a target tissue containing target cells, and the ultrasound energy may be applied in multiple treatment sessions that can be repeated without significant cell damage, inflammation, and / or death following the treatment sessions. The disclosed method may include applying or reapplying sonopore-forming treatment processes at different intervals, applying ultrasound to multiple sites in a target organ or tissue, and applying ultrasound energy with different parameters to safely and efficiently deliver nucleic acid payloads. Such ultrasound parameters may include applying with reduced power, applying with a reduced mechanical index, alternating between high and low mechanical indices, applying short flash pulses at a high mechanical index, and / or applying them during a short total treatment period to obtain beneficial results.

[0013] The present invention discloses a method for delivering a nucleic acid payload to target cells of a subject, the method comprising: applying a first treatment process to the subject, the first treatment process comprising: applying an amount of a first treatment composition to the subject, the first treatment composition comprising: i) a nucleic acid payload, and ii) multiple acoustic agents; applying ultrasonic energy to target cells at a first location in a target tissue of the subject; and applying ultrasonic energy to target cells at a second location in a target tissue of the subject, wherein the first location and the second location are different; and applying a second treatment process to the subject after the first treatment process, the second treatment process comprising: applying an amount of a second treatment composition to the subject, the second treatment composition comprising: i) a nucleic acid payload, and ii) multiple acoustic agents; applying ultrasonic energy to target cells at a third location in a target tissue of the subject; and applying ultrasonic energy to target cells at a fourth location in a target tissue of the subject, wherein the third location and the fourth location are different. In some embodiments, the method includes: administering a subsequent treatment to the subject after a second treatment process, the subsequent treatment process including: administering to the subject a quantity of a first treatment composition or a second treatment composition, the composition comprising: i) a nucleic acid payload, and ii) multiple acoustic agents; administering ultrasonic energy to target cells at a fifth location in the subject's target tissue; and administering ultrasonic energy to target cells at a subsequent location at a sixth location in the subject's target tissue, wherein the fifth and sixth locations are distinct. In some embodiments, the method includes, during the first treatment process, administering ultrasonic energy to target cells at a subsequent location in the subject's target tissue after administering ultrasonic energy to a second location. In some embodiments, the method includes, during the second treatment process, administering ultrasonic energy to target cells at a subsequent location in the subject's target tissue after administering ultrasonic energy to a fourth location. In some cases, administering the second treatment process increases the delivery or expression of the nucleic acid payload by at least 25%, 50%, 75%, 100%, 250%, 300%, 350%, 400%, 450%, or 500% compared to a method that includes administering the first treatment process without administering the second treatment process. In some cases, compared to methods that include administering a first treatment process without administering a second treatment process, or compared to methods that include administering a first treatment process and a second treatment process without administering a subsequent treatment process, administering a subsequent treatment process increases the delivery or expression of the nucleic acid payload by at least 100%, 250%, 300%, 350%, 400%, 450%, or 500%. In some cases, the first and second positions are adjacent. In some cases, the second and subsequent positions are adjacent. In some cases, the third and fourth positions are adjacent. In some cases, the fourth and subsequent positions are adjacent. In some cases, the fifth and sixth positions are adjacent.In some cases, the sixth position and subsequent positions are adjacent. In some cases, the fifth or sixth position is adjacent to any of the first through fourth positions. This document provides methods for targeted transfection and expression of nucleic acids into and in the cells, tissues, or organs of a subject using acoustic pore forming (e.g., processes involving applying ultrasonic acoustic energy to cells, tissues, or organs, such as to provide increased porosity in those cells, tissues, or organs). As used herein, in some embodiments, the subject is a mammal. In some embodiments, by way of non-limiting example, the mammal is a human, monkey, or other non-human primate. In some embodiments, the subject may be a rat, mouse, or another non-primate.

[0014] Some embodiments of this document provide methods for transfecting nucleic acid constructs into target cells or tissues (e.g., target cells or tissues of a subject) by applying a sonopore-forming treatment procedure (e.g., ultrasound-mediated gene delivery) to the subject, the treatment procedure comprising: applying a nucleic acid payload to the subject.

[0015] Some embodiments of this document provide methods for transfecting nucleic acid constructs into target cells or tissues (e.g., target cells or tissues of a subject) by applying a sono-induced pore-forming treatment process to the subject, the treatment process comprising: applying multiple microbubbles to the subject.

[0016] Some embodiments of this document provide a method for transfecting nucleic acid constructs into target cells or tissues (e.g., target cells or tissues of a subject) by applying a sonopore-forming treatment process to the subject, the treatment process comprising: applying ultrasonic energy near the target cells of the subject.

[0017] In some implementations, the methods provided herein include repeated treatment procedures.

[0018] In some implementations, the method provided herein includes repeating the treatment process at least once, more than 6 hours after the start of the first treatment procedure but within 10 days.

[0019] In some implementations, the method provided herein includes repeating the treatment process at least once more than 21 days after the start of the first treatment process.

[0020] In some implementations, the methods provided herein include repeated treatment processes, wherein repeated treatment processes increase the expression of the protein or amino acid sequence encoded by the nucleic acid payload by at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 250%, 500%, 750%, 1000%, or 2000% compared to a single dose.

[0021] In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 5%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 10%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 20%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 30%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 40%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 50%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 100%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 250%. In the implementation scheme, compared with a single dose, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 500%. In the implementation scheme, repeated treatment increases the expression of proteins encoded by the nucleic acid payload by at least 750% compared to a single dose. In the implementation scheme, repeated treatment increases the expression of proteins encoded by the nucleic acid payload by at least 1000% compared to a single dose. In the implementation scheme, repeated treatment increases the expression of proteins encoded by the nucleic acid payload by at least 2000% compared to a single dose.

[0022] In some embodiments, the treatment process is repeated at least once, more than 6 hours but within 10 days after the start of the first treatment. In some embodiments, the treatment process is repeated at least once, more than 21 days after the start of the first treatment. In some embodiments, the repeated treatment process includes reapplying ultrasound energy to the vicinity of target cells at a second location on the target organ. In some embodiments, the repeated treatment process includes reapplying ultrasound energy to the vicinity of target cells at the same location on the target organ. In some embodiments, the expression of the nucleic acid or amino acid sequence encoded by the nucleic acid payload persists at at least 50% of the peak expression level for at least 1 week. In some embodiments, the expression of the nucleic acid or amino acid sequence encoded by the nucleic acid payload persists at at least 50% of the peak expression level of the protein or mRNA for at least 1 week. In some embodiments, the repeated treatment process ensures that the production of the protein or mRNA encoded by the nucleic acid payload remains within 35% of the peak expression level between subsequent treatment processes.

[0023] In some embodiments, the treatment process is repeated at least once, more than 6 hours but within 10 days after the start of the first treatment. In some embodiments, the treatment process is repeated at least once, more than 21 days after the start of the first treatment. In some embodiments, the repeated treatment process includes applying ultrasound energy to a third and fourth location in the target tissue. In some embodiments, the repeated treatment process includes subsequent treatment processes, said subsequent treatment processes including applying ultrasound energy to a fifth and sixth location in the target tissue. In some embodiments, the expression of the nucleic acid or amino acid sequence encoded by the nucleic acid payload persists at at least 50% of the peak expression level for at least 1 week. In some embodiments, the expression of the nucleic acid or amino acid sequence encoded by the nucleic acid payload persists at at least 50% of the peak expression level of the protein or mRNA for at least 1 week. In some embodiments, the repeated treatment process will maintain the production of the protein or mRNA encoded by the nucleic acid payload within 35% of the peak expression level between subsequent treatment processes.

[0024] In some embodiments, the expression of the nucleic acid payload in the target cells is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks after the treatment process (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject). In some embodiments, the expression of the nucleic acid payload in the target cells is maintained for at least 1 week after the treatment process (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject). In some embodiments, the expression of the nucleic acid payload in the target cells is maintained for at least 2 weeks after the treatment process (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject). In some embodiments, the expression of the nucleic acid payload in the target cells is maintained for at least 3 weeks after the treatment process (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject). In some embodiments, the expression of the nucleic acid payload in the target cells is maintained for at least 4 weeks after the treatment process (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject). In some embodiments, the expression of the nucleic acid payload in the target cells is maintained for at least 5 weeks after the treatment process (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject).

[0025] In some embodiments, the expression of the nucleic acid payload in target cells remains at an elevated level for at least one week after treatment, compared to baseline expression levels (e.g., administering the nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the target cells of the subject, and optionally, administering ultrasound energy near target cells at a second location in the target tissue of the subject).

[0026] In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 5%, 10%, 20%, 30%, 40%, or 50% compared to a single dose. In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 5% compared to a single dose. In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 10% compared to a single dose. In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 20% compared to a single dose. In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 30% compared to a single dose. In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 40% compared to a single dose. In some embodiments, repeated treatment increases the expression of the protein encoded by the nucleic acid payload by at least 50% compared to a single dose.

[0027] When, for how long, and under what conditions can sonoporosis-based gene therapy be repeated to produce improved and durable gene expression in subjects that is superior to single-dose administration, and is safe for subjects and does not cause cellular-substantial inflammation, damage, and / or death, are important factors in the ability to repeat sonoporosis-based gene therapy. For example, Figure 3B This indicates that repeated sonoporotic gene therapy treatments, as described herein, can provide a difference of more than one order of magnitude in gene expression levels compared to single-dose administration; and even in repeated sonoporotic gene therapy, a difference of approximately half an order of magnitude in gene expression levels may exist, depending on the interval between treatment sessions. This article provides a method comprising a sonoporotic gene therapy treatment regimen to produce improved and durable gene expression in subjects that is superior to single-dose administration, and which is safe and effective for subjects.

[0028] In some embodiments, the treatment process is repeated within 72 hours after the first treatment. In some embodiments, the treatment process is repeated within 48 hours after the first treatment. In some embodiments, the treatment process is repeated twice within 24 hours after the first treatment. In some embodiments, the treatment process is repeated at least 30 days after the first treatment. In some embodiments, the treatment process is repeated at least 30 days after the first treatment. In some embodiments, re-application of sonoporotic gene therapy at the intervals described herein increases the efficacy of treatment, nucleic acid delivery, and gene expression without substantial cell damage, inflammation, and / or death due to the ultrasound treatment and gene transfection process.

[0029] In some embodiments, the treatment procedure includes administering the nucleic acid payload and acoustically active microstructure in a "pump" form over a short period of time. In some embodiments, the short period of time is less than 15 seconds. In some embodiments, the short period of time is at most 15 seconds. In some embodiments, the short period of time is at most 20 seconds. In some embodiments, the short period of time is at most 25 seconds. In some embodiments, the short period of time is at most 30 seconds. In some embodiments, the short period of time is at most 60 seconds. In some embodiments, the dose of the nucleic acid payload and acoustically active agent is delivered as a bolus injection to the target tissue immediately or shortly before ultrasound is applied to the target tissue, during the oscillation of the acoustically active agent and before the acoustically active agent breaks down, increasing the concentration of acoustically active agent and nucleic acid in the target tissue and near the target cells, thereby increasing the delivery of the nucleic acid payload to the target cells and / or the expression of the nucleic acid payload in the target cells.

[0030] In some embodiments, administering the amount of the first therapeutic composition includes administering a first dose of the first therapeutic composition and a second dose of the first therapeutic composition to the subject during a first treatment process. In some embodiments, during or after administering the second dose of the first therapeutic composition during the first treatment process, ultrasound energy is applied to a second location in the target tissue of the subject. In some embodiments, administering the amount of the first therapeutic composition during the first treatment process includes administering at least a third dose of the first therapeutic composition. In some embodiments, the method includes administering ultrasound energy to a subsequent location in the subject during or after administering the third dose during the first treatment process. In some embodiments, administering the amount of the second therapeutic composition during a second treatment process includes administering a first dose of the second therapeutic composition and a second dose of the second therapeutic composition to the subject. In some embodiments, during or after administering the second dose of the second therapeutic composition during the second treatment process, ultrasound energy is applied to a third location in the target tissue of the subject. In some embodiments, administering the amount of the second therapeutic composition during the second treatment process includes administering at least a third dose of the second therapeutic composition. In some embodiments, each dose of the first or second therapeutic composition is administered by intravenous injection. In some embodiments, intravenous injection is administered over discrete time intervals. In some implementations, the discrete time intervals do not exceed 60 seconds, 120 seconds, or 180 seconds.

[0031] In some embodiments, the treatment process includes administering two or more doses (“pumps”) of the DNA construct and acoustically active microstructure. In some embodiments, administering two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during the treatment process increases the expression of the protein encoded by the nucleic acid payload by at least 5% compared to a single dose. In a specific embodiment, administering two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during the treatment process increases the expression of the protein encoded by the nucleic acid payload by at least 10% compared to a single dose. In a specific embodiment, administering two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during the treatment process increases the expression of the protein encoded by the nucleic acid payload by at least 20% compared to a single dose. In a specific embodiment, administering two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during the treatment process increases the expression of the protein encoded by the nucleic acid payload by at least 30% compared to a single dose. In specific implementations, administration of two or more doses (“pumps”) of the DNA construct and acoustically active microstructure increases the expression of the protein encoded by the nucleic acid payload by at least 40% compared to a single dose. In specific implementations, administration of two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during treatment increases the expression of the protein encoded by the nucleic acid payload by at least 50% compared to a single dose. In specific implementations, administration of two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during treatment increases the expression of the protein encoded by the nucleic acid payload by at least 100% compared to a single dose. In specific implementations, administration of two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during treatment increases the expression of the protein encoded by the nucleic acid payload by at least 250% compared to a single dose. In specific implementations, administration of two or more doses (“pumps”) of the DNA construct and acoustically active microstructure during treatment increases the expression of the protein encoded by the nucleic acid payload by at least 500% compared to a single dose. In specific implementations, administration of two or more doses (“bolus”) of the DNA construct and acoustically active microstructure during treatment increases the expression of proteins encoded by the nucleic acid payload by at least 750% compared to a single dose. In specific implementations, administration of two or more doses (“bolus”) of the DNA construct and acoustically active microstructure during treatment increases the expression of proteins encoded by the nucleic acid payload by at least 1000% compared to a single dose.In a specific implementation plan, administering two or more doses (“bolus”) of the DNA construct and acoustically active microstructure during the treatment process, compared to a single dose, increases the expression of proteins encoded by the nucleic acid payload by at least 2000%.

[0032] The ultrasound protocol used in repetitive sonopore therapy is safe for use during repetitive sonopore therapy, thereby improving gene transfection and expression. This ultrasound protocol is a key factor in the efficacy of repetitive sonopore gene therapy. The aspects disclosed herein provide methods for repetitive sonopore gene therapy without inducing substantial inflammation of the target organ, tissue, or cells; substantial cell damage, inflammation, or death; or additionally inducing a significant increase in inflammatory or apoptotic cell biomarkers in the subject without causing cell damage, inflammation, or death. In some embodiments, the ultrasound energy (e.g., administered to the subject) is sufficient to disrupt microvessels. In some embodiments, the ultrasound energy (e.g., administered to the subject) is sufficient to allow nucleic acid payloads to enter target cells. In some embodiments, the ultrasound energy (e.g., administered to the subject) is sufficient to allow nucleic acid payloads to enter target cells, as demonstrated by the expression of nucleic acid or amino acid sequences.

[0033] In some embodiments, applying ultrasound energy may include the duration of a continuous treatment process involving the continuous application of ultrasound energy. In some embodiments, applying ultrasound energy includes a portion of the duration of a continuous treatment process involving the continuous application of ultrasound energy. In some embodiments, ultrasound energy may be applied continuously. In some embodiments, applying ultrasound energy includes applying multiple ultrasound flashes, wherein ultrasound energy is applied for a short duration with a high mechanical index before returning to applying ultrasound energy with a lower mechanical index. In some embodiments, these ultrasound flashes may be spaced apart from each other by several seconds. In some embodiments, continuous application of ultrasound energy includes an ultrasound transducer continuously sending and receiving ultrasound signals.

[0034] In some further embodiments, ultrasound energy can be continuously applied by alternating between ultrasound energy applied at a first mechanical index for a first duration and ultrasound energy applied at a second mechanical index for a second duration. In some further embodiments, ultrasound energy can be continuously applied by applying ultrasound energy at a first mechanical index for a first duration, applying ultrasound energy at a second mechanical index for a second duration, and then reapplying ultrasound energy at a first mechanical index for a duration of continuous application without stopping the application of ultrasound energy. In some further embodiments, ultrasound energy can be continuously applied by applying ultrasound energy at a first mechanical index for a first duration, applying ultrasound energy at a second mechanical index for a second duration, and then reapplying ultrasound energy at a first mechanical index for the duration of the treatment without stopping the application of ultrasound energy. In some embodiments, ultrasound energy can be applied at alternating mechanical indices for a duration of continuous application without stopping the application of ultrasound energy. In some embodiments, ultrasound energy can be applied at alternating mechanical indices for the duration of the treatment without stopping the application of ultrasound energy.

[0035] In some embodiments, ultrasonic energy is applied with a mechanical index ranging from 0.05 to 2.3. In some embodiments, ultrasonic energy is applied with a mechanical index ranging from 0.05 to 1.8. In some embodiments, ultrasonic energy is applied with any suitable mechanical index. In some embodiments, ultrasonic energy is applied with any suitable mechanical index to rupture microbubbles and facilitate payload entry into target cells. In some embodiments, ultrasonic energy is applied continuously in alternating pulses with lower and higher mechanical indices. In some embodiments, the lower mechanical index ranges from 0.05 to about 0.4. In some embodiments, the higher mechanical index ranges from 1.6 to about 2.3. In some embodiments, the alternating pulses occur at intervals ranging from 0.9 μs to 10 s. In some embodiments, the ultrasonic flashes may have a mechanical index increased relative to a first mechanical index. In some embodiments, applying ultrasonic energy includes applying at least four ultrasonic flashes with a mechanical index increased relative to a first mechanical index, the flashes being spaced less than 10 seconds apart. In some embodiments, applying ultrasonic energy includes applying ultrasonic flashes with a duration of about 0.9 μs to about 2.5 μs. In some embodiments, applying ultrasonic energy includes applying 10 pulses over 0.5 seconds, each pulse lasting approximately 2.2 μs. In some embodiments, applying ultrasonic energy includes applying ultrasound at a first MI of approximately 0.07 and then applying ultrasonic flashes at a second MI of approximately 0.8, lasting for 10 frames, spaced 4 seconds apart, for a total of 9 flashes. In some embodiments, applying ultrasonic energy includes applying ultrasound at a second MI within at least 9 flashes. In some embodiments, applying ultrasonic energy includes applying ultrasound at a second MI within at least 18 flashes. In some embodiments, applying ultrasonic energy includes applying ultrasound at a second MI within at least 27 flashes.

[0036] In some embodiments, ultrasonic energy is applied for 30, 60, 90, 120, 150, 180, 240, 300, or 360-1200 seconds. In some embodiments, ultrasonic energy is applied for 30 seconds. In some embodiments, ultrasonic energy is applied for 60 seconds. In some embodiments, ultrasonic energy is applied for 90 seconds. In some embodiments, ultrasonic energy is applied for 120 seconds. In some embodiments, ultrasonic energy is applied for 150 seconds. In some embodiments, ultrasonic energy is applied for 180 seconds. In some embodiments, ultrasonic energy is applied for 240 seconds. In some embodiments, ultrasonic energy is applied for 300 seconds. In some embodiments, ultrasonic energy is applied for 360 seconds.

[0037] In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 30, 60, 90, 120, 150, 180, 240, 300, or 360 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 30 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 60 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 90 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 120 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 150 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 180 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 240 seconds. In some embodiments, the treatment process, as determined by the length of the continuously applied ultrasound energy, lasts for 300 seconds. In some implementations, such as treatment procedures that last up to 360 seconds, the duration of which is determined by the length of the continuous application of ultrasound energy.

[0038] In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 5, 10, 15, 20, 25, 30, 35, or 40 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 5 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 10 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 15 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 20 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 25 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 30 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 35 minutes. In some embodiments, the treatment process, as determined by the length of continuous ultrasound energy application, lasts for 40 minutes.

[0039] As described herein, the application of ultrasound, including repeated ultrasound application during repeated sonopore treatments, can have undesirable effects on living cells or tissues. In some embodiments, the present invention provides methods for improving gene transfection that do not result in substantial DNA or cellular damage to target cells, tissues, or organs. In some embodiments, the methods do not cause substantial cellular damage to target cells. In some embodiments, the methods do not cause inflammation of the target tissue or result in an increase in apoptosis or inflammatory biomarkers. Apoptosis biomarkers can be used to detect cell damage. For example, in the liver, the detection of released hepatocyte transaminases (e.g., serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST)) can be an indicator of apoptotic hepatocytes. Other apoptosis biomarkers may include interleukin-6 (IL6) or B-cell lymphoma 2 (BCL2 or BCL2 apoptosis regulator). In some embodiments, no biomarkers of cell damage, inflammation, or death at the level of apoptosis are detected during or after the application of the sonopore treatment method disclosed herein. In some embodiments, no clinically elevated levels of biomarkers of cell damage are detected during or after the application of the methods disclosed herein. In some embodiments, repeated treatment procedures do not result in substantial cell damage, cell inflammation, or cell death. In some embodiments, repeated treatment procedures do not result in a significant increase in inflammatory biomarkers. In some embodiments, repeated sonopore treatment results in an increase in inflammatory biomarkers within 20% of the baseline level of the inflammatory biomarkers. Non-limiting examples of biomarkers of cell damage, inflammation, or apoptosis include ALT, AST, IL6, and BCL2. In some embodiments, repeated treatment procedures do not cause any other substantial, clinically significant increases in ALT, AST, IL6, or BCL2. In some embodiments, no apoptotic levels of the following biomarkers of cell damage are detected after the treatment procedure (e.g., administration of a nucleic acid payload to the subject; administration of multiple microbubbles to the subject; and application of ultrasound energy near the target cells of the subject, and optionally, application of ultrasound energy near target cells at a second location in the target tissue of the subject): ALT, AST, IL6, BCL2, or combinations thereof, and optionally, wherein the target cells are in the liver. In some embodiments, following treatment, the following biomarkers of cell damage do not show clinical elevation: ALT, AST, IL6, BCL2, or combinations thereof, and optionally, the target cells are located in the liver. In some embodiments, following treatment, the following biomarkers of cell damage do not show clinical elevation: urinary creatinine levels, urinary albumin to creatine ratio, blood creatinine levels, glomerular filtration rate, blood in urine, urinary protein levels, or urinary molar osmolality, and optionally, the target cells are located in the kidneys.In some implementations, the following biomarkers of cell damage do not rise clinically after the treatment course: blood troponin levels or creatine phosphokinase levels, and optionally, the target cells are located in the heart or skeletal muscle.

[0040] In some embodiments, the method includes percutaneously applying ultrasound energy to the vicinity of one or more target cells of a subject. In some embodiments, the one or more target cells are hepatocytes. In some embodiments, the one or more target cells are kidney cells. In some embodiments, the one or more target cells are pancreatic cells. In some embodiments, the one or more target cells are cardiac cells. In some embodiments, the one or more target cells are muscle cells. In some embodiments, the one or more target cells are neuronal cells. In some embodiments, the one or more target cells are brain cells. In some embodiments, the one or more target cells are blood cells (e.g., white blood cells). In some embodiments, the target cells are cancer cells.

[0041] In some embodiments, the one or more target cells are contained within a tissue. In some embodiments, the tissue is skeletal muscle tissue. In some embodiments, the tissue is smooth muscle tissue. In some embodiments, the tissue is connective tissue. In some embodiments, the tissue is lymphatic tissue. In some embodiments, the tissue is nerve tissue. In some embodiments, the tissue is diseased tissue, such as cancerous tissue, fibrotic tissue, or other tissue requiring gene therapy.

[0042] In some embodiments, the target tissue is contained within an organ. In some embodiments, the organ is the liver. In some embodiments, the organ is the kidney. In some embodiments, the organ is the pancreas. In some embodiments, the organ is the heart. In some embodiments, the organ is the brain.

[0043] In some embodiments, the one or more target cells are contained within a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a liquid tumor.

[0044] In some embodiments, ultrasonic energy is applied to target cells, tissues, or organs after the application of the nucleic acid construct and the acoustically active microstructure. In some embodiments, ultrasonic energy is applied to target cells, tissues, or organs during the application of the nucleic acid construct, the acoustically active microstructure, or both. In some embodiments, ultrasonic energy is applied to target cells, tissues, or organs both during and after the application of the nucleic acid construct, the acoustically active microstructure, or both.

[0045] In some embodiments, the method includes percutaneously applying an effective amount of ultrasound energy near target cells at a first location and a second location in the target tissue. In some embodiments, the first location and the second location are different regions of the target tissue (e.g., dorsal and ventral regions of the target tissue, or the first and second lobes of the target tissue). In some embodiments, the first location and the second location are in the same or similar regions of the target tissue (e.g., the first location and the second location are in the dorsal region of the target tissue, or the first location and the second location are in the same lobe of the target tissue).

[0046] In some embodiments, the first position and the third position in the target tissue are the same position. In some embodiments, the first position and the third position in the target tissue are different. In some embodiments, during the first treatment, the first position and the subsequent position in the target tissue are the same position. In some embodiments, during the first treatment, the first position and the subsequent position in the target tissue are different. In some embodiments, the second position and the fourth position in the target tissue are the same position. In some embodiments, the second position and the fourth position in the target tissue are different. In some embodiments, during the second treatment, the first position and the subsequent position in the target tissue are the same position. In some embodiments, during the second treatment, the third position and the subsequent position in the target tissue are different. In some embodiments, the fifth position is the same position as any of the first to fourth positions. In some embodiments, the sixth position is the same position as any of the first to fourth positions. In some cases, the first and second positions are adjacent positions of the target tissue. In some cases, the second and subsequent positions are adjacent positions of the target tissue. In some cases, the third and fourth positions are adjacent to the target tissue. In some cases, the fourth and subsequent positions are adjacent to the target tissue. In some cases, the fifth and sixth positions are adjacent to the target tissue. In some cases, the sixth and subsequent positions are adjacent to the target tissue. In some cases, the fifth or sixth position is adjacent to any one of the first through fourth positions. In some embodiments, applying ultrasound energy to the first and second positions of the subject includes moving an ultrasound probe through the subject's skin surface from the first position to the second position.

[0047] In some embodiments, the target tissue is the liver, and the first location is one of the right lobe, left lobe, caudate lobe, caudate lobe, or intermediate lobe, and the second location is in a lobe different from the first location. In some embodiments, the first location is one of the right lobe, left lobe, caudate lobe, or intermediate lobe, and the second location is in a different region of the same lobe as the first location (e.g., the first and second locations are different regions of the right lobe). In some embodiments, the first and second locations are in the same region of the same lobe. In some embodiments, the target cells and / or target tissue are in the liver. In some embodiments, the first location is the first lobe of the liver, and the second or subsequent location is the second or subsequent lobe of the liver. In some embodiments, the first lobe of the liver is the right lobe, the second lobe is the left lobe, and the subsequent lobe is one or both of the caudate lobe or quadrate lobe. In some embodiments, the nucleic acid payload comprises a therapeutic transgene, wherein the target cells are hepatocytes, and wherein at least 50% of the cells expressing the therapeutic transgene in the liver are hepatocytes.

[0048] In some embodiments, the target tissue is the kidney, and the first location is a side of the kidney, while the second location is a region different from the first location. In some embodiments, the second location is a central region of the kidney. In some embodiments, the second location or subsequent location is a longitudinal plane of the kidney. In some embodiments, the second location or subsequent location is a plane at least 45 degrees relative to an axis of rotation about the kidney. In some embodiments, the second location or subsequent location is a plane at approximately 90 degrees relative to an axis of rotation about the kidney.

[0049] In some embodiments, the target cells and / or target tissues are located in the kidney. In some embodiments, the first location is in a first region of the kidney, and the second location is in a second region of the kidney. In some embodiments, expression of the nucleic acid payload is induced in multiple cell types of the kidney. In some embodiments, the transgene encoded by the nucleic acid payload is expressed in cells in all regions of the kidney. In some embodiments, the transgene encoded by the nucleic acid payload is expressed in cells across four non-overlapping spatial regions of the kidney, which define the entire kidney.

[0050] In some implementations, the target tissue exhibits cystic lesions. In some implementations, the delivery and / or expression of nucleic acid payloads to target cells in the target tissue exhibiting cystic lesions is increased.

[0051] In some embodiments, the target tissue is the pancreas, and the first location is one of the head, neck, body, or tail regions, and the second location is in a different region from the first location. In some embodiments, the first location is one of the head, neck, body, or tail regions, and the second location is in a different region of the same region as the first location (e.g., the first and second locations are different regions of the head region). In some embodiments, the first and second locations are in the same region of the same area.

[0052] In some embodiments, the distance between the first location and the second or subsequent location in the target tissue is at least 25% of the maximum distance of the long axis of the organ including the target tissue. In some embodiments, the distance between the third location and the fourth or subsequent location in the target tissue is at least 25% of the maximum distance of the long axis of the organ including the target tissue. In some embodiments, the distance between the first location and the second or subsequent location in the target tissue is at least 1 cm, 2 cm, or 3 cm. In some embodiments, the distance between the third location and the fourth or subsequent location in the target tissue is at least 1 cm, 2 cm, or 3 cm. In some embodiments, applying ultrasound energy at the second or subsequent location in the target tissue increases microvascular perfusion of the target tissue. As used herein, “long axis of organ” is the maximum length of the longest line drawn from one end of the organ through the target organ to the other.

[0053] In some implementations, the distance between a first location in the target tissue and a second or subsequent location in the target tissue is at least the diameter of the focused ultrasound beam.

[0054] In some embodiments, the method further includes percutaneous application of ultrasound energy to target cells at a third location in the target tissue of the subject. In some embodiments, the method further includes intravenous administration of a third dose of i) the payload and ii) multiple acoustically active microstructures to the subject via a peripheral vein.

[0055] In some embodiments, compared to administering a single or two doses, administering a third dose and / or administering an effective amount of ultrasound energy at a third position will increase the expression of the nucleic acid or amino acid sequence encoded by the payload by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more. In some embodiments, compared to administering a single or two doses, administering a third dose and / or administering an effective amount of ultrasound energy at a third position will increase the expression of the nucleic acid or amino acid sequence encoded by the payload by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more within 24 hours. In some embodiments, compared to administering a single or two doses, administering a third dose and / or administering an effective amount of ultrasound energy at a third position will increase the expression of the nucleic acid or amino acid sequence encoded by the payload by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more within 48 hours. In some embodiments, compared to administering a single or two doses, administering a third dose and / or administering an effective amount of ultrasound energy at a third position increases the expression of the nucleic acid or amino acid sequence encoded by the payload by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more within 5 days. In some embodiments, compared to administering a single or two doses, administering a third dose and / or administering an effective amount of ultrasound energy at a third position increases the expression of the nucleic acid or amino acid sequence encoded by the payload by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more within 7 days.

[0056] In some cases, compared to methods that include administering a first treatment process without administering a second treatment process, administering a second treatment process increases the delivery or expression of the nucleic acid payload by at least 25%, 50%, 75%, 100%, 250%, 300%, 350%, 400%, 450%, or 500%. In some cases, compared to methods that include administering a first treatment process without administering a second treatment process, or compared to methods that include administering a first treatment process and a second treatment process without administering a subsequent treatment process, administering a subsequent treatment process increases the delivery or expression of the nucleic acid payload by at least 100%, 250%, 300%, 350%, 400%, 450%, or 500%.

[0057] In some embodiments, the first therapeutic composition and the second therapeutic composition are the same therapeutic composition. In some embodiments, the first therapeutic composition and the second therapeutic composition contain different doses of nucleic acid payload. In some embodiments, the first therapeutic composition and the second therapeutic composition contain different doses of acoustic active agent or different acoustic active agents. In some embodiments, applying ultrasound energy to a first location and a second location on a subject includes moving an ultrasound probe through the subject's skin from the first location to the second location.

[0058] In some embodiments, the nucleic acid payload comprises a non-endogenous gene. In some embodiments, the transgene comprises a detectable biomarker. In some embodiments, the transgene comprises luciferase. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the expression of luciferase. In some embodiments, the transgene comprises green fluorescent protein. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the expression of green fluorescent protein.

[0059] In some embodiments, the nucleic acid payload is configured for gene enhancement, gene replacement, gene editing, base editing, base knockdown, gene knockdown, or gene knockout. In some embodiments, delivering the nucleic acid payload to target cells of a subject increases or decreases gene expression in the target cells. In some embodiments, the nucleic acid payload may be a therapeutic payload. In some embodiments, the nucleic acid construct is a microplasmid containing the nucleic acid payload. In some embodiments, the nucleic acid payload contains a transgene. In some embodiments, the transgene may be an endogenous transgene. In some embodiments, the transgene may be a non-endogenous transgene. In some embodiments, the transgene is a therapeutic transgene.

[0060] In some embodiments, the transgene comprises therapeutic RNA. In some embodiments, the therapeutic RNA is mRNA. In some embodiments, the therapeutic RNA is an RNA interference (RNAi) agent, such as double-stranded RNA, single-stranded RNA, microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), or a triple-stranded oligonucleotide. In some embodiments, the therapeutic RNA is a catalytically active RNA molecule (ribozyme). In some embodiments, the therapeutic RNA is transfer RNA (tRNA). In some embodiments, the therapeutic RNA contains one or more chemical modifications (e.g., one or more modified nucleobases, nucleosides, or nucleotides).

[0061] In some embodiments, the transgene comprises one or more components of a gene editing system. In some embodiments, the payload comprises a nuclease or engineered nuclease suitable for gene editing. In some embodiments, the nuclease is delivered as a polypeptide. In some embodiments, the nuclease is delivered as a nucleic acid encoding the nuclease. In some embodiments, the gene editing system is a CRISPR / Cas system. In some embodiments, the payload comprises gRNA or a nucleic acid molecule encoding gRNA (e.g., a plasmid encoding gRNA). In some embodiments, the payload comprises a Cas protein or its homologs or variants, or a nucleic acid molecule encoding a Cas protein or its homologs or variants. In some embodiments, the payload comprises TALEN or a nucleic acid molecule encoding TALEN. In some embodiments, the payload comprises a zinc finger nuclease (ZFN) or a nucleic acid encoding ZFN. In some embodiments, the nuclease is an engineered nuclease. In some embodiments, the engineered nuclease is non-catalytically active. In some embodiments, the engineered nuclease is a fusion protein comprising an engineered nuclease, a regulatory protein, or an enzyme or its functional domain (e.g., a nuclease fused to a transcriptional regulatory domain or a nuclease fused to a deaminase). In some implementations, the payload may also include a template DNA molecule suitable for knock-in into the subject's genome via non-homologous end joining (NHEJ) or homologous directed repair (HDR).

[0062] In some embodiments, the payload contains nucleic acids exceeding the size limits of conventional gene therapy vectors. In some embodiments, the payload exceeds the size limits of adeno-associated virus vectors (AAV). In some embodiments, the payload is greater than about 4.7 kb. In some embodiments, the payload is greater than about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, or about 13 kb.

[0063] In some embodiments, the transgene includes a therapeutic transgene. In some embodiments, the therapeutic transgene includes: GLP-1, INS, Reg3g, MafA, PDX-1, NUEROG3, NGN3, DRYK, DYRK1A, DYRK1B, factor VIII (FVIII), factor IX (FIX), or combinations thereof. In some embodiments, the therapeutic transgene contains GLP-1. In some embodiments, the therapeutic transgene contains INS. In some embodiments, the therapeutic transgene contains REg3g. In some embodiments, the therapeutic transgene contains MafA. In some embodiments, the therapeutic transgene contains PDX-1. In some embodiments, the therapeutic transgene contains NUEROG3. In some embodiments, the therapeutic transgene contains NGN3. In some embodiments, the therapeutic transgene contains DYRK1A. In some embodiments, the therapeutic transgene contains DYRK1B. In some embodiments, the therapeutic transgene includes factor VIII. In some embodiments, the therapeutic transgene includes factor IX.

[0064] In some embodiments, the therapeutic transgene includes: PKD1, PKD2, COL4A3, COL4A4, COL4A5, Klotho, Smad7, TGF-β, SLC7A1, SLC3A9, UMOD, REN, HNF1B, MUC1, SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, NPHS1, NPHS2, CTNS, or combinations thereof. In some embodiments, the therapeutic transgene comprises PKD1. In some embodiments, the therapeutic transgene comprises PKD2. In some embodiments, the therapeutic transgene comprises COL4A3. In some embodiments, the therapeutic transgene comprises COL4A4. In some embodiments, the therapeutic transgene comprises COL4A5. In some embodiments, the therapeutic transgene comprises Klotho. In some embodiments, the therapeutic transgene comprises Smad7. In some embodiments, the therapeutic transgene comprises TGF-β. In some embodiments, the therapeutic transgene comprises SLC7A1. In some embodiments, the therapeutic transgene comprises SLC7A9. In some embodiments, the therapeutic transgene comprises SLC12A1. In some embodiments, the therapeutic transgene comprises UMOD. In some embodiments, the therapeutic transgene comprises REN. In some embodiments, the therapeutic transgene comprises HNF1B. In some embodiments, the therapeutic transgene comprises MUC1. In some embodiments, the therapeutic transgene comprises KCNJ1. In some embodiments, the therapeutic transgene comprises CLCNKA. In some embodiments, the therapeutic transgene comprises CLCNKB. In some embodiments, the therapeutic transgene comprises BSND. In some embodiments, the therapeutic transgene comprises NPHS1. In some embodiments, the therapeutic transgene comprises NPHS2. In some embodiments, the therapeutic transgene comprises CNTS.

[0065] In some embodiments, the payload comprises a therapeutic transgene coupled to one or more gene regulatory elements. In some embodiments, the gene regulatory element may be a promoter, enhancer, ribosome binding site, or transcription termination signal. In some embodiments, the nucleic acid payload comprises a therapeutic transgene coupled to a promoter. In some embodiments, the nucleic acid payload comprises a regulatory element, such as a promoter (e.g., APOE-ATT). In some embodiments, the nucleic acid payload comprises a therapeutic transgene coupled to a promoter other than the CMV promoter or the ubiquitin C (UbC) promoter. In some embodiments, the nucleic acid construct comprises a regulatory element, such as a promoter, enhancer, ribosome binding site, or transcription termination signal. Examples of promoters considered herein include, but are not limited to, for example, the CMV promoter, the UbC promoter, the CAG promoter, the EF-1α promoter, the ApoE promoter, the AAT promoter, the ApoE-AAT1 promoter, the 3XSERP promoter, the P3-heterozygous promoter, or combinations thereof. In some embodiments, the nucleic acid construct comprises a promoter sequence containing CAG. In some embodiments, the nucleic acid construct contains a promoter sequence containing ApoE. In some embodiments, the nucleic acid construct contains a promoter sequence containing SERP. In some embodiments, the nucleic acid construct contains a promoter sequence containing P3.

[0066] In some embodiments, the nucleic acid payload and multiple microbubbles are applied at a volume ratio of at least 1 part nucleic acid payload solution to 4 parts microbubble solution. In some embodiments, the nucleic acid payload and multiple microbubbles are applied at a volume ratio of approximately 1 part nucleic acid payload solution to 3 parts microbubble solution. In some embodiments, the nucleic acid payload and multiple microbubbles are applied at a volume ratio of approximately 1 part nucleic acid payload solution to 2 parts microbubble solution. In some embodiments, the nucleic acid payload and multiple microbubbles are applied at a volume ratio of approximately 1 part nucleic acid payload solution to 1 part microbubble solution.

[0067] In some embodiments, the nucleic acid constructs and sonoactive microstructures are administered intravenously. In some embodiments, intravenous administration is via a peripheral vein. In some embodiments, a peripheral vein is any vein outside the trunk. Exemplary peripheral veins include, but are not limited to, the jugular vein, brachiocephalic vein, saphenous vein, and veins in the forearm, head, and foot. Compared to other administration methods (e.g., intravenous injection in the portal vein or inferior vena cava), the benefit of peripheral intravenous administration is the generation of lower hydrostatic pressure and peak negative pressure levels within the target tissue / organ. Peripheral intravenous injection can facilitate continuous injection within an invasive pathway and can facilitate the repetitive sonopore-forming methods disclosed herein. However, administration via a peripheral vein may not result in delivery of microbubbles or nucleic acid payloads to the target tissue or cells, or may result in significantly reduced delivery. When the methods disclosed herein are used for intravenous administration of nucleic acid payloads and microbubbles via a peripheral vein, the sonopore-forming treatment process can be repeated without resulting in substantial cellular damage, inflammation, and / or death following the treatment process. When the methods disclosed herein are used to administer nucleic acid payloads and microbubbles intravenously via peripheral veins during treatment, transfection of the nucleic acid payloads into cells can be increased, and expression of nucleic acid or amino acid sequences encoded by the nucleic acid payloads can be increased.

[0068] In some embodiments, inducing the expression of a nucleic acid payload includes inducing the production of a nucleotide sequence encoded by the payload. In some embodiments, inducing the expression of a nucleic acid payload includes inducing the production of RNA encoded by the payload. In some embodiments, inducing the expression of a nucleic acid payload includes inducing the production of messenger RNA (mRNA) encoded by the payload. In some embodiments, inducing the expression of a nucleic acid payload includes inducing the production of small interfering RNA (siRNA) encoded by the payload. In some embodiments, inducing the expression of a nucleic acid payload includes inducing the production of small hairpin RNA (shRNA) encoded by the payload.

[0069] In some embodiments, inducing the expression of a nucleic acid payload includes inducing the production of a protein encoded by the payload. In some embodiments, regulating the expression of a nucleic acid payload includes regulating the production of RNA encoded by the payload. In some embodiments, regulating the expression of a nucleic acid payload includes regulating the production of a protein encoded by the nucleic acid payload, or regulating the production of a protein encoded by mRNA encoded by the nucleic acid payload.

[0070] The methods described herein can be used to treat subjects requiring gene therapy or enzyme replacement therapy. In some aspects, this disclosure provides methods for treating subjects with liver conditions. In some embodiments, the liver conditions treated are: Wilson's disease, progressive familial intrahepatic cholestasis, von Willebrand disease, hemophilia A, hemophilia B, factor 5 deficiency, alpha-mannoside storage disease, Gaucher disease (glucocerebrosidase deficiency, glucocerebrosidase storage disease), Niemann-Pick disease A / B, carbamoyl phosphate synthase I deficiency, type III glycogen storage disease, cystinosis, A1AT deficiency, and type I and II citrullinemia.

[0071] The sonoporization process described herein can be used to treat subjects requiring gene therapy or enzyme replacement therapy. On the other hand, this disclosure provides methods for treating subjects with liver conditions. In some embodiments, the liver conditions treated are: Wilson's disease, progressive familial intrahepatic cholestasis, von Willebrand disease, hemophilia A, hemophilia B, factor 5 deficiency, alpha-mannoside storage disease, Gaucher's disease (glucocerebrosidase deficiency, glucocerebrosidase storage disease), Niemann-Pick disease A / B, carbamoyl phosphate synthase I deficiency, type III glycogen storage disease, cystinosis, A1AT deficiency, and type I and II citrullinemia.

[0072] In some embodiments, this disclosure provides a method for treating a subject with liver disease symptoms having a therapeutic transgene. In some embodiments, the therapeutic transgene encodes one or more of the following: ATP7B; ABCB11; ABCB4; ATP8B1; TJP2; VWF; FVIII; FIX; F5; MAN2B1; GBA; SMPD1; CPS1; GDE / AGL; CTNS; SERPINA1; ASS1 and / or SLC25A13.

[0073] In some embodiments, this disclosure provides a method of treating a subject with a liver condition having a therapeutic transgene. In some embodiments, the liver condition is Wilson's disease, and the therapeutic transgene encodes ATP7B. In some embodiments, the liver condition is cholestasis, progressive familial intrahepatic cholestasis (PFIC1-4), and the therapeutic transgene encodes one or more of ABCB11, ABCB4, ATP8B1, and / or TJP2. In some embodiments, the liver condition is von Willebrand disease, and the therapeutic transgene encodes VWF. In some embodiments, the liver condition is hemophilia A, and the therapeutic transgene encodes FVIII. In some embodiments, the liver condition is hemophilia B, and the therapeutic transgene encodes FIX. In some embodiments, the liver condition is factor V deficiency, and the therapeutic transgene encodes F5. In some embodiments, the liver condition is alpha-mannosinolate storage disease, and the therapeutic transgene encodes MAN2B1. In some embodiments, the liver condition is Gaucher disease (glucocerebrosidase deficiency, glucocerebrosidosis), and the therapeutic transgene encodes GBA. In some embodiments, the liver condition is Manpic disease A / B, and the therapeutic transgene encodes SMPD1. In some embodiments, the liver condition is carbamoyl phosphate synthase I deficiency, and the therapeutic transgene encodes CPS1. In some embodiments, the liver condition is type III glycogen storage disease, and the therapeutic transgene encodes GDE / AGL. In some embodiments, the liver condition is cystinemia, and the therapeutic transgene encodes CTNS. In some embodiments, the liver condition is A1AT deficiency, and the therapeutic transgene encodes SERPINA1. In some embodiments, the liver condition is type I and type II citrullinemia, and the therapeutic transgene encodes one or more of ASS1 and / or SLC25A13.

[0074] The methods described herein can be used to treat subjects requiring gene therapy or enzyme replacement therapy. In some aspects, this disclosure provides a method for treating subjects with liver disease. In some implementations, liver conditions are treated by transfection with therapeutic transgenes, wherein the therapeutic transgenes (e.g., one or more transgenes) are: ATP7B (1465AA); ABCB11 (1321AA); ABCB4 (1286AA); ATP8B1 (1251AA); TJP2 (1190AA); VWF (2813AA); FVIII (2351AA); FIX (415AA); F5 (2224AA); MAN2B1 (1011AA); GBA (536AA); SMPD1 (631AA); CPS1 (1500AA); GDE / AGL (1532AA); CTNS (367AA); SERPINA1 (418AA); ASS1 (412AA); SLC25A13 (675AA).

[0075] In some embodiments, this disclosure provides a method for treating a subject with a liver condition using a payload containing a therapeutic transgene. In some embodiments, the liver condition is Wilson's disease, and the therapeutic transgene encodes ATP7B (1465 aa). In some embodiments, the liver condition is cholestasis, progressive familial intrahepatic cholestasis (PFIC1-4), and the therapeutic transgene encodes one or more of ABCB11 (1321 aa), ABCB4 (1286 aa), ATP8B1 (1251 aa), and / or TJP2 (1190 aa). In some embodiments, the liver condition is von Willebrand disease, and the therapeutic transgene encodes VWF (2813 aa). In some embodiments, the liver condition is hemophilia A, and the therapeutic transgene encodes FVIII (2351 aa). In some embodiments, the liver condition is hemophilia B, and the therapeutic transgene encodes FIX (415 aa). In some embodiments, the liver symptom is factor V deficiency, and the therapeutic transgene encodes F5 (2224 aa). In some embodiments, the liver symptom is alpha-mannosin storage disease, and the therapeutic transgene encodes MAN2B1 (1011 aa). In some embodiments, the liver symptom is Gaucher's disease (glucocerebrosidase deficiency, glucocerebrosidosis), and the therapeutic transgene encodes GBA (536 aa). In some embodiments, the liver symptom is Niemann-Pick disease A / B, and the therapeutic transgene encodes SMPD1 (631 aa). In some embodiments, the liver symptom is carbamoyl phosphate synthase I deficiency, and the therapeutic transgene encodes CPS1 (1500 aa). In some embodiments, the liver symptom is type III glycogen storage disease, and the therapeutic transgene encodes GDE / AGL (1532 aa). In some embodiments, the liver symptom is cystinosis, and the therapeutic transgene encodes CTNS (367 aa). In some embodiments, the liver symptom is A1AT deficiency, and the therapeutic transgene encodes SERPINA1 (418 aa). In some embodiments, the liver symptom is type I and type II citrullinemia, and the therapeutic transgene encodes one or more of ASS1 (412 aa) and / or SLC25A13 (675 aa). In some embodiments, this document provides a method for treating a subject with liver symptom, comprising providing the subject with a treatment procedure, wherein the treatment procedure includes: administering a nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the subject's target cells; and repeating the treatment procedure at least once more than 6 hours but within 10 days after the start of the first treatment procedure.

[0076] In some embodiments, this document provides a method for treating a subject with hemophilia A, comprising providing the subject with a treatment procedure, wherein the treatment procedure includes: administering a nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the subject's target cells; and repeating the treatment procedure at least once more than 6 hours but within 10 days after the start of the first treatment procedure. In some embodiments, the therapeutic transgene is operatively linked to a liver-specific promoter. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding factor VIII. In some embodiments, the nucleic acid payload and / or multiple microbubbles are delivered systemically. In some embodiments, the nucleic acid payload and / or multiple microbubbles are delivered intravenously.

[0077] In some embodiments, this document provides a method for treating a subject with Wilson's disease, comprising providing the subject with a treatment procedure, wherein the treatment procedure includes: administering a nucleic acid payload to the subject; administering multiple microvesicles to the subject; and administering ultrasound energy near the subject's target cells; and repeating the treatment procedure at least once more than 6 hours but within 10 days after the start of the first treatment procedure. In some embodiments, the therapeutic transgene is operatively linked to a liver-specific promoter. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding ATP7B. In some embodiments, the nucleic acid payload and / or multiple microvesicles are delivered systemically. In some embodiments, the nucleic acid payload and / or multiple microvesicles are delivered intravenously.

[0078] On the one hand, using the acoustic perforation method described herein, this disclosure provides a method for treating subjects with kidney conditions. In some embodiments, the kidney condition being treated is acute kidney injury (AKI), Alport syndrome, or autosomal dominant polycystic kidney disease (ADPKD).

[0079] On the one hand, using the acoustic pore-forming method described herein, this disclosure provides a method for treating subjects with kidney disease symptoms. In some embodiments, the payload comprises a therapeutic transgene encoding one or more of the following: COL4A3 (1670 aa), COL4A4 (1690 aa), COL4A5 (1685 aa), PKD1 (4303 aa), and / or PKD2 (968 aa).

[0080] In some embodiments, this disclosure provides a method for treating a subject with a kidney condition having a therapeutic transgene. In some embodiments, the kidney condition is Abbott syndrome, and the therapeutic transgene encodes one or more of COL4A3, COL4A4, and / or COL4A5. In some embodiments, the kidney condition is autosomal dominant polycystic kidney disease, and the therapeutic transgene encodes one or more of PKD1 and / or PKD2. In some embodiments, this document provides a method for treating a subject with a kidney condition comprising providing the subject with a treatment procedure, wherein the treatment procedure includes: administering a nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the subject's target cells; and repeating the treatment procedure at least once more than 6 hours but within 10 days after the start of the first treatment procedure. In some embodiments, this document provides a method for treating a subject with Abbott syndrome, comprising providing the subject with a treatment procedure, wherein the treatment procedure includes: administering a nucleic acid payload to the subject; administering multiple microbubbles to the subject; and administering ultrasound energy near the subject's target cells; and repeating the treatment procedure at least once more than 6 hours but within 10 days after the start of the first treatment procedure. In some embodiments, the therapeutic transgene is operatively linked to a liver-specific promoter. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding COL4A3. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding COL4A4. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding COL4A5. In some embodiments, the nucleic acid payload and / or multiple microvesicles are delivered systemically. In some embodiments, the nucleic acid payload and / or multiple microvesicles are delivered intravenously.

[0081] In some embodiments, this document provides a method for treating a subject with autosomal dominant polycystic kidney disease, comprising providing the subject with a treatment procedure, wherein the treatment procedure includes: administering a nucleic acid payload to the subject; administering multiple microvesicles to the subject; and administering ultrasound energy near the subject's target cells; and repeating the treatment procedure at least once more than 6 hours but within 10 days after the start of the first treatment procedure. In some embodiments, the therapeutic transgene is operatively linked to a liver-specific promoter. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding PKD1. In some embodiments, the nucleic acid payload and / or multiple microvesicles are delivered systemically. In some embodiments, the nucleic acid payload and / or multiple microvesicles are delivered intravenously.

[0082] In some embodiments, applying ultrasound energy near the acoustically active microstructure increases microvascular perfusion of the target tissue. In some embodiments, applying ultrasound energy at a second location within the target tissue increases microvascular perfusion of the target tissue. In some embodiments, repeated acoustic perforation treatment increases microvascular perfusion of the target tissue. In some embodiments, the target cells are located in target tissue exhibiting cystic lesions. In some embodiments, the target cells are located in target tissue exhibiting cystic lesions, wherein delivery of nucleic acids to the target cells in the tissue exhibiting cystic lesions is increased. In some embodiments, the target cells are included in a subject with polycystic kidney disease, wherein the therapeutic transgene includes a sequence encoding PKD1 or PDK2, wherein delivery of the therapeutic transgene to the target cells is increased, thereby treating polycystic kidney disease.

[0083] In some embodiments, the nucleic acid construct is a microplasmid. As used herein, the term "microplasmid (mpDNA)" refers to a nucleic acid construct that is smaller than a conventional plasmid or pDNA in size, excluding therapeutic transgenes, promoters, and regulatory elements (i.e., contains fewer base pairs (bp)). In some embodiments, the mpDNA construct contains a backbone of less than 1 kb. In some embodiments, the mpDNA construct is less than 1000 bp and does not include an expression cassette. In some embodiments, the mpDNA construct contains a backbone of less than 500 bp. In some embodiments, the mpDNA construct is less than 500 bp and does not include an expression cassette. In some embodiments, the microplasmid does not contain a bacterial origin of replication. As used herein, the term "Nanoplasmid™" (e.g., Nanoplasmid from Aldevron, Fargo, South Dakota.) refers to a small mpDNA construct containing a plasmid backbone of less than 500 bp and not containing an antibiotic resistance gene.

[0084] Microplasmid DNA constructs can be used to deliver expression cassettes, transgenes, or non-endogenous genes to cells in target cell types, tissues, or organs. In some embodiments, the mpDNA construct contains fewer than 500 base pairs and does not include an expression cassette. In some embodiments, the mpDNA contains fewer than 1000 base pairs and does not include an expression cassette. In some embodiments, the microplasmid contains fewer than 500 base pairs and does not include an expression cassette. In some embodiments, the mpDNA does not contain an antibiotic resistance gene. In some embodiments, the mpDNA construct does not contain a nucleotide sequence encoding a bacterial gene. In some embodiments, the mpDNA construct does not contain a bacterial genome. In some embodiments, the mpDNA construct contains a therapeutic transgene and / or regulatory element. In some embodiments, the mpDNA construct is a nanoplasmid (e.g., Nanoplasmid™ derived from Aldevron, Fargo, and South Dakota). In some embodiments, when used in conjunction with claimed methods and ultrasonic spectroscopy, the microplasmid construct enhances the expression of non-endogenous genes or therapeutic transgenes. In some embodiments, the nanoplasmid construct enhances the expression of non-endogenous genes or therapeutic transgenes. In some embodiments, the persistence of protein expression encoded by the nucleic acid payload can be increased relative to the expression of the same protein in a larger plasmid (e.g., a plasmid longer than 2 kb, excluding transgenes). In some embodiments, the persistence of protein expression encoded by the nucleic acid payload can be increased relative to the expression of the same protein in another nucleic acid construct.

[0085] In some implementations, the nucleic acid construct is a microplasmid coupled to a nucleic acid payload (e.g., a construct containing a backbone of less than 1000 bp or less than 500 bp).

[0086] In some embodiments, the nucleic acid payload comprises a HALO DNA construct. In some embodiments, the nucleic acid payload comprises a double-stranded, covalently closed circular DNA construct. In some embodiments, the nucleic acid payload comprises a DNA construct containing structural elements that promote nuclear entry. In some embodiments, the nucleic acid payload comprises a DNA construct containing structural elements that promote persistent gene expression after gene transfection.

[0087] In some embodiments, the nucleic acid payload is a small linear DNA construct. In some embodiments, the small linear DNA construct lacks a bacterial-derived backbone. In some embodiments, the small linear DNA construct is a non-viral, uncoated vector. In some embodiments, the small linear DNA construct is an open linear DNA molecule. In some embodiments, the small linear DNA construct is a partially closed linear DNA molecule (e.g., the linear DNA construct is covalently closed at a first end but not covalently closed at a second end). In some embodiments, the small linear DNA construct is a closed linear DNA molecule (e.g., covalently closed). In some embodiments, the small linear DNA construct includes a double-stranded region containing a DNA sequence of interest (e.g., a transgenic, non-endogenous gene, or other heterologous sequence), flanked by a hairpin, stem loop, or single-stranded loop at one or both ends.

[0088] In some embodiments, the nucleic acid payload is a closed linear DNA (“clDNA”) molecule containing a stem region comprising a double-stranded DNA sequence of interest covalently closed at both ends by hairpin loops. In some embodiments, the clDNA molecule is a single-stranded, covalently closed DNA molecule that forms a “dumbbell” or “dog bone” structure under conditions allowing nucleotide hybridization. Thus, although clDNA is formed from a single-stranded DNA molecule, the “dumbbell” structure formed by the hybridization of two complementary sequences within the same molecule produces a structure consisting of a double-stranded intermediate segment flanked by two single-stranded loops.

[0089] In some embodiments, the clDNA molecule is a capless linear double-stranded DNA molecule formed from a continuous DNA strand with covalently closed ends. In some embodiments, the clDNA molecule contains a DNA sequence of interest (e.g., a transgene, a non-endogenous gene, or other heterologous sequence) flanked by a 5′ inverted terminal repeat (ITR) sequence and a 3′ ITR sequence. In some embodiments, the 5′ ITR and 3′ ITR may have a symmetrical three-dimensional structure relative to each other (e.g., symmetrical or substantially symmetrical). In some embodiments, the 5′ ITR and 3′ ITR may have a different three-dimensional structure relative to each other (e.g., asymmetric ITR). In some embodiments, the clDNA molecule also contains a nucleotide sequence encoding an inhibitor of an immune response. In some embodiments, the inhibitor suppresses an innate immune response.

[0090] In some implementations, clDNA can be divided into at least three parts. The first and third parts include some complementarity, enabling these parts to pair bases and form a double helix or stem structure under appropriate or physiological conditions. The double helix is ​​a result of self-complementary sequences within the polynucleotide. The second part lies between the first and third parts and includes the sequence of the linear single-stranded nucleic acid for delivery. Because the second part is located between two complementary sequences, it typically forms a “loop” of nucleic acid that begins and terminates at the first and third parts that form the double helix. It is usually represented as single-stranded because, in order for the delivery vector to function as intended, regions complementary to other sequences within the polynucleotide would not be intentionally included. However, it is well known that single-stranded nucleic acids are assumed to have some secondary structure, so the second part can be any possible conformation, including one or more of hairpins, loops, and pseudoknots, including portions of the linear nucleic acid.

[0091] In some embodiments, the nucleic acid payload is a partially closed linear DNA molecule. In some embodiments, the partially closed linear DNA molecule comprises a linear portion of a double-stranded DNA molecule, wherein the first end of the molecule is closed (or covalently closed, e.g., by a hairpin, stem loop, or single-stranded loop), and the second end of the molecule is open (e.g., having exposed DNA ends). In some embodiments, the double-stranded region contains a DNA sequence of interest (e.g., a transgenic, non-endogenous gene, or other heterologous sequence). In some embodiments, the partially closed linear DNA product contains one or more nuclease-resistant nucleotides at its open end. In some embodiments, an open end refers to at least 5, at least 10, at least 15, or at least 20 base pairs closest to the open end of the DNA product. In some embodiments, the partially closed linear DNA product contains at least 5 nuclease-resistant nucleotides in the open-end region. A nuclease-resistant nucleotide is any suitable nucleotide that provides or enhances resistance to nuclease digestion (e.g., exonucleases or endonucleases). In some embodiments, the nuclease-resistant nucleotide is a modified nucleotide. In some implementations, the modified nucleotide is a thiophosphorylated nucleotide (e.g., 2'-deoxynucleotide-5'-(α-thio)-triphosphate). A "thiophosphorylated nucleotide" refers to a nucleotide with a modified phosphate backbone in which the sugar moieties are linked by thiophosphate bonds. In the phosphate backbone of the oligonucleotide sequence, the thiophosphate bond contains a sulfur atom as a substitute for a non-bridging oxygen atom. This modification makes the internucleotide linkage resistant to nuclease degradation.

[0092] In some embodiments, the nucleic acid payload is a circular RNA (circRNA) molecule. Circular RNA is a 3-5' covalently closed RNA loop. In some embodiments, circRNA exhibits improved resistance to exonuclease-mediated degradation compared to comparable linear RNA. In some embodiments, circRNA exhibits improved stability compared to comparable linear RNA. In some embodiments, circRNA includes an internal ribosome entry site (IRES).

[0093] In some embodiments, any nucleic acid payload described herein comprises one or more modified nucleotides. In some embodiments, any nucleic acid payload described herein comprises at least two modified nucleotides. In some embodiments, one or more modified nucleotides improve transfection efficiency, expression efficiency, stability, bioavailability, functional persistence, resistance to degradation, overall functional performance, or a combination thereof. "Modified nucleotide" is any nucleotide that has been chemically modified (by modifying a base, sugar, or phosphate ester group) or incorporated with a non-natural portion into its structure (e.g., adenosine, guanosine, cytidine, and thymidine). Thus, depending on the modification, the modified nucleotide can be naturally occurring or non-naturally occurring. In some embodiments, the modified nucleotide is resistant to nuclease activity (e.g., endonucleases or exonucleases).

[0094] In some embodiments, the total amount of DNA administered to the subject for sonoforming purposes can range from about 1 microgram (μg) to about 200 mg. In some embodiments, the total amount of DNA administered to the subject is from 20 mg to 100 mg. In some embodiments, the total amount of DNA administered to the subject is about 20 mg. In some embodiments, the total amount of DNA administered to the subject is about 50 mg.

[0095] In some embodiments, the nucleic acid construct is administered at a dose of at least 0.4 mg / kg. In some embodiments, the nucleic acid construct is administered at a dose of up to 500 mg / kg. In some embodiments, the nucleic acid construct is administered at a dose of about 0.5 mg / kg body weight to about 500 mg / kg body weight. In some embodiments, the nucleic acid construct is administered at a dose of 2 mg / kg to 5.5 mg / kg. In some embodiments, the nucleic acid construct is administered at a dose of about 3.5 μg / μl. In some embodiments, about 2 × 10^13 to about 3 × 10^13 copies of the nucleic acid construct are administered to the subject.

[0096] The acoustically active microparticles considered herein (also referred to as acoustically active microstructures, acoustic microspheres, or microbubbles) include, but are not limited to, those used as contrast agents in ultrasound imaging. In some embodiments, the acoustically active microstructures include phospholipid-stabilized microstructures. In some embodiments, the phospholipid-stabilized microstructures comprise a high-molecular-weight gas core or a perflutran core. Examples of acoustically active microstructures include, but are not limited to, OPTISON (GE Healthcare), Sonazoid (GE Healthcare), or DEFINITY and Definity RT (Lantheus Medical Imaging, Inc.). In some embodiments, the acoustically active microstructure is LUMASON (Bracco) (sulfur hexafluoride lipid-type A microspheres). In some embodiments, the acoustically active microstructure is SonoVue (sulfur hexafluoride microbubbles). In some embodiments, the acoustically active microstructures include protein-stabilized microstructures. In some embodiments, the acoustically active microstructure is Optison microbubbles.

[0097] In some embodiments, the acoustic active agent (e.g., acoustic active microstructure or microbubble) comprises a phospholipid-stabilized microstructure. In some embodiments, the acoustic active agent (e.g., acoustic active microstructure or microbubble) comprises a phospholipid-stabilized shell. In some embodiments, the acoustic active agent (e.g., acoustic active microstructure or microbubble) comprises a lipid-stabilized shell. In some embodiments, the acoustic active agent (e.g., acoustic active microstructure or microbubble) comprises a protein-stabilized shell. In some embodiments, the acoustic active agent (e.g., acoustic active microstructure or microbubble) comprises an albumin-stabilized shell. In some embodiments, the phospholipid-stabilized microstructure comprises a high-molecular-weight gas core, such as a perfluoropropane core. Examples of acoustic active agents (e.g., acoustic active microstructures or microbubbles) include, but are not limited to, OPTISON (GE Healthcare), Sonazoid (GE Healthcare), or Definity and Definity RT (Lantheus Medical Imaging, Inc.). In some embodiments, the acoustic active agent (e.g., acoustic active microstructure or microbubble) is a Sonazoid microbubble, a Definity microbubble, or a Definity RT microbubble. In some embodiments, the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is Optison microbubbles. In some embodiments, the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is SonoVue (sulfur hexafluoride microbubbles). In some embodiments, the acoustic active agent (e.g., acoustic active microstructures or microbubbles) comprises protein-stabilizing microstructures. In some embodiments, the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is Optison microbubbles.

[0098] The acoustic active agent (e.g., acoustic active microstructures or microbubbles) may be applied before, after, or simultaneously with the application of the nucleic acid construct (or nucleic acid payload) (e.g., co-application). In some embodiments, the nucleic acid construct and the acoustic active agent (e.g., acoustic active microstructures or microbubbles) are applied co-administered. In some embodiments, the application of the nucleic acid construct and the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is performed continuously, in parallel, sequentially, or continuously. In some embodiments, the application of the nucleic acid construct and the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is performed continuously. In some embodiments, the application of the nucleic acid construct and the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is performed in parallel. In some embodiments, the application of the nucleic acid construct and the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is performed sequentially. In some embodiments, the application of the nucleic acid construct and the acoustic active agent (e.g., acoustic active microstructures or microbubbles) is performed continuously during the ultrasound treatment procedure (e.g., always intravenously).

[0099] As used herein, the concentration of microstructures per mL refers to the concentration of the acoustic active agent (e.g., acoustic microstructures or microbubbles) in the pharmaceutical composition prior to administration to a subject. In some embodiments, the acoustic active microstructure is administered at a concentration of about 5 x 10^8 to about 1.2 x 10^10 microstructures per mL, for example, 1 x 10^9 microstructures per mL of Definity RT. In some embodiments, the acoustic active microstructure is administered at a dose of about 1-50 mL, for example, 1 mL of protein-stabilized acoustic active microstructures (e.g., Optison). In some embodiments, the protein-stabilized acoustic active microstructures (e.g., Optison) have a diameter of 3-4.5 micrometers. The acoustic active microstructure can be administered at a concentration of about 500 M (millions) to about 800 M microstructures per mL. In some embodiments, 1 x 10^9 phospholipid-stabilized acoustic active microstructures (e.g., Sonazoid) are administered. In some embodiments, the phospholipid-stabilized acoustic active microstructures (e.g., Sonazoid) have a diameter of 1-5 micrometers. In some embodiments, the acoustically active microstructure is administered at a dose of about 0.1 to about 0.8 mg microstructure / kg body weight. In some embodiments, the acoustically active microstructure is administered at a dose of about 0.1 to about 1.0 ml / kg body weight. In some embodiments, the acoustically active microstructure is administered at a concentration of about 10^9 microstructures / ml. In some embodiments, the acoustically active microstructure is administered at a concentration of at least 5 x 10^8 microstructures / mL. In some embodiments, the acoustically active microstructure is administered at a concentration of up to 1.2 x 10^10 microstructures / mL. In some embodiments, the acoustically active microstructure is administered at a concentration of 5 x 10^8 to 8 x 10^8 microstructures / mL.

[0100] In some implementations, the nucleic acid construct and the acoustic active agent (e.g., acoustically active microstructures or microbubbles) are mixed before co-administration. In some cases, the acoustically active microstructure is mixed with the nucleic acid construct and additional buffers or reagents (such as saline or other biocompatible solutions with different electrostatic charges, surface chemical structures, and ligands) before administration to the subject. For example, Optison acoustically active microstructures can be mixed with nanoparticles and saline and administered together.

[0101] This disclosure provides an ultrasound system including a computer system programmed to implement the methods of this disclosure. The ultrasound system 200 may be operatively connected to one or more ultrasound transducers 211 controlled by a computer system 201 and one or more computer processors 204. The computer processor 204 may include one or more computer-readable media 205, which include instructions configured to cause the ultrasound system to perform the methods of this disclosure. The ultrasound system 200 and / or the computer processor 204 may communicate with a cloud 207 or other remote server, enabling remote operation and control of the ultrasound system 200 and the execution of the methods disclosed herein. The computer system 201 may be a user's electronic device or a computer system remotely located relative to an electronic device. The electronic device may be a mobile electronic device. The computer system includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor"), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system also includes memory or memory location 206 (e.g., random access memory, read-only memory, flash memory), electronic storage units (e.g., hard disks), communication interfaces (e.g., network adapters) for communicating with one or more other systems, and peripheral devices such as cache, other memory, data storage, and / or electronic display adapters. The memory, storage units, interfaces, and peripheral devices communicate with the CPU via a communication bus (solid line) such as a motherboard. Storage units may be data storage units (or data repositories) for storing data. The computer system may be operatively coupled to a computer network (“network”) via a communication interface. A network may be the Internet, the Internet of Things, and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, the network is a telecommunications and / or data network. A network may include one or more computer servers that can enable distributed computing, such as cloud computing. In some cases, the network may enable peer-to-peer networking, which allows devices coupled to the computer system to act as clients or servers.

[0102] Aspects disclosed herein provide a system (e.g., an ultrasound system) that includes: an ultrasound transducer configured to apply ultrasonic acoustic energy to a subject at a plurality of mechanical indices; a computer system including a computer processor and a computer-readable medium, wherein the computer system is configured to implement a method of applying ultrasonic acoustic energy to target cells of the subject, the method including: applying ultrasonic acoustic energy to the target cells at a first mechanical index (MI) of up to 0.4 (e.g., 0 < MI ≤ 0.4); and applying ultrasonic acoustic energy to the target cells at a second MI greater than 0.4 and up to 2.0 (e.g., 0.4 < MI ≤ 2.0), wherein the subject has been administered a nucleic acid construct comprising a nucleic acid payload and a plurality of sonoactive microstructures, and wherein the nucleic acid construct is a minicircle plasmid. In some embodiments, the ultrasound transducer that applies the ultrasonic acoustic energy to the target cells is in continuous contact with the subject's tissue and continuously (1) applies ultrasonic acoustic energy to the subject or (2) receives reflected ultrasonic energy from the subject. In some embodiments, the nucleic acid construct is a plasmid that does not include an expression cassette and has a length less than or equal to 500 base pairs, or wherein the nucleic acid construct is a minicircle plasmid. In some embodiments, applying the ultrasonic acoustic energy at the first MI and applying the ultrasonic acoustic energy at the second MI are repeated at least two times. In some embodiments, applying the ultrasonic acoustic energy at the first MI and applying the ultrasonic acoustic energy at the second MI are repeated 4 to 18 times. In some embodiments, applying the ultrasonic acoustic energy at the first MI and applying the ultrasonic acoustic energy at the second MI are repeated 6 to 12 times. In some embodiments, applying the ultrasonic acoustic energy at the first MI and applying the ultrasonic acoustic energy at the second MI are repeated 8 to 10 times. In some embodiments, the second MI ranges from about 1.4 to about 2.0. In some embodiments, applying the ultrasonic acoustic energy at the second mechanical index induces the formation of pores in the membrane of the cells. In some embodiments, applying the ultrasonic acoustic energy at the first mechanical index induces the formation of intercellular gaps or interendothelial gaps. In some embodiments, the ultrasound transducer transmits ultrasonic acoustic energy or receives reflected ultrasonic acoustic energy for at least 95% of the period during which the ultrasound transducer is in continuous contact with the subject. In some embodiments, applying the ultrasonic acoustic energy in d. includes applying the ultrasonic acoustic energy at the second MI using pulses. In some embodiments, applying the ultrasonic acoustic energy at the second MI includes applying the ultrasonic acoustic energy at the second MI using pulses having a duration of from about 1 μs to about 500 μs. In some embodiments, applying the ultrasonic acoustic energy at the second MI includes applying the ultrasonic acoustic energy at the second MI using pulses having a duration of up to 200 μs.In some embodiments, applying the ultrasonic energy at the second MI comprises applying the ultrasonic energy at the second MI using a pulse for a duration of up to 500 μs. In some embodiments, applying the ultrasonic energy at the second MI comprises applying the ultrasonic energy at the second MI using a pulse for a duration of about 1 μs to about 200 μs. In some embodiments, applying the ultrasonic energy at the second MI comprises applying the ultrasonic energy at the second MI using a pulse for a duration of about 2.3 μs. In some embodiments, the method comprises repeatedly applying the ultrasonic energy at the first MI and applying the ultrasonic energy at the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy at the first MI for a duration sufficient to allow reperfusion of the acoustically active microstructure in the tissue containing the target cells. In some embodiments, the repetition comprises applying the ultrasonic energy at the first MI for 1-30 seconds before repeatedly applying the ultrasonic energy at the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy at the first MI for 5-15 seconds before applying the ultrasonic energy at the second MI. In some embodiments, the repetition includes applying the ultrasonic energy for 10 seconds at the first MI before applying the ultrasonic energy at the second MI.

[0103] The systems disclosed herein (e.g., ultrasound systems) can be controlled or operated by a computer including a computer-readable medium configured to perform a method of applying ultrasonic acoustic energy to target cells of a subject, the method comprising: applying ultrasonic acoustic energy to the target cells with a first mechanical index (MI) of up to 0.4; and applying ultrasonic acoustic energy to the target cells with a second MI greater than 0.4 and up to 2.0, wherein the subject has been administered (1) a nucleic acid construct comprising a nucleic acid payload, wherein the nucleic acid construct is a microplasmid, and (2) a plurality of acoustically active microstructures. In some embodiments, an ultrasonic transducer applying the ultrasonic acoustic energy to the target cells is in continuous contact with the subject's tissue and continuously (1) applies ultrasonic acoustic energy to the subject or (2) receives reflected ultrasonic energy from the subject. In some embodiments, the microplasmid is less than or equal to 500 base pairs in length and does not include an expression cassette. In some embodiments, the application of the ultrasonic acoustic energy with the first MI and the application of the ultrasonic acoustic energy with the second MI are repeated at least twice. In some embodiments, the application of ultrasonic energy at the first MI and the application of ultrasonic energy at the second MI are repeated 4 to 18 times. In some embodiments, the application of ultrasonic energy at the first MI and the application of ultrasonic energy at the second MI are repeated 6 to 12 times. In some embodiments, the application of ultrasonic energy at the first MI and the application of ultrasonic energy at the second MI are repeated 8 to 10 times. In some embodiments, the second MI ranges from about 1.4 to about 2.0. In some embodiments, the application of ultrasonic energy at the second mechanical index induces the formation of pores in the cell membrane. In some embodiments, the application of ultrasonic energy at the first mechanical index induces the formation of intercellular or interendothelial space. In some embodiments, the ultrasonic transducer transmits ultrasonic energy or receives reflected ultrasonic energy for at least 95% of the time period during which the ultrasonic transducer is in continuous contact with the subject. In some embodiments, applying the ultrasonic energy at the second MI includes applying the ultrasonic energy at the second MI using a pulse. In some embodiments, applying the ultrasonic acoustic energy with the second MI includes applying the ultrasonic acoustic energy with a pulse of duration from about 1 μs to about 500 μs with the second MI. In some embodiments, applying the ultrasonic acoustic energy with the second MI includes applying the ultrasonic acoustic energy with a pulse of duration from about 200 μs with the second MI. In some embodiments, applying the ultrasonic acoustic energy with the second MI includes applying the ultrasonic acoustic energy with a pulse of duration from about 1 μs to about 200 μs with the second MI.In some embodiments, applying the ultrasonic energy at the second MI includes applying the ultrasonic energy at the second MI using a pulse for a duration of about 2.3 μs. In some embodiments, the specification includes repeating the application of the ultrasonic energy at the first MI and the application of the ultrasonic energy at the second MI. In some embodiments, the repetition includes applying the ultrasonic energy at the first MI for a duration sufficient to allow reperfusion of the acoustically active microstructure in the tissue containing the target cells. In some embodiments, the repetition includes applying the ultrasonic energy at the first MI for 1-30 seconds before repeating the application of the ultrasonic energy at the second MI. In some embodiments, the repetition includes applying the ultrasonic energy at the first MI for 5-15 seconds before applying the ultrasonic energy at the second MI. In some embodiments, the repetition includes applying the ultrasonic energy at the first MI for 10 seconds before applying the ultrasonic energy at the second MI.

[0104] A CPU can execute a series of machine-readable instructions, which can be embodied in a program or software. These instructions can be stored in a memory location, such as memory. The instructions can be directed to the CPU, which can then be programmed or otherwise configured to implement the methods disclosed herein. Examples of operations performed by the CPU can include reading, decoding, executing, and writing back.

[0105] A CPU can be part of a circuit, such as an integrated circuit. One or more other components of the system can be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0106] Storage units can store files, such as drivers, libraries, and saved programs. Storage units can also store user data, such as user preferences and user programs. In some cases, a computer system may include one or more additional data storage units located outside the computer system, such as on a remote server that communicates with the computer system via an intranet or the Internet.

[0107] A computer system can communicate with one or more remote computer systems via a network. For example, a computer system can communicate with a user's remote computer system (e.g., a handheld device). Examples of remote computer systems include personal computers (e.g., portable PCs), tablet PCs (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, smartphones (e.g., Apple® iPhone, Android-enabled devices, Blackberry®), or personal digital assistants. Users can access the computer system via a network.

[0108] The methods described herein can be implemented using machine-executable code (e.g., a computer processor) stored in an electronic storage location (e.g., a memory or electronic storage unit) of a computer system. The machine-executable code, or machine-readable code, can be provided in software form. During use, the code can be executed by the processor. In some cases, the code can be retrieved from the storage unit and stored in memory for processor-ready access. In some cases, the electronic storage unit can be excluded, and the machine-executable instructions are stored in memory.

[0109] The code can be pre-compiled and configured for use with machines that have processors suitable for executing the code, or it can be compiled during runtime. The code can be provided in a programming language, and the programming language can be selected to enable the code to be executed either pre-compiled or compile-time.

[0110] The aspects of the systems and methods provided herein, such as computer systems, can be embodied in programming. These aspects of the technology can be considered "products" or "artifacts," typically in the form of machine (or processor) executable code and / or associated data carried or embodied on a type of machine-readable medium. Machine-executable code can be stored in electronic storage units, such as memory (e.g., read-only memory, random access memory, flash memory) or hard disks. "Storage" media can include any or all tangible memory of computers, processors, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication enables the loading of software from one computer or processor to another, e.g., from a management server or host computer platform to an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as light waves, radio waves, and electromagnetic waves used through physical interfaces between local devices, via wired and optical terrestrial networks, and via various air links. Physical elements carrying such waves (such as wired or wireless links, optical links, etc.) can also be considered as media carrying software. As used herein, unless limited to non-transitory, tangible "storage" media, the term "readable medium" for a computer or machine refers to any medium involved in providing instructions to a processor for execution.

[0111] Therefore, machine-readable media, such as computer-executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical discs or disks, any storage device such as any computer, and such as those shown in the attached figures that can be used to execute databases, etc. Volatile storage media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wires and optical fibers, including the wires that form buses within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Therefore, common forms of computer-readable media include, for example: floppy disks, floppy disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card tape, any other physical storage media with a perforated pattern, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chips or cassette tapes, carriers for transmitting data or instructions, cables or links for transmitting such carriers, or any other media from which a computer can read programming code and / or data. Many of these forms of computer-readable media can be involved in bringing one or more sequences of one or more instructions to a processor for execution.

[0112] A computer system may include or communicate with an electronic display, which includes a user interface (UI) for providing, for example, the concentration of an analyte of interest. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0113] The methods and systems disclosed herein can be implemented using one or more algorithms. These algorithms can be implemented in software when executed by a central processing unit.

[0114] In some aspects, this disclosure provides quality control methods or methods for assessing risks associated with the presence of food, hospitals, clinics, or bacteria at any other location that poses a risk to one or more subjects. In many cases, the systems, platforms, software, networks, and methods described herein include digital processing devices or their uses. In further embodiments, the digital processing device includes one or more hardware central processing units (CPUs), i.e., processors that perform device functions, such as the automated sequencing apparatus disclosed herein or a computer system for analyzing multiple nucleic acid sequencing reads from samples originating from food processing facilities or any other facility (such as a hospital, clinic, or others). In even further embodiments, the digital processing device also includes an operating system configured to execute executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Internet, enabling it to access the World Wide Web. In even further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device. In other embodiments, the digital processing device may be deployed locally or remotely in the cloud.

[0115] Based on the description herein, suitable digital processing devices, as non-limiting examples, include server computers, desktop computers, laptop computers, notebook computers, mini-notebook computers, netbook computers, internet tablet computers, set-top computers, handheld computers, internet devices, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those skilled in the art will recognize that many smartphones are suitable for use with the systems described herein. Those skilled in the art will also recognize that optional televisions, video players, and digital music players with optional computer network connectivity are suitable for use with the systems described herein. Suitable tablet computers include those with catalogs, tablets, and convertible configurations known to those skilled in the art. In many respects, this disclosure contemplates any suitable digital processing device that can be deployed in, or used within, food processing facilities to process and analyze various nucleic acids from a variety of samples.

[0116] In some embodiments, the digital processing device includes an operating system configured for executing executable instructions. The operating system is, for example, software comprising programs and data that manages the device's hardware and provides services for executing applications. Those skilled in the art will recognize, as non-limiting examples, suitable server operating systems include FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those skilled in the art will recognize, as non-limiting examples, suitable personal computer operating systems include Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Those skilled in the art will also recognize, as non-limiting examples, suitable mobile smartphone operating systems include Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.

[0117] In some embodiments, the digital processing device includes a storage device and / or a memory device. A storage device and / or memory device is one or more physical means for temporarily or permanently storing data or programs. In some embodiments, the device is volatile memory and requires power to maintain the stored information. In some embodiments, the device is non-volatile memory and retains the stored information when the digital processing device is not powered. In further embodiments, the non-volatile memory includes flash memory. In some embodiments, the non-volatile memory includes dynamic random access memory (DRAM). In some embodiments, the non-volatile memory includes ferroelectric random access memory (FRAM). In some embodiments, the non-volatile memory includes phase-change random access memory (PRAM). In other embodiments, the device is a storage device, including, as non-limiting examples, CD-ROMs, DVDs, flash memory devices, disk drives, tape drives, optical disc drives, and cloud-based storage. In further embodiments, the storage device and / or memory device is a combination of devices such as those disclosed herein.

[0118] In some embodiments, the digital processing device includes a display that sends visual information to a user. In some embodiments, the display is a cathode ray tube (CRT). In some embodiments, the display is a liquid crystal display (LCD). In a further embodiment, the display is a thin-film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light-emitting diode (OLED) display. In various further embodiments, the OLED display is a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In even further embodiments, the display is a combination of devices such as those disclosed herein.

[0119] In some embodiments, the digital processing device includes an input device for receiving information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device, including, as non-limiting examples, a mouse, trackball, trackpad, joystick, game controller, or stylus. In some embodiments, the input device is a touchscreen or multi-touchscreen. In other embodiments, the input device is a microphone for capturing voice or other sound input. In other embodiments, the input device is a camera for capturing motion or visual input. In further embodiments, the input device is a combination of devices such as those disclosed herein.

[0120] In some embodiments, the digital processing device includes a digital camera. In some embodiments, the digital camera captures digital images. In some embodiments, the digital camera is an autofocus camera. In some embodiments, the digital camera is a charge-coupled device (CCD) camera. In further embodiments, the digital camera is a CCD camcorder. In other embodiments, the digital camera is a complementary metal-oxide-semiconductor (CMOS) camera. In some embodiments, the digital camera captures still images. In other embodiments, the digital camera captures video images. In various embodiments, suitable digital cameras include cameras with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and higher megapixel resolutions, including increments therein. In some embodiments, the digital camera is a standard definition camera. In other embodiments, the digital camera is an HD camcorder. In further embodiments, the HD camera captures images having at least about 1280 × about 720 pixels or at least about 1920 × about 1080 pixels. In some embodiments, the digital camera captures color digital images. In other embodiments, the digital camera captures grayscale digital images. In various embodiments, the digital images are stored in any suitable digital image format. As non-limiting examples, suitable digital image formats include Joint Picture Experts Group (JPEG), JPEG 2000, Exif (Exif), Tagged Image File Format (TIFF), RAW, Portable Web Graphics (PNG), Graphics Interchange Format (GIF), Windows® Bitmap (BMP), Portable Image (PPM), Portable Grayscale (PGM), Portable Bitmap File Format (PBM), and WebP. In various embodiments, the digital images are stored in any suitable digital video format. As non-limiting examples, suitable digital video formats include AVI, MPEG, Apple® QuickTime®, MP4, AVCHD®, Windows Media®, DivX™, Flash Video, OggTheora, WebM, and RealMedia.

[0121] In many respects, the systems, platforms, software, networks, and methods disclosed herein include one or more non-transitory computer-readable storage media encoded with a program comprising instructions executable by an operating system of an optionally networked digital processing device. For example, in some aspects, the method includes creating a data file associated with multiple sequencing reads from multiple samples related to a food processing facility. In further embodiments, the computer-readable storage medium is a tangible component of the digital processing device. In even further embodiments, the computer-readable storage medium is optionally removable from the digital processing device. In some embodiments, as non-limiting examples, the computer-readable storage medium includes CD-ROMs, DVDs, flash memory devices, solid-state storage, disk drives, magnetic tape drives, optical disc drives, cloud computing systems and services, etc. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitory encoded on the medium.

[0122] In some embodiments, the systems, platforms, software, networks, and methods disclosed herein include at least one computer program. The computer program includes a sequence of instructions executable in the CPU of a digital processing device, the sequence of instructions being written to perform a specified task. Based on the disclosure provided herein, those skilled in the art will recognize that the computer program can be written in various versions of various languages. In some embodiments, the computer program contains a single sequence of instructions. In some embodiments, the computer program includes multiple sequences of instructions. In some embodiments, the computer program is provided from one location. In other embodiments, the computer program is provided from multiple locations. In various embodiments, the computer program includes one or more software modules. In various embodiments, the computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plugins, extensions, add-ons, or attachments, or combinations thereof.

[0123] In some embodiments, the computer program includes a web application. Based on the disclosure provided herein, those skilled in the art will recognize that, in various embodiments, the web application utilizes one or more software frameworks and one or more database systems. In some embodiments, the web application is created on a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, the web application utilizes one or more database systems, including, as non-limiting examples, relational database systems, non-relational database systems, object-oriented database systems, relational database systems, and XML database systems. In further embodiments, suitable relational database systems include, as non-limiting examples, Microsoft® SQL Server, MySQL™, and Oracle®. Those skilled in the art will also recognize that, in various embodiments, the web application is written in one or more versions of one or more languages. The web application can be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, the web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or Extensible Markup Language (XML). In some embodiments, the web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some implementations, the web application is written to some extent in a client-side scripting language, such as Asynchronous JavaScript and XML (AJAX), Flash® Actionscript, JavaScript, or Silverlight®. In some implementations, the web application is written to some extent in a server-side coding language, such as ActiveServer Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In some implementations, the web application is written to some extent in a database query language such as Structured Query Language (SQL). In some implementations, the web application integrates with enterprise server products, such as IBM® Lotus Domino®. In some implementations, the web application used to provide artists with a career development network that allows artists to upload information and media files includes media player components.In various further embodiments, the media player element utilizes one or more of a number of suitable multimedia technologies, including, as a non-limiting example, Adobe® Flash®, HTML5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

[0124] In some embodiments, the computer program includes a mobile application provided to a mobile digital processing device. In some embodiments, the mobile application is provided to the mobile digital processing device at the time of manufacture. In other embodiments, the mobile application is provided to the mobile digital processing device via a computer network described herein.

[0125] Given the disclosure provided herein, mobile applications are created using techniques known to those skilled in the art, utilizing hardware, languages, and development environments known in the art. Those skilled in the art will recognize that mobile applications are written in several languages. As non-limiting examples, suitable programming languages ​​include C, C++, C#, Objective-C, Java™, Javascript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML / HTML, or combinations thereof, with or without CSS.

[0126] Suitable mobile application development environments are available from several sources. Commercially available development environments, as non-limiting examples, include AirplaySDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are freely available, including, as non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Additionally, mobile device manufacturers distribute software development kits, including, as non-limiting examples, the iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.

[0127] Those skilled in the art will recognize that several business forums can be used to distribute mobile applications, including, as non-limiting examples, the Apple® App Store, Android™ Marketplace, BlackBerry® App World, Palm device app store, webOS app catalog, Windows® Mobile Marketplace, Nokia® Ovi Store, Samsung® App Store, and Nintendo® DSi Store.

[0128] In some implementations, the computer program includes a standalone application, which is a program that runs as an independent computer process, rather than an add-on to an existing process, such as a plugin. Those skilled in the art will recognize that standalone applications are typically compiled. A compiler is a computer program that translates source code written in a programming language into binary object code (such as assembly language or machine code). Suitable programming languages ​​for compilation, as non-limiting examples, include C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB.NET, or combinations thereof. Compilation is typically performed at least partially to create an executable program. In some implementations, the computer program includes one or more executable compiled applications.

[0129] In some embodiments, after the first body of the 3D object is generated, the movable stage is removed from the actuator system. The 3D object can then proceed to further processing steps, such as the infusion sequence described herein. In various embodiments, the systems, platforms, software, networks, and methods disclosed herein include software, server, and database modules. In view of the disclosure provided herein, software modules are created using machines, software, and languages ​​known in the art, employing techniques known to those skilled in the art. The software modules disclosed herein are implemented in a variety of ways. In various embodiments, the software module includes files, code segments, programming objects, programming structures, or combinations thereof. In further embodiments, the software module includes multiple files, multiple code segments, multiple programming objects, multiple programming structures, or combinations thereof. In various embodiments, as non-limiting examples, the one or more software modules include web applications, mobile applications, and standalone applications. In some embodiments, the software module resides within a single computer program or application. In other embodiments, the software module resides within more than one computer program or application. In some embodiments, the software module is hosted on a single machine. In other embodiments, the software module is hosted on more than one machine. In a further embodiment, the software module is hosted on a cloud computing platform. In some implementations, the software module is hosted on one or more machines in one location. In other implementations, the software module is hosted on one or more machines in more than one location.

[0130] definition As used in the specification and claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. For example, the term “sample” includes multiple samples, including mixtures thereof.

[0131] As used herein, the term “approximately” refers to a number plus or minus 10% of that number. The term “approximately” refers to a range minus 10% of its lowest value and plus 10% of its highest value.

[0132] As used in this article, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that function as both conjunctions and adversative words in practice. For example, the expressions “at least one of A, B, and C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “one or more of A, B, or C,” and “A, B, and / or C” each mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0133] As used herein, “or” can mean “and,” “or,” or “and / or,” and can be used exclusively and inclusively. For example, the term “A or B” can mean “A or B,” “A but not B,” “B but not A,” and “A and B.” In some cases, the context may specify a particular meaning.

[0134] Example The following examples are provided to further illustrate some embodiments of this disclosure, but are not intended to limit the scope of this disclosure; by their exemplary nature it will be understood that other procedures, methods or techniques known to those skilled in the art may be used alternatively.

[0135] Example 1: Acoustic pore formation in a mouse liver model using a repetitive acoustic pore formation scheme, comparing different repetition intervals. Laboratory animals and protocols There were seven experimental groups, each consisting of four BALB / c mice, except for the baseline group which consisted of three BALB / c mice. Prior to the experiments, a jugular vein catheter (JVC) was implanted into each mouse, through which the acoustically active microstructures and nucleotide constructs were administered.

[0136] Mice in this experiment received at least one dose of a microplasmid DNA (mpDNA) construct and a mixture of acoustically active microstructures. The mpDNA construct used in this experiment was the Nanoplasmid™ construct. All groups, except the control group (baseline group), received a second dose of ultrasound after delivery of the first dose. Except for the “6h no US” control group, ultrasound (US) energy was delivered percutaneously with each dose to transfect the target organ, the liver. In the five experimental groups, the second dose of ultrasound and US was delivered at 6h, 24h, 48h, or 72h after the first dose, respectively. These experimental protocols are described below. Figure 1 As shown.

[0137] Figure 1 Illustrative experimental protocols are provided in which two doses of DNA constructs and acoustically active microstructures are delivered to mice along with ultrasound (two-dose acoustic pore formation assay). In these protocols, mice receive at least one dose of a mixture of microplasmid DNA (mpDNA) constructs and acoustically active microstructures. Ultrasound focused on a target region or target cell is delivered to the mouse along with the mixture of microplasmid DNA constructs and acoustically active microstructures. The microplasmid DNA constructs, such as nanoplasmid DNA constructs, may contain nucleic acid payloads, such as therapeutic transgenes coupled to promoter sequences or other regulatory element sequences.

[0138] Figure 1Several two-dose sono-induced pore-forming protocols are described. Large dots along the horizontal line represent approximate delivery times of the mpDNA and sono-active microstructure mixture dose and US. The top (“6h without US”, lightest gray) and bottom (“baseline”, darkest gray) arrows represent control experiments where no ultrasound (US) was delivered to mice or no second dose was delivered. In other experiments, the second dose was delivered at 6h, 24h, 48h, or 72h after the first dose (from second from the top arrow to second from the bottom arrow, from light gray to dark gray, respectively). One dose of the nanoparticle and sono-active microstructure mixture contains 250 μg of the nanoparticle construct. The nanoparticle construct contains nucleic acid encoding a luciferase gene coupled to the ApoE promoter.

[0139] The time period for the first measurement of the expression of the nucleic acid payload (e.g., luciferase) delivered by mpDNA using IVIS fluorescence imaging in mice is indicated by a vertical solid arrow (24 h after dose 1). This measurement may include baseline fluorescence measurements. Subsequent IVIS fluorescence imaging procedures at 24 h, 48 h, 72 h, and 1 week after the second dose (dose 2) are indicated by vertical dashed lines. IVIS fluorescence imaging was performed on all mice in each group at each time point.

[0140] Prepare a dose of sonoactive microstructure and DNA solution by first preparing the sonoactive microstructure according to the label instructions: remove from 4C storage and roll between your palms for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 μL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space is included in the calculation). Using the same needle and syringe, draw 75 μL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space (approximately 50 μL). Then replace the 18G needle with a 25G blunt needle for injection into the JVC.

[0141] Each dose comprises three 100 μl doses of mpDNA and acoustically active microstructure mixture in PBS, each dose delivered via a jugular vein catheter. The microplasmid DNA construct contains nucleic acid encoding a luciferase gene coupled to the ApoE promoter. Acoustically active microstructure Optison™ microbubbles were delivered to mice at a 1:4 volume ratio (mpDNA solution: acoustically active microstructure mixture, including needle dead space dispersed in the mixture).

[0142] Following the application of microbubbles and nucleic acid payloads, in these experiments, ultrasound energy was delivered to the liver region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the liver at a low mechanical index (MI) of 0.07 using B-mode ultrasound imaging. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 0.8, without interruption of ultrasound energy application at any point during the treatment process. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the application of the nine pulses was repeated three times. The duration of the high MI pulse was approximately 0.82 microseconds. The ultrasound application lasted less than 30 seconds.

[0143] IVIS fluorescence irradiance imaging was performed in all groups 24 h after delivery of the first dose, at which time the fluorescence indicated the expression level of the luciferase payload. Figure 1 (Vertical solid arrow, "24h after dose 1"). This measurement constitutes the baseline fluorescence measurement. Subsequent IVIS fluorescence imaging was performed at 24h, 48h, 72h, and 1 week after delivery of the second dose (dose 2) for each group. Figure 1 (vertical dashed line). IVIS fluorescence imaging was performed on all mice in each group at each time point. Figure 1 The seven dual-dose sono-forming protocols used in this experiment are illustrated. Large dots along the horizontal line represent approximate delivery times for the mpDNA and sono-active microstructure doses.

[0144] result Using IVIS, mice were imaged 24 h after delivery of the first dose (dose 1) of mpDNA and acoustically active microstructures during the first sonopore-forming treatment, and then re-imaged at 24 h, 48 h, and 72 h after delivery of the second dose during the second sonopore-forming treatment. Figure 2 Mice in the "6h no ultrasound" control group (without ultrasound delivery) showed weaker fluorescence signals at baseline at each measurement compared to mice in the ultrasound-delivered group. Figure 2 Comparing the leftmost column with the other columns, it shows a lack of gene transfection and expression due to sonoporation treatment. Mice not treated with ultrasound showed a decrease in gene transfection and expression 1 week after dose 2 ( Figure 2 The very low fluorescence emissivity (see bottom left image) indicates a lack of gene transfection and expression due to the absence of sonopore-forming therapy. In all other groups, the fluorescence emissivity persisted for 1 week after the dose-2 time point. Figure 2 (bottom row), indicating that the two-dose sono-induced pore formation protocol can induce persistent expression of nucleic acid payloads.

[0145] Figure 2 It shows in such as Figure 1 Fluorescence images collected by IVIS fluorescence imaging in the two-dose experiment described herein. Mice were imaged using IVIS at 24 h after delivery of the first dose (dose 1) of mpDNA and sonoactive microstructures with ultrasound, and then re-imaged at 24 h, 48 h, 72 h, and 1 week after delivery of the second dose with ultrasound (shown in the first to fifth rows of the images, respectively). Five groups of mice received the second dose of mpDNA and sonoactive microstructures, or (1) 6 h without ultrasound, (2) 6 h with ultrasound, (3) 24 h with ultrasound, (4) 48 h with ultrasound, or (5) 72 h with ultrasound (shown in the leftmost to rightmost columns of the images, respectively). The rightmost column (baseline) shows the control group including mice that did not receive the second dose. For mice that did not receive ultrasound, the fluorescence signal was weaker at each time point than that of mice in the group that received ultrasound (compare the leftmost column and other columns), indicating the efficacy of the sonoporogenic gene transfection process. In mice that did not receive US, very low fluorescence radiance was measured 1 week after dose 2 (bottom left panel). In all other mouse groups, fluorescence radiance persisted 1 week after dose 2 (bottom row), indicating that the two-dose sonopore formation protocol can induce persistent expression of nucleic acid payloads.

[0146] IVIS fluorescence radiometric imaging of mice in the baseline group, 6h ultrasound group, 6h no ultrasound group, 24h group, 48h group, and 72h group provides the maximum mean fluorescence radiometric values ​​of mice at 24h, 24h, 48h, 72h, and 1 week after dose 1. The mean fluorescence radiometric values ​​of mice in each group are considered. Figure 3A The bar chart shows the mean values ​​of the baseline group, the 6h US group, the 6h US-free group, the 24h group, the 48h group, and the 72h group at 24h, 48h, 72h, and 1 week after dose 2. Figure 3A This is a bar graph representing the mean fluorescence irradiance measurements of mice in each of the baseline, 6h with US, 6h without US, 24h, 48h, and 72h groups, collected via IVIS fluorescence imaging (n=4 mice per group). These data demonstrate the ability of the dual-dose sonopore formation protocol to induce persistent expression of nucleic acid payloads. The values ​​in this graph correspond to... Figure 2 The fluorescence image shown is illustrated. The dots in the bar graph represent fluorescence values ​​measured from a single mouse, while the height of the bar represents the mean fluorescence value for that group at the time of measurement. Error bars represent the standard deviation. The mean values ​​at 24 hours after dose 1 are also shown for the 24h, 48h, and 72h groups. Individual measurements for each mouse are represented by dots. These values ​​correspond to… Figure 2The fluorescence images shown are illustrated. The fluorescence values ​​observed in the baseline group (delivery of only one dose) and the 6-h no-sonication group (delivery of two doses, without sonication) were lower than those in the other groups where two doses were delivered with sonication. In the group receiving both doses and sonication, high mean fluorescence radiance values ​​were measured one week after the second dose delivery, indicating the ability of the two-dose sonication pore-forming protocol to induce persistent expression of nucleic acid payloads.

[0147] Figure 3B The results of fluorescence radiance measurements show that subjects who underwent repeated sono-induced pore formation therapy exhibited nucleic acid payload expression that was more than an order of magnitude higher than those who underwent only a single therapy. Figure 3B Line graphs show the mean fluorescence emissivity measurements of mice in the baseline group, 6h ultrasound group, 6h no ultrasound group, 24h group, 48h group, and 72h group. These values ​​correspond to... Figure 2 The fluorescence image shown and Figure 3A The bar chart shown. According to... Figure 3A The data shown indicate that the fluorescence values ​​observed in the baseline group (delivered only 1 dose, indicated by arrows labeled "Single Dose / Baseline Only") and the 6-h no-sonication group (delivered 2 doses, no sonication, with the lowest measured data, indicated by arrows labeled "6-h no sonication") were lower than those in other groups that delivered two doses with sonication. In the groups receiving both doses and sonication, high mean fluorescence radiance values ​​were measured one week after the second dose delivery, indicating the ability of the two-dose sonication protocol to induce persistent expression of nucleic acid payloads. These data indicate the ability of the two-dose sonication protocol to induce persistent expression of nucleic acid payloads. The values ​​in this figure correspond to... Figure 2 The fluorescence image shown and Figure 3A The bar graph shown. The markers represent the mean fluorescence values ​​of the groups at the time of measurement. The error bars represent the standard deviation. The line indicating the baseline group data is indicated by an arrow labeled "Single Dose / Baseline Only," and the line indicating the 6-hour ultrasound-free group data is indicated by an arrow labeled "6-hour Ultrasound-Free."

[0148] Figure 4The percentage change in mean fluorescence radiance measured 24 h after the first dose is shown in three mouse groups that received the second dose. These groups were: the group receiving the second dose 24 h after the first dose (circle marker), the group receiving the second dose 48 h after the first dose (square marker), and the group receiving the second dose 72 h after the first dose (triangle marker). These data suggest that a two-dose sonopore treatment protocol administering the second dose more than 24 h after the first dose can induce higher levels of nucleic acid payload expression. Error bars represent standard deviation. Surprisingly, the group receiving the second dose 48 h after the first dose (square marker) was observed to exhibit the highest levels of gene expression, which persisted for days and weeks after sonopore treatment, at levels half an order of magnitude higher (e.g., approximately 5-fold) than the group receiving the second dose 72 h after the first dose (diamond marker).

[0149] Example 2: Acoustic pore formation in a mouse liver model using a triple-repetition acoustic pore formation scheme compared to single-repetition and double-repetition schemes. Laboratory animals and protocols There were four experimental groups, each consisting of four BALB / c mice. Prior to the experiment, a jugular vein catheter (JVC) was implanted into each mouse, through which the acoustically active microstructure and nucleic acid construct were administered.

[0150] To prepare a dose of sonoactive microstructure and DNA solution, first follow the label instructions to prepare the sonoactive microstructure: remove from 4C storage and roll between your fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 uL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space (approximately 50 microliters (uL)) is included in the calculation). Using the same needle and syringe, draw 75 uL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0151] Each dose comprises three 100 μl doses of mpDNA and a sonoactive microstructure mixture in PBS, each dose delivered via a jugular vein catheter. The microplasmid DNA construct contains nucleic acid encoding a luciferase gene coupled to the ApoE promoter. Sonoactive microstructures Optison™ microbubbles or Sonazoid™ microbubbles were delivered to mice at a 1:4 volume ratio (mpDNA solution: sonoactive microstructure mixture, including needle dead space dispersed in the mixture).

[0152] Following the application of microbubbles and nucleic acid payloads, in these experiments, ultrasound energy was delivered to the liver region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the liver at a low mechanical index (MI) of 0.07 using B-mode ultrasound imaging. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 0.8, without interruption of ultrasound energy application at any point during the treatment process. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the application of the nine pulses was repeated three times. The duration of the high MI pulse was approximately 0.82 microseconds. The ultrasound application lasted less than 30 seconds.

[0153] Figure 5 Illustrative experimental protocols are provided in which multiple doses of DNA constructs and acoustically active microstructures are delivered to mice along with ultrasound (multi-dose acoustic pore formation assay). In these protocols, mice receive one to three doses of a mixture of microplasmid DNA (mpDNA) constructs and acoustically active microstructures. Ultrasound focused on a target region or target cell is delivered to the mouse along with the mixture of microplasmid DNA constructs and acoustically active microstructures. The microplasmid DNA constructs, such as nanoplasmid DNA constructs, may contain genetic payloads, such as therapeutic transgenes coupled to promoter sequences or other regulatory element sequences.

[0154] Figure 5Four multi-dose sonopore-forming protocols are described. Large dots along the horizontal line represent approximate delivery times for the mpDNA and sonoactive microstructure doses. The top arrow (“G1”) depicts a group in which a single 250 μg dose of a 1:4 volume ratio mpDNA and Optison microbubble mixture is delivered to mice. The second arrow from the top (“G2”) depicts a group in which two 250 μg doses of a 1:4 volume ratio mpDNA and Optison microbubble mixture are delivered to mice, with a 48-hour interval between each dose delivery. The third arrow from the top (“G3”) depicts a group in which three 250 μg doses of a 1:4 volume ratio mpDNA and Optison microbubble mixture are delivered to mice, with a 48-hour interval between each dose delivery. The fourth arrow from the top (“G4”) depicts a group in which three 250 μg doses of a 1:2 volume ratio mpDNA and Sonazoid microbubble mixture are delivered to mice, with a 48-hour interval between each dose delivery. Approximate times of dose delivery for all groups are indicated by black vertical arrows. Expression of the genetic payload (e.g., luciferase) delivered by mpDNA was measured by IVIS fluorescence imaging in mice at 24 h, 48 h, 72 h, and 1 week after final dose delivery. Measurement times are indicated by black vertical lines. IVIS fluorescence imaging was performed on all mice in each group at each time point.

[0155] In this experiment, mice received at least one dose of a mixture of microplasmid DNA and sonoactive microstructures. Mice in group 1 (G1) received a single dose of a 1:4 volume ratio mixture of nanoplasmids and Optison microbubbles. Mice in group 2 (G2) received two doses of a 1:4 volume ratio mixture of nanoplasmids and Optison microbubbles, 48 ​​hours apart. Mice in group 3 (G3) received three doses of a 1:4 volume ratio mixture of nanoplasmids and Optison microbubbles, 48 ​​hours apart. Mice in group 4 (“G4”) received three 250 μg doses of a 1:2 volume ratio mixture of nanoplasmids and Sonazoid microbubbles, delivered to the mice, 48 hours apart. Ultrasound (US) was applied percutaneously to the liver with each dose. These experimental protocols are described below. Figure 5 As shown.

[0156] One dose of the nanoparticle and acoustically active microstructure mixture contained 250 μg of the nanoparticle construct. Therefore, during the experiment, a total of 250 μg of DNA was delivered to G1, 500 μg of DNA to G2, 750 μg of DNA to G3, and 750 μg of DNA to G4. The nanoparticle construct contained nucleic acid encoding a luciferase gene coupled to the ApoE promoter. IVIS fluorescence radiometric imaging was performed on all groups at 24 h, 48 h, 72 h, and 1 week after the last dose. IVIS fluorescence imaging was performed on all mice in each group at each time point. Figure 5 Four multi-dose sono-forming pore-forming schemes used in this experiment are shown. Large dots along the horizontal line represent approximate delivery times for the mpDNA and sono-active microstructure doses. Figure 5 (Vertical solid arrow).

[0157] Serum ALT, IL-6, and AST levels were measured 24 hours after the first sonopore treatment to assess systemic inflammation, cellular inflammation, and hepatic cellular damage induced by sonopore treatment. Figure 8 As shown, no changes in liver enzymes were observed 24 hours after delivery (compare the rightmost 0 μg bar with the other bars), indicating a good response to sonoporosis treatment, which did not result in any significant cellular damage or other cellular inflammatory response to the liver.

[0158] result Figure 6 It shows in such as Figure 5 Fluorescence images collected by IVIS fluorescence imaging during the multi-dose experiment described herein. Mice were imaged using IVIS at 24 h, 48 h, 72 h, and 1 week after delivery of the final dose (shown in columns 1 through 7 of the images, respectively). The experimental protocol for the mice in the top row (“Optison 3 doses”) corresponds to… Figure 5 The third protocol shown is described above. In this protocol, mice were administered three doses of the mpDNA and Optison microbubble mixture, with each administration 48 hours apart. The experimental protocol for the mice in the second row from the top (“Sonazoid 3 doses”) corresponds to... Figure 5 The fourth protocol shown is described. In this protocol, mice were administered three doses of the mpDNA and Sonazoid microbubble mixture, with each administration 48 hours apart. The protocol used for the mice in the third row from the top (“Optison 2 doses”) corresponds to... Figure 5The second protocol shown is described. In this protocol, mice are administered two doses of the mpDNA and Optison microbubble mixture, 48 hours apart. The experimental protocol for bottom-stranded mice (“Optison 1 dose”) corresponds to... Figure 5 The first protocol shown. In this protocol, a dose of mpDNA and Optison microbubble mixture was administered to mice.

[0159] Mice were imaged using IVIS fluorescence imaging at 24 h, 48 h, 72 h, and 1 week after final dose delivery. Figure 6 (Columns 1 through 3 are shown). IVIS fluorescence irradiance measurements for all groups indicated luciferase genetic payload expression measured in the initial 24 hours to 1 week after final dose delivery. However, based on fluorescence measured by IVIS one week after final dose delivery, mice in groups receiving one or two doses (bottom two rows) showed stronger luciferase expression for a longer duration. These data suggest that a multi-dose sonopore-forming protocol can be used to achieve long-duration expression of the genetic payload.

[0160] Figure 7A This is a bar graph representing the average fluorescence emissivity measurements of mpDNA and acoustically active microstructures in mice that received multiple doses, collected via IVIS fluorescence imaging. These values ​​correspond to... Figure 6 The fluorescence images shown are illustrated below. The dots in the bar graph represent fluorescence values ​​measured from a single mouse, while the height of the bar represents the mean fluorescence value for that group at the time of measurement. Groups include mice that received one dose (top left), two doses (top right), or three doses (bottom). The error bars represent the standard deviation. The initial mean fluorescence radiance values ​​observed in the three-dose group (bottom) were higher than those in the one-dose group (top left) or two-dose group (top right). The duration of the fluorescence signal was longer in the one-dose or two-dose group than in the three-dose group.

[0161] Figure 7B This is a grouped bar graph representing the mean fluorescence emissivity measurements of mpDNA and acoustically active microstructures in mice collected via IVIS fluorescence imaging after receiving multiple doses. These values ​​correspond to... Figure 6 The fluorescence image shown and Figure 7AThe bar graph is shown below. The dots in the bar graph represent fluorescence values ​​measured from a single mouse, while the height of the bar represents the mean fluorescence value for that group at the time of measurement. Each group included mice receiving one dose (leftmost bar, lightest gray), two doses (second leftmost bar, second light gray), three doses of Optison microbubbles (third leftmost bar, third light gray), or three doses of Sonazoid microbubbles (rightmost bar, dark gray). The error bars represent standard deviation. The initial mean fluorescence radiance values ​​of the groups receiving three doses (the two rightmost bars in each group) were higher than those of the groups receiving one dose (leftmost bar in each group) or two doses (second bar from the left in each group). This increase in fluorescence signal appears to be proportional to the dose. In the groups receiving one or two doses, the duration of the fluorescence signal was longer than in the groups receiving three doses. The duration of the fluorescence signal is also proportional to the number of doses. In early measurements, Sonazoid microbubbles showed a significantly stronger signal than Optison microbubbles. At 24h, 48h, and 72h time points, the initial fluorescence values ​​of the groups receiving one or two doses (triangular markers) were lower than those of the groups receiving three doses (circular and square markers). However, the duration of protein expression (as indicated by the presence of fluorescence signal) was longer in the groups receiving two or three doses than in the group receiving one dose. These data demonstrate a positive correlation between the number of doses delivered and the initial amount of protein expression. Figure 7C Explanation Figure 6 The mean fluorescence emissivity measurements for each group of n=4 subjects are shown, where repeated sonoporosis gene therapy treatment in the subjects resulted in an order of magnitude increase in the measured fluorescence emissivity.

[0162] Example 3: Acoustic pore formation in an NHP model using a triple-repeated acoustic pore formation scheme Laboratory animals and protocols Three experimental animals, each a male cynomolgus monkey, were used. Two of the cynomolgus monkeys were administered a mixture of nanoparticle genetic payloads and acoustically active microstructures, along with ultrasound (US) energy. The third cynomolgus monkey was not used in the experiment and did not receive any intravenous injection of microbubbles or plasmids, nor was it subjected to external ultrasound at any time. The nucleic acid payloads used are summarized below.

[0163]

[0164] Before beginning the experimental procedure, the IV catheter was inserted into the saphenous vein. A dose of sonoactive microstructure and DNA solution was prepared by first preparing the sonoactive microstructure according to the label instructions: remove from 4C storage and roll between the fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; pass a 25G needle through the rubber gasket to provide a pressure outlet; and draw 12 mL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space is included in the calculation). Using the same needle and syringe, 8 mL of DNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenized. The DNA + Optison solution was withdrawn from the needle dead space. The 18G needle was then replaced with a 25G blunt needle for injection into the IV catheter.

[0165] After administering a mixture of 0.5 mL microbubbles and nucleic acid payload, ultrasound energy is delivered to the subject's heart, kidneys (e.g., unilateral or bilateral), liver, muscle, or a combination thereof using an M5Sc probe positioned perpendicular to the subject's location. The depth of focus is set to 3.75–4 cm, and the zoom setting is 0. Ultrasound is delivered continuously, alternating between a low mechanical index (MI) of 0.09 and a high MI of 2.3, without interruption of ultrasound energy delivery at any point during the treatment. Approximately 20 seconds after administering a bolus injection of the acoustically active microstructure and DNA solution, nine high MI ultrasound flashes of 2.3 are delivered, spaced approximately 5 seconds apart, and this process is repeated three times for nine pulses. The high MI pulse duration is approximately 2.25 microseconds. Subsequently, a bolus injection of the acoustically active microstructure and DNA solution is administered approximately every 30 seconds, during which the ultrasound probe is moved to a new location on the subject's liver, where ultrasound is delivered at a low mechanical index (MI) of 0.09 to induce microbubble oscillation, and at a high MI of 2.3 to induce microbubble rupture. Approximately 0.5–1.0 mL of acoustically active microstructure and DNA solution was continuously injected into each subject every 30 seconds until 20 mL of DNA and microbubble solution was completely applied to the target organ, with a total treatment time of approximately 20 minutes. The same treatment procedure was repeated at 48-hour intervals, for a total of three treatment cycles per target organ.

[0166] The same procedure was repeated for each target organ (liver, kidneys x2, heart, skeletal muscle (quadriceps)). For the liver, approximately 9 high-MI flashes were performed at each lobe, including the right, left, quadrate, and caudate lobes. For the kidneys, approximately 9 high-MI flashes were applied in both cross-sectional and longitudinal views, alternating between views until the infusion was complete. For the skeletal muscle, after each bolus of the sonoactive microstructure and DNA solution, the ultrasound probe was moved approximately 1 inch to the next position.

[0167] result To image target organs and tissues of interest. Figure 10 Quantitative results of fluorescence in a multi-dose experiment using non-human primate subjects are shown. Kidneys of NHP01 and NHP02 were observed as green fluorescence color-coded yellow-orange to indicate the intensity of the fluorescence signal across the entire surface of the organ, including transverse and longitudinal cross-sections. Green fluorescence color-coded yellow-orange was also observed to indicate the intensity of the fluorescence signal across the peripheral boundary of the liver, across the leftmost portion of the heart, and along the periphery of the muscles.

[0168] Figure 10 Quantitative results showed approximately 5*10^6 p / s / cm in the liver. 2 / sr, approximately 1*10^8 p / s / cm in the kidneys 2 / sr, approximately 1*10^8 p / s / cm in the heart 2 Approximately 1*10^7 p / s / cm in sr and skeletal muscle 2 Average emissivity of / sr.

[0169] These data demonstrate that ultrasound-mediated gene delivery can induce the expression of genetic payloads using a variety of administration regimens in the kidneys, liver, heart, and muscles of non-human primates.

[0170] Example 4: Acousto-induced pore formation in a mouse liver model using a triple-repetition acoustic pore formation scheme compared to single-repetition and double-repetition schemes. Laboratory animals and protocols There were two experimental groups, each consisting of four RAG2 knockout mice. Prior to the experiment, a jugular vein catheter (JVC) was implanted into each mouse, through which the acoustically active microstructure and nucleic acid construct were administered.

[0171] In this experiment, mice received a total of four sonopore treatments, each consisting of three bolus injections of microplasmid DNA (mpDNA) and acoustically active microstructures. The first sonopore treatment was 16 weeks apart from subsequent treatments, with a total of three subsequent treatments starting 16 weeks after the initial treatment. The third and fourth treatments were 48 hours apart. The mpDNA construct used in this experiment was the Nanoplasmid™ construct. Ultrasonic (US) energy was delivered percutaneously with each dose to transfect the target organ, the liver. Within each treatment, there was a 48-hour interval between sonopore treatments containing the stated dose of the mpDNA construct and the acoustically active microstructure mixture. These experimental protocols are described below. Figure 11 As shown. Group 1 received three 228 μg doses of DNA payload, delivering a total of 684 μg of DNA per treatment cycle, and Group 3 received three 100 μg doses of DNA payload, delivering a total of 300 μg of DNA per treatment cycle. The volume ratio of DNA payload solution to acoustically active microstructure solution in each group was 1:4.

[0172] Prepare a dose of sonoactive microstructure and DNA solution by first preparing the sonoactive microstructure according to the label instructions: Remove the microstructure from the 4C storage and roll it between your palms for 20 seconds; remove the protective plastic and aluminum capping from the Optison vial; insert a 25G needle through the rubber gasket of the Optison vial to provide a pressure outlet; and draw 12 mL of Optison into the syringe using a 1.5-inch 18G needle (the dead space of the needle is included in the calculation). Using the same needle and syringe, draw 228 μg or 100 μg of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the IV catheter.

[0173] Each procedure consists of three 228 μg or three 100 μg doses of mpDNA and acoustically active microstructure mixture in PBS, each dose delivered via a jugular catheter. The microplasmid DNA construct contains nucleic acid encoding a luciferase gene coupled to the ApoE promoter. Acoustically active microstructure Optison™ microbubbles are delivered to mice at a 1:4 volume ratio (mpDNA solution: acoustically active microstructure mixture, including needle dead space dispersed in the mixture).

[0174] Following the application of microbubbles and nucleic acid payloads, in these experiments, ultrasound energy was delivered to the liver region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the liver at a low mechanical index (MI) of 0.07 using B-mode ultrasound imaging. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 0.8. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the application of the nine pulses was repeated three times. The high MI pulse duration was approximately 0.82 microseconds. The ultrasound application time was less than 120 seconds. Subjects were re-administered 16 weeks after the initial treatment during a subsequent sonopore treatment, and then again 48 hours and 96 hours after the subsequent sonopore treatment delivered at 16 weeks.

[0175] Following the first treatment, IVIS fluorescence radiometry imaging was performed on all groups at 24, 48, and 72 hours, and then repeated weekly for 16 weeks. Following subsequent treatments, IVIS fluorescence radiometry imaging was performed on all groups at 24 and 72 hours, and then at 1 and 2 weeks after the subsequent treatment. Figure 11 (Vertical arrow). IVIS fluorescence imaging was performed on all mice in each group at each time point. Figure 11 The large dots along the horizontal line represent the approximate delivery time of the mpDNA and acoustically active microstructure doses.

[0176] result Mice were imaged using IVIS at 24 h, 48 h, and 72 h after the first treatment cycle and subsequent treatment cycles, and then weekly. The mean fluorescence irradiance measured by IVIS indicated luciferase expression in the target organ, the liver. Durability of luciferase expression was observed up to 16 weeks after the first treatment cycle (Figure 12 A and Figure 12 B).

[0177] The mean fluorescence radiance measured after the second process in both groups of mice (mice that received 228 ug or 100 ug mpDNA in each process) was greater than the mean fluorescence radiance measured after the first process. Figure 12A and Figure 12B Following a 16-week follow-up treatment course, between 16 weeks after the first course and 2 weeks after the second course, the mean fluorescence radiance measured in the group receiving 228 μg DNA increased nearly 20-fold. Figure 12A and Figure 12B ). Figure 12A and Figure 12BThe error bars in the figure represent standard deviations. Further observations showed that mice treated with the 228 ug or 100 ug mpDNA groups as described herein exhibited stable luciferase expression for the duration of the experimental study following sonopore treatment, and showed a stable increase in expression at elevated levels for at least 2 weeks after subsequent sonopore treatment.

[0178] Example 5: Delivery of FVIII in a mouse liver model using a triple-repeated sono-porousization scheme In this experiment, four experimental groups of five RAG2 mice were evaluated: 1) a group receiving the acoustically active microstructure and 300 μg of a nucleic acid construct encoding FVIII coupled to the APOE-AAT promoter; 2) a group receiving the acoustically active microstructure and 750 μg of a nucleic acid construct encoding FVIII coupled to the APOE-AAT promoter; 3) a group receiving the acoustically active microstructure and 750 μg of a nucleic acid construct encoding FVIII coupled to the APOE-AAT promoter without ultrasound; and 4) a group receiving the acoustically active microstructure but without the nucleic acid payload and underwent ultrasound. Prior to the experiment, a jugular vein catheter (JVC) was implanted in each mouse through which the acoustically active microstructure and nucleic acid construct were administered.

[0179] In this experiment, mice underwent a total of three sonopore-forming processes, each consisting of three boluses of microplasmid DNA (mpDNA) encoding FVIII coupled to the APOE-AAT promoter, administered in each treatment session. The intervals between the first and second sonopore-forming processes were 48 hours, and between the second and third processes were 72 hours. The mpDNA construct used in this experiment was the Nanoplasmid™ construct. Ultrasonic (US) energy was delivered percutaneously with each dose to transfect the target organ, the liver. Group 1 received three 100 μg doses of DNA payload, delivering a total of 300 μg of DNA per treatment session, and Group 2 received three 250 μg doses of DNA payload, delivering a total of 750 μg of DNA per treatment session. The volume ratio of DNA payload solution to sonoactive microstructure solution was 1:4 for each group. The sonoactive microstructure used in this experiment was the phospholipid-stabilized microbubble Sonazoid®.

[0180] To prepare a dose of the sonoactive microstructure and DNA solution, first follow the label instructions: Remove the injectable powder from the manufacturer's packaging by twisting the top of the ampoule. Place the syringe directly into the ampoule without using the cannula. Add 2 mL of phosphate-buffered saline from the syringe to the vial and shake for one minute to ensure the product is homogeneous. Aspirate the product into the syringe and re-inject it back into the vial. Shake the vial immediately before injection to reconstitute the product and aspirate it into the syringe. Using the same needle and syringe, aspirate 250 μg or 100 μg of DNA payload into the syringe to combine the DNA and Sonozoid® sonoactive agent. Mix the Sonozoid® sonoactive agent and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogeneous. Withdraw the DNA + Sonozoid® sonoactive agent solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the IV catheter.

[0181] Each procedure consists of three 250 μg or three 100 μg doses of mpDNA and a sonoactive microstructure mixture in PBS, each dose delivered via a jugular catheter. The microplasmid DNA construct contains nucleic acids encoding the FVIII gene coupled to the ApoE-AAT promoter. The sonoactive microstructure Sonozoid® sonoactive agent is delivered to mice at a 1:4 volume ratio (mpDNA solution: sonoactive microstructure mixture, including needle dead space dispersed in the mixture).

[0182] Following each bolus injection of the DNA payload and acoustic active agent, in these experiments, ultrasound energy was delivered to the liver region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the liver at a low mechanical index (MI) of 0.07 using B-mode ultrasound imaging. The depth setting was set to 2 cm, and the zoom setting to 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 2.3. Eighteen high MI ultrasound flashes of 2.3 were delivered, with a 4-second interval between each flash, and this was repeated three times for a total of 18 pulses. The high MI pulse duration was approximately 0.82 microseconds. Ultrasound administration was less than 120 seconds per bolus injection. After each bolus injection of the DNA payload and acoustic active agent, the ultrasound probe was moved to different locations on the subject's liver. Subjects were re-administered the same treatment protocol at 48 and 72 hours after the initial treatment in subsequent acoustic perforation treatments.

[0183] result The level of transgenic FVIII in mouse plasma was measured using an MSD assay. In short, the capture antibody (GMA-8024) was loaded into 96-well plates and incubated overnight at 4°C. The plates were then washed three times with wash buffer and incubated with blocking buffer for 30 minutes at room temperature. Eight-point serial dilution standards were prepared using Xinta® in the range of 0.92 IU / ml to 0.01 IU / ml. Two-fold diluted samples and standards were added to the wells of the 96-well plates. The plates were incubated at room temperature for 2 hours and washed three times. Detection was performed within 2 hours after the three washes by incubating the samples with the GMA-8023 antibody. Signals were generated by Sulfo-TAG and detected by the MSD instrument.

[0184] Following the first and second treatment cycles, the mean FVIII levels in mice after the first and second cycles (mice receiving 300 μg or 750 μg mpDNA in each cycle, respectively) were shown in... Figure 34A In the second repeated sonopore-forming treatment regimen, FVIII increased (approximately three-fold) from approximately 0.01 IU / mL to approximately 0.03 IU / mL. The mean FVIII levels measured at 72 hours and 1 week after the third treatment cycle in mice that underwent the first and second cycles (mice receiving 300 μg or 750 μg mpDNA in each cycle, respectively) are shown in... Figure 34A In the mean, the expression was approximately 0.1 IU / ML, with an increase of approximately 10-fold after the first treatment and approximately 3-fold after the second treatment. Surprisingly and unexpectedly, the results presented in this paper demonstrate that the multi-organ localization and repeated treatment regimen described herein provides a significantly greater than additive effect in increasing FVIII delivery and expression to mouse liver, with each subsequent sonopore treatment resulting in an approximately 3-fold increase in gene delivery and expression.

[0185] Example 6: Sonoporation in a rat liver model using measured dose-response method Laboratory animals and protocols There were four experimental groups, each consisting of four BALB / c mice. Prior to the experiment, a jugular vein catheter (JVC) was implanted into each mouse, through which the acoustically active microstructure and nucleic acid construct were administered.

[0186] To prepare a dose of sonoactive microstructure and DNA solution, first follow the label instructions to prepare the sonoactive microstructure: remove from 4C storage and roll between your fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 uL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space (approximately 50 microliters (uL)) is included in the calculation). Using the same needle and syringe, draw 75 uL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0187] Each treatment session consisted of three boluses of mpDNA and acoustically active microstructure mixture in PBS, totaling 200 μL, delivered via a jugular vein catheter. The microplasmid DNA construct contained nucleic acid encoding a luciferase gene coupled to the ApoE promoter. Acoustically active microstructure Optison™ microbubbles were delivered to mice at a 1:4 volume ratio (mpDNA solution: acoustically active microstructure mixture, including needle dead space dispersed in the mixture). Mice in each group were administered doses containing 5 μg, 50 μg, 100 μg, or 250 μg of DNA (see [reference to relevant documentation]). Figure 13 (The 1x, 10x, 20x, and 50x DNA groups).

[0188] After applying microbubbles and nucleic acid payloads, in these experiments, ultrasound energy was delivered to the liver region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the liver using B-mode ultrasound imaging at a low mechanical index (MI) value of 0.07. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) value of 0.07 and a high MI value of 0.8. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the delivery of the nine pulses was repeated three times. The high MI pulse duration was approximately 0.82 microseconds. The ultrasound delivery time was less than 120 seconds.

[0189] Following administration of sonoporosis treatment and mpDNA, IVIS fluorescence radiometric imaging was first performed on all groups 24 hours after dose delivery. Subsequent IVIS fluorescence imaging was performed on each group at 48 hours, 1 week, and 2 weeks after dose delivery. Figure 13 IVIS fluorescence imaging was performed on all mice in each group at each time point.

[0190] result Mice were imaged using IVIS fluorescence imaging at 24 h, 48 h, 1 week, and 2 weeks after administration of the dose. Figure 13 IVIS fluorescence irradiance measurements across all groups indicated that the luciferase genetic payload was expressed 24 hours post-delivery, with the strongest fluorescence observed in the group receiving the highest dose of DNA and the lowest fluorescence observed in the group receiving the lowest dose of DNA. These data suggest an early positive correlation between the expression level of the genetic payload and the dose of mpDNA delivered.

[0191] Figure 13 The values ​​represent the mean fluorescence emissivity measurements of mice administered different doses of mpDNA, collected via IVIS fluorescence imaging. Error bars indicate standard deviation. The initial mean fluorescence emissivity values ​​observed in the groups receiving higher doses of mpDNA (50x DNA and 20x DNA) were higher than those in the groups receiving lower doses (1x DNA and 10x DNA). The higher initial mean fluorescence emissivity measured in the groups receiving more DNA persisted for at least 2 weeks after dose administration. The duration and stability of the fluorescence signal were greater in the groups receiving more mpDNA than in the groups receiving less mpDNA. These data suggest that the expression of the genetic payload can be titrated by adjusting the dose of delivered genetic material.

[0192] Example 7: Sono-induced pore formation in a rat kidney model for measuring dose-response Laboratory animals and protocols There were three experimental groups, each consisting of four BALB / c mice. Prior to the experiment, a jugular vein catheter (JVC) was implanted into each mouse, through which the acoustically active microstructure and nucleic acid constructs were administered. Mice in each group were administered doses containing 25 μg, 50 μg, and 100 μg mpDNA (see [link to relevant documentation]). Figure 14 (The 1x, 2x, and 4x DNA groups).

[0193] To prepare a dose of sonoactive microstructure and DNA solution, first follow the label instructions to prepare the sonoactive microstructure: remove from 4C storage and roll between your fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 uL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space (approximately 50 microliters (uL)) is included in the calculation). Using the same needle and syringe, draw 75 uL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0194] Each treatment session consisted of three boluses of mpDNA and a sonoactive microstructure mixture in PBS, delivered via a jugular vein catheter. The microplasmid DNA construct contained nucleic acid encoding a luciferase gene coupled to the CAG promoter. Sonoactive microstructures (Optison™ microbubbles) were delivered to mice at a 1:4 volume ratio (mpDNA solution: sonoactive microstructure mixture, including needle dead space dispersed in the mixture). Mice in each group were administered doses containing 25 μg, 50 μg, and 100 μg of mpDNA (see [reference to previous treatments]). Figure 14 (The 1x, 2x, and 4x DNA groups).

[0195] After applying microbubbles and nucleic acid payloads, in these experiments, ultrasound energy was delivered to the kidney region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the kidney using B-mode ultrasound imaging at a low mechanical index (MI) value of 0.07. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) value of 0.07 and a high MI value of 0.8. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the delivery of the nine pulses was repeated three times. The high MI pulse duration was approximately 0.82 microseconds. The ultrasound delivery time was less than 120 seconds.

[0196] Following administration of sonoporosis treatment and mpDNA, IVIS fluorescence radiometric imaging was first performed on all groups 24 hours after dose delivery. Subsequent IVIS fluorescence imaging was then performed on each group at 48 hours, 72 hours, 9 days, and 14 days after dose delivery. Figure 14 IVIS fluorescence imaging was performed on all mice in each group at each time point.

[0197] result Mice were imaged using IVIS fluorescence imaging at 24 h, 48 h, 72 h, 9 days, and 14 days after administration of the dose. Figure 14 IVIS fluorescence radiance measurements in all groups indicated that the luciferase genetic payload was expressed 24 hours post-delivery, and the measured fluorescence was similar across all groups.

[0198] Figure 14 The values ​​represent mean fluorescence emissivity measurements of mice administered different doses of mpDNA, collected via IVIS fluorescence imaging. Error bars indicate standard deviation. Although the expression levels of the genetic payload were initially similar across all groups, starting at 48 hours post-dose delivery, the mean fluorescence emissivity values ​​observed in the groups receiving higher doses of mpDNA (2x DNA and 4x DNA groups) were higher than those in the group receiving lower doses of mpDNA (1x DNA group). The higher mean fluorescence emissivity observed in the groups receiving more DNA persisted at least 2 weeks post-dose delivery. The duration and stability of the fluorescence signal were greater in the groups receiving more mpDNA than in the groups receiving less mpDNA. The fluorescence signal was stronger at 2 weeks post-dose delivery in the 2x and 4x DNA groups than at 24 hours post-dose delivery.

[0199] Example 8: Acoustic perforation in a rat kidney model using a multi-dose and multiple ultrasound localization scheme Laboratory animals and protocols There were three experimental groups, each consisting of four BALB / c mice. Prior to the experiment, a jugular vein catheter (JVC) was implanted into each mouse, through which acoustically active microstructures and nucleic acid constructs were administered. The groups consisted of mice that received one, two, or three treatment cycles of the acoustically active microstructure and DNA solution. Figure 15 ).

[0200] To prepare a dose of sonoactive microstructure and DNA solution, first follow the label instructions: remove from 4C storage and roll between your fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 uL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space (approximately 50 microliters (uL) is included in the calculation)). Using the same needle and syringe, draw 75 uL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0201] Each treatment session consisted of one dose of mpDNA and acoustically active microstructure mixture in PBS, totaling 150 μL, delivered via a jugular vein catheter. The microplasmid DNA construct contained nucleic acid encoding a luciferase gene coupled to the CAG promoter. Acoustically active microstructure Optison™ microbubbles were delivered to mice at a 1:4 volume ratio (mpDNA solution: acoustically active microstructure mixture, including needle dead space dispersed in the mixture). Each dose, containing 100 μg of DNA, was administered to mice in each group.

[0202] These groups consisted of mice that received one, two, or three treatment cycles of acoustically active microstructures and DNA solutions. Figure 15 When multiple treatment cycles were administered, a 6-hour interval was maintained between cycles. For each treatment cycle, one dose of the acoustically active microstructure and nucleic acid construct was administered. Following the administration of the microbubbles and nucleic acid payload, in these experiments, ultrasound energy was delivered to the kidney region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the kidney at a low mechanical index (MI) of 0.07 using B-mode ultrasound imaging. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 0.8, without interruption of ultrasound energy application at any point during the treatment cycle. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the administration of the nine pulses was repeated three times. The duration of the high MI pulse was approximately 0.82 microseconds. The ultrasound administration lasted less than 30 seconds.

[0203] Following administration of sonoporosis treatment and mpDNA, IVIS fluorescence radiometric imaging was performed on all groups 24 hours after delivery of the last dose. Subsequent IVIS fluorescence imaging was performed on each group 48 hours and 1 week after delivery of the last dose. Figure 15 IVIS fluorescence imaging was performed on all mice in each group at each time point.

[0204] result Mice were imaged using IVIS fluorescence imaging at 24 h, 48 h, and 1 week after the last dose delivery. Figure 15 IVIS fluorescence emissivity measurements across all groups indicated that the luciferase genetic payload was expressed 24 hours post-delivery, with the strongest fluorescence in the group receiving the highest dose of DNA (3 dose groups) and the lowest fluorescence in the group receiving the lowest dose of DNA (1 dose group). These data suggest a positive correlation between the expression level of the genetic payload and the number of mpDNA doses delivered.

[0205] Figure 15 The values ​​represent mean fluorescence emissivity measurements collected by IVIS fluorescence imaging in mice treated with different doses of mpDNA and sonopore therapy. Error bars indicate standard deviation. The initial mean fluorescence emissivity values ​​observed in the groups receiving higher doses of mpDNA (3-dose groups and 2-dose groups) were higher than those in the group receiving lower doses of mpDNA (1-dose group). This higher initial mean fluorescence emissivity, measured in the groups receiving even higher doses of mpDNA, persisted for at least 2 weeks after dose administration. These data suggest that the expression of the genetic payload can be titrated by adjusting the dose of delivered genetic material.

[0206] Example 9: Acousto-induced perforation in a mouse kidney model using a multi-dose and ultrasound-guided treatment localization protocol. Laboratory animals and protocols Three groups of BALB / c mice were administered acoustically active microstructures and nanoparticles encoding luciferase via intravenous delivery through a peripheral venous catheter. During treatment, mice were administered one, two, or three doses (“pumps”) of the DNA payload (nanoparticles) and acoustically active microstructures (“microbubbles”). Groups receiving multiple doses of the DNA payload received ultrasound application to subsequent sites on the kidneys after subsequent doses. The microbubbles were administered at concentrations of 5 × 10⁸ and approximately 8 × 10⁸ microbubbles / mL. A solution of a total of 250 μg of nanoparticles, mixed with a solution of acoustically active microstructures dispersed in one, two, or three doses, was administered to each group over a 15-second time interval. Group 1 received a single bolus injection of 250 μg of nanoparticles and microbubble solution, along with a total injection volume of approximately 200 μL. Group 2 received two bolus injections of 250 μg of nanoparticles and microbubble solution, along with two intravenous injections of approximately 100 μL each. Group 3 received 250 μg of nanoparticles and microbubble solution via two bolus injections, followed by three intravenous injections of approximately 100 μL each. After each administration, continuous ultrasound energy was percutaneously applied to the liver: one bolus group at one location, two bolus groups at two locations, and three bolus groups at three locations. Each ultrasound administration consisted of nine ultrasound pulses spaced 4 seconds apart. Luciferase expression was measured by IVIS at 1, 2, 5, 7, or 14 days post-administration. Figure 16 An illustrative scheme as described in this embodiment is provided.

[0207] result Using IVIS, mice were imaged 1, 2, 5, 7, or 14 days after the administration of nanoparticles, microbubbles, and ultrasonic energy. Figure 17 The original IVIS images of mice that received one bolus and ultrasound energy at one location (left column), two boluses and ultrasound energy at two locations (middle column), and three boluses and ultrasound energy at three locations (right column) are shown.

[0208] like Figure 17 and Figure 18 As shown, luciferase expression was observed in all three experimental groups 24 hours after administration and persisted for at least 14 days. For the first week after administration, expression levels appeared to correlate with the number of bolus injections of the same dose of DNA payload, with the highest mean expression levels observed in the groups receiving three bolus injections and sonication energy at three locations, and the lowest expression levels observed in mice receiving one dose and sonication energy at one location. Figure 18 Expression was still detected in all experimental groups 14 days after administration.

[0209] Example 12: Acoustic perforation in an NHP kidney model using a multiple injection and multiple ultrasound localization scheme Animals and programs Three experimental animals, each a male cynomolgus monkey, were used. Two of the cynomolgus monkeys were administered a mixture of nanoparticle genetic payloads and acoustically active microstructures, along with ultrasound (US) energy. The third cynomolgus monkey was not used in the experiment and did not receive any intravenous injection of microbubbles or plasmids, nor was it subjected to external ultrasound at any time. The nucleic acid payloads used are summarized below.

[0210]

[0211] Before beginning the experimental procedure, the IV catheter was inserted into the saphenous vein. A dose of sonoactive microstructure and DNA solution was prepared by first preparing the sonoactive microstructure according to the label instructions: remove from 4C storage and roll between the fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; pass a 25G needle through the rubber gasket to provide a pressure outlet; and draw 12 mL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space is included in the calculation). Using the same needle and syringe, 8 mL of DNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenized. The DNA + Optison solution was withdrawn from the needle dead space. The 18G needle was then replaced with a 25G blunt needle for injection into the IV catheter.

[0212] After administering a mixture of 0.5 mL microbubbles and nucleic acid payload, ultrasound energy was delivered to the kidney region of the subject (e.g., unilateral or bilateral) using an M5Sc probe perpendicular to the subject's positioning. Depth of focus was set to 3.75–4 cm, and zoom was set to 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) value of 0.09 and a high MI value of 2.3, without interruption of ultrasound energy delivery at any point during the treatment. Approximately 20 seconds after administering the bolus injection of the acoustically active microstructure and DNA solution, nine high MI ultrasound flashes of 2.3 were delivered, with approximately 5 seconds between each high MI flash, and this was repeated three times for nine pulses. The high MI pulse duration was approximately 2.25 microseconds. Subsequently, approximately every 30 seconds, the bolus injection of the acoustically active microstructure and DNA solution was administered, with approximately nine high MI flashes delivered in both cross-sectional and longitudinal views, alternating between views, until the infusion was completed. The same treatment procedure was repeated at 48-hour intervals, for a total of three treatment cycles per kidney.

[0213] At the end of the experiment, NHP kidney sections were prepared. Two sections of each experimental animal's kidney and one section of a non-experimental animal's kidney were analyzed using RNAscope to evaluate transfection of kidney cells using ultrasound-mediated gene delivery (UMGD).

[0214] Kidney samples were analyzed using the RNAscope™ assay (RNAscope™ 2.5 LSx Red kit; catalog number 322750). Optimal pretreatment conditions for maximizing the signal-to-noise ratio were established based on evaluation of tissue samples using reference positive and negative control probes. The following standard RNAscope™ 2.5 LSx Red assay pretreatment conditions were used: (i) Epitope retrieval 2: 95°C for 15 minutes. Protease III: 40°C for 15 minutes.

[0215] The control probe used is: (i) ACD positive control probe ( Crab-eating macaques The probes used include: peptidyl prolyl isomerase B (cyclophilin B); Mfa-PPIB (catalog number 424148); and ACD negative control probe (Bacillus subtilis dihydropyridine dicarboxylic acid reductase; dapB; catalog number 312038). The target probe is (enhanced green fluorescent protein (EGFP), mRNA; EGFP-04; catalog number 538858; GenBack accession number MN623123.1). These probes are summarized below.

[0216]

[0217] Visual scoring was performed, assigning a single score to each sample based on the dominant staining pattern throughout the sample. Staining intensity had no effect on the score. Dots in the histological image were correlated with the number of individual RNA molecules. The percentage of positive cells was visually scored and categorized based on the number of cells with more than one dot per cell (i.e., 0%, 1–25%, 26–25%, 51–75%, 76–99%, 100%).

[0218] result Five kidney samples were evaluated using RNAscope™ 2.5 LSx Red. All samples passed quality control checks, had moderate PPIB positive control staining, and little or no dapB background staining. RNAscope™ 2.5 LSx Red assays were performed in all samples to assess EGFP expression in NHP kidney samples. EGFP was detected in all four kidney samples from experimental animals, but not in kidney samples from non-experimental animals. Figure 23A Examples of histological images of samples are provided (arrows indicate EGFP-positive cells). Figure 23BThe percentage of EGFP-positive glomeruli in NHP kidney sections is provided. Minimal EGFP expression was detected in samples from untreated NHP (negative control), while EGFP expression was detected in over 70% of glomeruli in one experimental sample. These results demonstrate that UMGD efficiently delivers genetic payloads to NHP kidneys, and that repetitive sonopore formation using a multi-organ localization protocol efficiently delivers and drives genetic payload expression in NHP kidneys.

[0219] Example 13: Sonoscopic perforation in an NHP kidney model using a multiple injection and multiple ultrasound localization protocol, demonstrating delivery to glomerular and non-glomerular regions. Animals and programs Three experimental animals, each a male cynomolgus monkey, were used. Two of the cynomolgus monkeys were administered a mixture of nanoparticle genetic payloads and acoustically active microstructures, along with ultrasound (US) energy. The third cynomolgus monkey was not used in the experiment and did not receive any intravenous injection of microbubbles or plasmids, nor was it subjected to external ultrasound at any time. The nucleic acid payloads used are summarized below.

[0220]

[0221] Before beginning the experimental procedure, the IV catheter was inserted into the saphenous vein. A dose of sonoactive microstructure and DNA solution was prepared by first preparing the sonoactive microstructure according to the label instructions: remove from 4C storage and roll between the fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; pass a 25G needle through the rubber gasket to provide a pressure outlet; and draw 12 mL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space is included in the calculation). Using the same needle and syringe, 8 mL of DNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenized. The DNA + Optison solution was withdrawn from the needle dead space. The 18G needle was then replaced with a 25G blunt needle for injection into the IV catheter.

[0222] After administering a mixture of 0.5 mL microbubbles and nucleic acid payload, ultrasound energy was delivered to the kidney region of the subject (e.g., unilateral or bilateral) using an M5Sc probe perpendicular to the subject's positioning. Depth of focus was set to 3.75–4 cm, and zoom was set to 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) value of 0.09 and a high MI value of 2.3, without interruption of ultrasound energy delivery at any point during the treatment. Approximately 20 seconds after administering the bolus injection of the acoustically active microstructure and DNA solution, nine high MI ultrasound flashes of 2.3 were delivered, with approximately 5 seconds between each high MI flash, and this was repeated three times for nine pulses. The high MI pulse duration was approximately 2.25 microseconds. Subsequently, approximately every 30 seconds, the bolus injection of the acoustically active microstructure and DNA solution was administered, with approximately nine high MI flashes delivered in both cross-sectional and longitudinal views, alternating between views, until the infusion was completed. The same treatment procedure was repeated at 48-hour intervals, for a total of three treatment cycles per kidney.

[0223] At the end of the experiment, NHP kidney sections were prepared. One section from each experimental animal's kidney and one section from a non-experimental animal's kidney were analyzed using RNAscope to evaluate transfection of specific kidney cell types in the glomerular and non-glomerular regions using ultrasound-mediated gene delivery (UMGD).

[0224] Kidney samples were analyzed using RNAscope™ assays (RNAscope™ 25 LS Multiplex Fluorescent ISH Kit; catalog number 322800; and RNAscope™ LS 4-Plex Ancillary Kit for Multiplex Fluorescent ISH Kit; catalog number 322830). Optimal pretreatment conditions for maximizing the signal-to-noise ratio were established based on evaluation of tissue samples using reference positive and negative control probes. The following standard RNAscope™ Multiplex FL assay pretreatment conditions were used: (i) Epitope retrieval 2: 15 min at 95°C and Protease III: 15 min at 40°C.

[0225] The target probe is: (1) Enhanced green fluorescent protein (EGFP), mRNA; EGFP-04; catalog number 538858; GenBack accession number MN623123.1; (2) Crab-eating macaques Nephrotic 2-podopsin (NPHS2), mRNA; Mfa-NPHS2-C2; catalog number 870598-C2; GenBack accession number NM_005540114.2; (3) Crab-eating macaquesPlatelet / endothelial cell adhesion molecule 1 (PECAM1), mRNA; Mfa-PECAM1-C1-C3; catalog number 434498-C3; GenBack accession number NM_005584700.1; and (4) Homo sapiens Channel non-voltage-gated 1α (SCNN1A), mRNA; Hs-SCNN1A-C4; catalog number 480978-C4; GenBack accession number NM_001038.5. These probes are summarized below.

[0226]

[0227] The target probe indicated that: (1) EGFP: payload expression in renal cells; (2) NPHS2: podocytes; (3) SCNN1a: renal tubular epithelial cells; and (4) PECAM1: endothelial cells. Figure 24 ).

[0228] Visual scoring was performed, assigning a single score to each sample based on the dominant staining pattern throughout the sample. Staining intensity had no effect on the score. Dots in the histological image were correlated with the number of individual RNA molecules. The percentage of positive cells was visually scored and categorized based on the number of cells with more than one dot per cell (i.e., 0%, 1–25%, 26–25%, 51–75%, 76–99%, 100%).

[0229] result Three NHP kidney samples were evaluated using RNAscope™ 25 LS multifluorescent ISH. All samples had previously passed quality control checks (see Example 12). RNAscope™ 25 LS multifluorescent ISH was performed to assess the expression of EGFP-04 mRNA and NPHS2, SCNN1A, and PECAM1 mRNA in the three kidney samples. EGFP-04 expression indicates the expression of the genetic payload in kidney cells. SCNN1A-positive cells indicate the renal tubular epithelial cell type. NPHS2-positive cells indicate the podocyte type. PECAM1 expression indicates the endothelial cell type. Figure 24 Examples of tissue sections are provided. EGFP-04 mRNA expression was observed in both treated kidney samples, but not in the untreated samples. NPHS2, SCNN1A, and PECAM1 mRNA expression were also observed in all kidney samples. EGFP-04 mRNA expression was observed in all treated samples, but not in the untreated samples. Various cellular markers were also observed in all samples.

[0230] Figure 25AExamples of histological samples stained with EGFP, NPHS2, PECAM1, and SCNN1A in non-glomerular regions of NHP kidneys are provided. Figure 25B This study provides quantification of the percentages of EGFP-04-positive endothelial cells (PECAM1-positive), renal tubular epithelial cells (SCNN1A-positive), and podocytes (NPHS2-positive) in both treated and untreated samples outside the glomerular region. EGFP-04 expression was lowest in all three cell types in untreated animals, while in treated animals, EGFP-04 was expressed in all three cell types, with the highest expression observed in renal tubular cells outside the glomerular region.

[0231] Figure 26A Examples of histological samples stained with EGFP, NPHS2, PECAM1, and SCNN1A in the glomerular region of NHP kidneys are provided. Figure 26B This study provides quantification of the percentages of EGFP-04-positive endothelial cells (PECAM1-positive), renal tubular epithelial cells (SCNN1A-positive), podocytes (NPHS2-positive), and unknown cells in both treated and untreated samples from the glomerular region. EGFP-04 expression was lowest in all three cell types in the untreated animals, while in the treated animals, EGFP-04 was expressed in all three cell types, with the highest expression observed in endothelial cells and podocytes in the non-glomerular region.

[0232] These results demonstrate that UMGD effectively delivers genetic payloads to endothelial cells, tubular epithelial cells, and podocytes in both glomerular and non-glomerular regions of NHP kidneys, and that repetitive sonopore formation using a multi-organ localization protocol effectively delivers and drives the expression of genetic payloads in NHP kidneys.

[0233] Example 14: Acoustophoresis in a mouse kidney model using a multiple injection and multiple ultrasound localization scheme, demonstrating delivery to glomerular and non-glomerular regions. Animals and programs Two experimental groups of Balb / C mice were included. One group received a mixture of nanoparticle genetic payloads and acoustically active microstructures, along with ultrasound (US) energy, in two animals. The second experimental group consisted of untreated mice that did not receive any intravenous injection of microbubbles or plasmids and were not subjected to ultrasound at any time. Prior to the experiment, a jugular vein catheter (JVC) was implanted into the treated mice, through which the acoustically active microstructures and nucleic acid constructs were administered.

[0234] Prepare a dose of sonoactive microstructure and DNA solution by first preparing the sonoactive microstructure according to the label instructions: Activate DEFINITY RT by shaking the vial for 45 seconds. After activation, but no more than 15 minutes later, place the activated DEFINITY RT vial upright and inject 1.4 mL of PBS into the vial. Quickly rotate the upright vial for 10 seconds to mix the contents and use a; then aspirate 150 μL of Definity RT microbubbles (phospholipid-stabilized) into the syringe using a 1.5-inch 18G needle. Using the same needle and syringe, aspirate 50 μL of DNA payload into the syringe to combine the DNA and DEFINITY RT. Mix the DEFINITY RT microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogeneous. Withdraw the DNA + DEFINITY RT solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0235] Each treatment session consisted of three doses of a mixture of mpDNA (nanoplasmid) and acoustically active microstructures administered via bolus injection in PBS, each dose delivered through a jugular vein catheter. Each dose of mpDNA was 275 μg (116 μmol), and a total of 825 μg (348 μmol) of mpDNA was delivered during the treatment. After each bolus injection, the ultrasound probe was moved from a cross-sectional view to a longitudinal view (e.g., rotated approximately 90 degrees around the mouse), and the views were alternated until the infusion was complete. The microplasmid DNA construct contained nucleic acid encoding a luciferase gene coupled to the CAG promoter (Np-CAG-Fluc).

[0236] Three doses were administered to mice via three bolus injections. For each dose, following the administration of the microbubbles and nucleic acid payload, ultrasound energy was delivered to the kidney region of the mice using an L6-24 probe and ultrasound imaging at a low mechanical index (MI) of 0.09. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 0.8, without interruption of ultrasound energy application at any point during the treatment process. Nine flashes of high MI ultrasound at 0.8 were delivered, with 2-second intervals between each flash, and the administration of the nine pulses was repeated three times. The duration of the high MI pulse was approximately 0.82 microseconds. Each bolus injection was administered with ultrasound for less than 30 seconds.

[0237] At the end of the experiment, right and left kidney sections from both treated and untreated mice were prepared. RNAscope was used to analyze two sections from the treated animals and one section from the untreated animals to evaluate transfection of specific kidney cell types in both glomerular and non-glomerular regions using ultrasound-mediated gene delivery (UMGD).

[0238] Kidney samples were analyzed using RNAscope™ assays (RNAscope™ 25 LS Multiplex Fluorescent ISH Kit; catalog number 322800; and RNAscope™ LS 4-Plex Ancillary Kit for Multiplex Fluorescent ISH Kit; catalog number 322830). Optimal pretreatment conditions for maximizing the signal-to-noise ratio were established based on evaluation of tissue samples using reference positive and negative control probes. The following standard RNAscope™ Multiplex FL assay pretreatment conditions were used: (i) Epitope retrieval 2: 15 min at 95°C and Protease III: 15 min at 40°C.

[0239] Positive and negative control probes are provided below.

[0240]

[0241] The target probe is: (1) Enhanced green fluorescent protein (EGFP), mRNA; EGFP-04; catalog number 538858; GenBack accession number MN623123.1; (2) House mouse Nephrotic 2-podopsin (NPHS2), mRNA; Mm-NPHS2-C2; catalog number 507058-C2; GenBack accession number NM_130456.4; (3) House mouse Platelet / endothelial cell adhesion molecule 1 (PECAM1), mRNA; Mm-PECAM1-C4; catalog number 316728-C4; GenBack accession number NM_001032378.1; and (4) House mouse Channel non-voltage-gated 1α (SCNN1A), mRNA; Mm-SCNN1A-C3; catalog number 441398-C3; GenBack accession number NM_011324.2.

[0242] Target probe indications: (1) EGFP: payload expression in renal cells; (2) NPHS2: podocytes; (3) SCNN1a: renal tubular epithelial cells; and (4) PECAM1: endothelial cells. Figure 27 These probes are summarized below.

[0243]

[0244] Visual scoring was performed, assigning a single score to each sample based on the dominant staining pattern throughout the sample. Staining intensity had no effect on the score. Dots in the histological image were correlated with the number of individual RNA molecules. The percentage of positive cells was visually scored and categorized based on the number of cells with more than one dot per cell (i.e., 0%, 1–25%, 26–25%, 51–75%, 76–99%, 100%).

[0245] Single-nuclear RNA sequencing (snRNAseq) was also performed on kidney samples to analyze gene expression in kidney cells, thereby identifying tubular cells, endothelial cells, and podocytes.

[0246] result Six mouse kidney samples (two left and two right kidney samples from treated mice, and one left and one right kidney sample from untreated mice) were evaluated using RNAscope™ 25 LS multiplex fluorescent ISH. Mm-Ppib, Mm-Polr2a, Mm-Ubc, and Mm-Hprt were used as positive control biomarkers for sample quality control and to assess RNA quality in the tissue samples. The bacterial gene dapB was used as a negative control. Optimization was performed to establish the optimal signal-to-noise ratio.

[0247] RNAscope™ 25 LS multiplex fluorescent ISH was performed to assess the expression of EGFP-04 mRNA, as well as NPHS2, SCNN1A, and PECAM1 mRNA, in six kidney samples. EGFP-04 expression indicates the expression of genetic payload in kidney cells. SCNN1A-positive cells indicate renal tubular epithelial cell type. NPHS2-positive cells indicate podocyte type. PECAM1 expression indicates endothelial cell type. Figure 27 Examples of tissue sections are provided. EGFP-04 mRNA expression was observed in all treated kidney samples, but not in untreated samples. NPHS2, SCNN1A, and PECAM1 mRNA expression were also observed in kidney samples. EGFP-04 mRNA expression was observed in all treated samples, but not in untreated samples. Various cellular markers were also observed in all samples.

[0248] Figure 28A Examples of histological samples stained with EGFP, NPHS2, PECAM1, and SCNN1A in the non-glomerular region of mouse kidneys are provided. Figure 28BThis study provides quantification of the percentages of EGFP-04-positive endothelial cells (PECAM1-positive), renal tubular epithelial cells (SCNN1A-positive), and podocytes (NPHS2-positive) in both treated and untreated samples outside the glomerular region. EGFP-04 expression was lowest in all three cell types in the untreated animals, while it was observed in endothelial and tubular cells of the treated animals, with the highest expression observed in endothelial cells outside the glomerular region.

[0249] Figure 29A Examples of histological samples stained with EGFP, NPHS2, PECAM1, and SCNN1A in the glomerular region of mouse kidneys are provided. Figure 29B This study provides quantification of the percentages of EGFP-04-positive endothelial cells (PECAM1-positive), renal tubular epithelial cells (SCNN1A-positive), podocytes (NPHS2-positive), and unknown cells in both treated and untreated samples from the glomerular region. EGFP-04 expression was lowest in all three cell types in the untreated animals, while it was observed in endothelial and podocyte cells of the treated animals, with the highest expression observed in endothelial cells of the glomerular region.

[0250] These results demonstrate that UMGD effectively delivers genetic payloads to endothelial cells, tubular epithelial cells, and podocytes in both glomerular and non-glomerular regions of the mouse kidney.

[0251] The distribution of endothelial cells, renal tubular epithelial cells, and podocytes in mouse kidneys was determined using snRNA-seq. Figure 30A The study provides spatial distributions of endothelial cells, renal tubular epithelial cells, and podocytes in mouse kidneys, as detected by snRNAseq. Figure 30B A comparison of the percentage of total cells (endothelial cells, renal tubular epithelial cells, podocytes, or unknown cells) in mouse kidneys, as determined using snRNAseq (left) and RNAscope (right), is provided. Although there were more unknown cells in RNAscope than in snRNAseq, both methods showed that the highest percentage was for renal tubular endothelial cells, followed by endothelial cells, and then the lowest percentage was for podocytes.

[0252] These results demonstrate that repetitive sonopore formation using a multi-organ localization scheme effectively delivers and drives the expression of genetic payloads in multiple cell types of mouse kidneys.

[0253] Example 15: Acoustic pore formation in a rat liver model using a multiple injection and multiple ultrasound localization scheme Animals and programs Two groups of two experimental animals were used, each being a C57 mouse. One experimental group was administered a mixture of nanoparticle genetic payload and acoustically active microstructure, along with ultrasound (US) energy. The second experimental group was unexperimented and did not receive any intravenous injection of microbubbles or plasmids, nor was it subjected to externally applied ultrasound at any time.

[0254] Prior to the experiment, a jugular vein catheter (JVC) was implanted into the treated mice, through which acoustically active microstructures and nucleic acid constructs were administered.

[0255] To prepare a dose of sonoactive microstructure and DNA solution, first follow the label instructions: remove from 4C storage and roll between your fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 uL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space (approximately 50 microliters (uL) is included in the calculation)). Using the same needle and syringe, draw 75 uL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0256] Each treatment session consists of three doses of a mixture of mpDNA (nanoplasmid) and acoustically active microstructures in PBS, administered via bolus injection, with each dose delivered through a jugular vein catheter. Each dose of mpDNA is approximately 83 μg, and a total of approximately 250 μg of mpDNA is delivered. The microplasmid DNA construct contains nucleic acid encoding a luciferase gene coupled to the CAG promoter (Np-CAG-Fluc).

[0257] Three doses were administered to mice, each administered as a single bolus. For each dose, following the administration of the microbubbles and nucleic acid payload, ultrasound energy was delivered to the liver region of the mice using an L6-24 probe and ultrasound imaging at a low mechanical index (MI) of 0.09. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 1.5, without interruption of ultrasound energy delivery at any point during the treatment process. Nine flashes of high MI ultrasound at 1.5 were delivered, with a 4-second interval between each flash, and the administration of the nine pulses was repeated three times. The duration of the high MI pulse was approximately 0.82 microseconds. Each bolus injection was administered with less than 30 seconds of ultrasound.

[0258] At the end of the experiment, liver sections from both treated and untreated mice were collected. Two sections from the livers of the treated animals and two sections from the kidneys of the untreated animals were analyzed using RNAscope to evaluate transfection of specific hepatocyte types using ultrasound-mediated gene delivery (UMGD).

[0259] Liver samples were analyzed using RNAscope™ assays (RNAscope™ 25 LS Multiplex Fluorescent ISH Kit; catalog number 322800; and RNAscope™ LS 4-Plex Ancillary Kit for Multiplex Fluorescent ISH Kit; catalog number 322830). Optimal pretreatment conditions for maximizing the signal-to-noise ratio were established based on evaluation of tissue samples using reference positive and negative control probes. The following standard RNAscope™ Multiplex FL assay pretreatment conditions were used: (i) Epitope repair 2: 15 min at 95°C and Protease III: 15 min at 40°C.

[0260] Positive and negative control probes are provided below.

[0261]

[0262] The target probe is: (1) Enhanced green fluorescent protein (EGFP), mRNA; EGFP-04; catalog number 538858; GenBack accession number MN623123.1; (2) House mouse Desialyl glycoprotein receptor 1 (ASGR1), mRNA; Mm-Asgr1-C2; catalog number 313738-C2; GenBack accession number NM_009714.2; (3) House mouse Melanoma cell adhesion molecule 1 (MCAM1), mRNA; Mm-MCAM-C3; catalog number 406328-C3; GenBack accession number NM_023061.2; (4) House mouse Adhesion G protein-coupled receptor E1 (ADGRE1), mRNA; Mm-Adgre1-C4; catalog number 460658-C4; GenBack accession number NM_010130.4.

[0263] Target probe indications: (1) EGFP: payload expression in hepatocytes; (2) NPHS2: podocytes; (3) SCNN1a: renal tubular epithelial cells; and (4) PECAM1: endothelial cells. Figure 27 These probes are summarized below.

[0264]

[0265] Visual scoring was performed, assigning a single score to each sample based on the dominant staining pattern throughout the sample. Staining intensity had no effect on the score. Dots in the histological image were correlated with the number of individual RNA molecules. The percentage of positive cells was visually scored and categorized based on the number of cells with more than one dot per cell (i.e., 0%, 1–25%, 26–25%, 51–75%, 76–99%, 100%).

[0266] Liver samples were also subjected to single-nuclear RNA sequencing (snRNAseq) to analyze gene expression in hepatocytes, thereby identifying duct cells, endothelial cells, and podocytes.

[0267] result Four mouse liver samples (two from treated mice and two from untreated mice) were evaluated using RNAscope™ 25 LS multiplex fluorescent ISH. Mm-Ppib, Mm-Polr2a, Mm-Ubc, and Mm-Hprt were used as positive control biomarkers for sample quality control and to assess RNA quality in the tissue samples. The bacterial gene dapB was used as a negative control. Optimization was performed to establish the best signal-to-noise ratio.

[0268] RNAscope™ 25 LS multiplex fluorescent ISH was performed to assess the expression of EGFP-04 mRNA, as well as MCAM, ADGRE1, and ASGR1 mRNA, in four liver samples. EGFP-04 expression indicates the expression of the genetic payload in hepatocytes. MCAM-positive cells indicate the hepatic sinusoidal endothelial cell (LSEC) type. Asgr1-positive cells indicate the hepatocyte (HC) type. Adgre1 expression indicates the Kupffer cell (KC) type. Figure 31A Examples of tissue sections are provided. EGFP-04 mRNA expression was observed in all treated liver samples, but not in untreated samples. MCAM, ADGRE1, and ASGR1 mRNA expression were also observed in whole liver samples, including both treated and untreated groups. EGFP-04 mRNA expression was observed in all treated samples, but not in untreated samples. Various cellular markers were also observed in all samples.

[0269] Figure 31A Examples of histological samples stained with MCAM, ADGRE1, and ASGR1 in mouse livers are provided. Figure 31BThe percentages of LSECs (MCAM-positive), hepatocytes (Asgr1-positive), and Kupffer cells (NPHS2-positive) that were also EGFP-04-positive in both treated and unexperimented liver samples were quantified. EGFP-04 expression was lowest in all three cell types in unexperimented animals, while it was observed in all three cell types in treated animals, with the highest expression observed in hepatocytes. Approximately 60% of the relatively EGFP+ cells in mouse livers are hepatocytes.

[0270] These results demonstrate that UMGD effectively delivers genetic payloads to LSEC, hepatocytes, and Kupffer cells in the mouse liver.

[0271] The distribution of LSEC, hepatocytes, and Kupffer cells in mouse livers was determined using snRNA-seq. Figure 32A Spatial distributions of LSECs, hepatocytes, and Kupffer cells in mouse livers, as detected by snRNAseq, are provided. Figure 32B A comparison of the percentages of LSEC, hepatocytes, and Kupffer cells or unknown cells in the total number of cells in mouse livers, as determined using snRNA-seq (left) and RNAscope (right), is provided. EGFP-04 expression was observed in all three cell types in the treated animals, with the highest expression observed in hepatocytes. Approximately 78.8% of all relatively EGFP+ cells in the mouse liver were hepatocytes, as measured by snRNA-seq. Approximately 74.3% of all relatively EGFP+ cells in the mouse liver were hepatocytes, as measured by snRNA-seq. RNAscope and snRNA-seq methods showed the highest percentage for hepatocytes, followed by LSEC cells, and then the lowest percentage for Kupffer cells.

[0272] Example 16: Expression of genetic payload in NHP kidney cells Animals and programs Following the methods described in this paper, ultrasound-mediated gene delivery (UMGD) was performed on non-human primates (NHPs) to deliver a genetic payload of enhanced green fluorescent protein (EGFP). NHP kidney sections were analyzed using RNAscope to evaluate the transfection of kidney cells using UMGD.

[0273] Kidney samples from (5) nonhuman primates (NHPs) were analyzed using the RNAscope™ assay (RNAscope™ 2.5 LSx Red kit; catalog number 322750). Optimal pretreatment conditions for maximizing the signal-to-noise ratio were established based on evaluation of tissue samples using reference positive and negative control probes. The standard RNAscope™ 2.5 LSx Red assay pretreatment conditions were as follows: (i) Epitope repair 2: 15 min at 95 °C. Protease III: 15 min at 40 °C.

[0274] The control probe used is: (i) ACD positive control probe ( Crab-eating macaques The target probe is (peptidyl prolyl isomerase B (cyclophilin B)); Mfa-PPIB; catalog number 424148); ACD negative control probe (Bacillus subtilis dihydropyridine dicarboxylic acid reductase; dapB; catalog number 312038). The target probe is (Mycobacterium tuberculosis H37Rv plasmid pTYGi9EGFP-04; catalog number 538858; GenBack accession number MN623123.1).

[0275] Visual scoring was performed, assigning a single score to each sample based on the dominant staining pattern throughout the sample. Staining intensity had no effect on the score. Dots in the histological image were correlated with the number of individual RNA molecules. The percentage of positive cells was visually scored and categorized based on the number of cells with more than one dot per cell (i.e., 0%, 1–25%, 26–25%, 51–75%, 76–99%, 100%).

[0276] result All samples passed quality control checks, exhibiting moderate PPIB positive control staining and little or no dapB background staining. RNAscope™ 2.5 LSx Red assays were performed in all samples to assess EGFP expression in NHP kidney samples. EGFP was detected in the kidney samples. Figure 23A Examples of histological images of samples are provided (arrows indicate EGFP-positive cells). Figure 23B The percentage of EGFP-positive glomeruli in NHP kidney sections is provided. These results indicate that UMGD effectively delivers genetic payloads to NHP kidneys.

[0277] Example 17: DNA transfection via sonoporosis using a multi-organ localization and repeat protocol a in a mouse model of polycystic kidney disease. Animals and programs In this embodiment, four experimental groups were conducted to evaluate the transfection of the Fluc reporter gene into a mouse model of polycystic kidney disease. The test mice were C57, wild-type, or Nek8 mice, which are preferred for inducing the development of polycystic kidney disease.jck Mice that mutated to homozygous form. For Nek8 jck Mice with homozygous mutations exhibited late cystic lesions. A jugular venous catheter was implanted in subjects, followed by sonopore treatment to deliver firefly luciferase to the kidneys. Subjects received the jugular venous catheter for infusion of nucleic acid encoding firefly luciferase coupled to the CAG promoter, and either saline or protein-stabilized sonoactive microstructures, depending on the experimental group. The protein-stabilized sonoactive microstructures used were Optison protein-stabilized microspheres, prepared according to the instructions on the label as described in previous embodiments herein. Each subject received three doses of the nucleic acid and microbubble solution, administered via bolus injection, followed by three applications of ultrasound energy to each kidney after administration of either the bolus injection of saline or the sonoactive microstructure and nucleic acid. With ultrasound energy application, the interval between repeated administrations of nucleic acid and saline or the sonoactive microstructure was 4 hours. The ultrasound energy was applied to the distal cortex of the kidney, alternating between cross-sectional and longitudinal views. Using an L6-24 ultrasound probe, ultrasonic energy was applied with an alternating mechanical index between a low MI of 0.9 and a high MI of 1.3, delivered via 27 high MI flashes with a flash duration of less than 2 seconds. Approximately 150 μL of saline or protein-stabilized acoustically active microstructures and approximately 50 μL of saline containing nucleic acids were administered per dose, for a total infusion volume of 200 μL. Subjects underwent IVIS analysis on days 1, 2, 3, and 7 post-treatment, followed by 1 / 3 N BF fixation and drying of renal tissue prior to rapid freezing and storage, and subsequent evaluation using immunohistochemistry and qPCR readings. Experimental conditions are summarized below.

[0278]

[0279]

[0280] result Using IVIS, mice were imaged 1, 2, 3 and 7 days after administration of nucleic acid, microbubbles and sonic energy. Figure 19 The mean radioactivity of IVIS images from different experimental groups of mice is shown at the indicated time points. For the wild-type and C57 groups, the mean radioactivity measurements ranged from 10^6 to 10^7, and for the Nek8 group... jck The mutation exceeded 10^7. The negative control group, which was administered saline alone, showed a radioactivity signal only around 10^4, consistent with background noise.

[0281] like Figure 19As shown, luciferase expression was observed in all experimental groups 24 hours after administration and persisted for at least 7 days. Sonoformation revealed initial differences between experimental groups. The control group, which did not receive sonoformation microstructures, exhibited a low but detectable signal. Compared to wild-type or C57 mice, for Nek8... jck Mice that mutated to homozygosity exhibited cystic lesion bands, increased kidney size, and showed increased gene transfection and expression levels.

[0282]

[0283] Immunofluorescence assays were performed on the expression of luciferase in the kidneys from frozen tissue. Frozen kidney tissue was obtained and slides were prepared. Before staining, the slides were stored at -80°C. After staining, the slides were stored at 4°C. The staining protocol is as follows: Frozen sectioning protocol • Allow the frozen kidney slices to reach room temperature (approximately 30 minutes). • Rinse in PBS for 5 minutes at room temperature • Fix in 4% paraformaldehyde for 5 minutes at room temperature • Wash in PBS for 5 minutes (2x) at room temperature. • Wash for 5 minutes in PBT (0.25% Triton X-100) at room temperature. • Wash in PBS for 5 minutes at room temperature. • Block in BlockAid blocking solution for 1 h at room temperature • In a BlockAid closed solution (humidified chamber) O / N at 4°C, the primary Ab was subjected to the following conditions. • Wash each item separately for 3 minutes in PBS (x5) at room temperature the next morning. • Allow the secondary Ab to incubate in BlockAid-blocked solution in PBS at room temperature for 2 hours (protected from light). • Wash each item separately in PBS (x5) at room temperature for 3 minutes (protected from light). • Equipped with Vector H-1200 and DAPI • Store glass slides at 4°C Slides were observed using a Nikon Eclipse E600 microscope, and images were captured using VisiView (version 4.5.0.0, license number 1646) software. As expected, staining with the GTX antibody GTX20498 produced no signal in DKD mice without any gene delivery via sonopores. A luciferase (green) signal was obtained in the glomeruli of the kidneys with gene delivery via sonopores. The immunofluorescence staining method for luciferase was optimized for use on frozen kidney sections. A luciferase antibody (from a previous internal study) that produced no background signal in the frozen kidneys was identified. Luciferase signal was detected in the glomeruli, confirmed by synaptopodin (IF) staining. The results of IF staining are shown below. Figure 33 As shown.

[0284] Example 18: Long-term dose response from multiple payload doses and multiple ultrasound administrations Laboratory animals and protocols Four BALB / c mice in each of the four groups were administered acoustically active microstructures and nanoparticles encoding luciferase via intravenous delivery through a jugular catheter. Group 1 received 5 μg of nucleic acid, Group 2 received 50 μg of nucleic acid, Group 3 received 100 μg of nucleic acid, and Group 4 received 250 μg of nucleic acid; each was suspended in 50 μL of phosphate-buffered saline.

[0285] Mice were administered three doses of bolus-injected nanoparticles of nucleic acid and acoustically active microstructures (“microbubbles”). Over a 15-second time interval, each group was administered a corresponding dose of 50 μL of nucleic acid and 200 μL of acoustically active microstructure suspension in PBS, with the nucleic acid and acoustically active microstructure defining a single dose. Following administration of the nucleic acid and acoustically active microstructure, continuous ultrasound energy was percutaneously applied to the liver at alternating low and high MI (Mechanical Index). Ultrasound was applied with a low mechanical index of 0.07 and a high mechanical index of 0.8 in nine flashes over 4-second intervals. After ultrasound energy application, the acoustically active microstructure and nucleic acid were re-administered, and ultrasound was re-applied. Three doses of nanoparticles, microbubbles, and ultrasound energy were administered during one treatment cycle. Luciferase expression was measured by IVIS at 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks and 26 weeks after treatment.

[0286] result Using IVIS, mice were imaged at 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks and 26 weeks after treatment. Figure 22 The mean IVIS radiation rate values ​​of the treated mice are shown, with IVIS analysis performed at the indicated time points.

[0287] like Figure 22 As shown, luciferase expression was observed in the experimental groups 24 hours after administration and persisted for at least 16 weeks, and for the 5, 50, and 500 μg groups for 26 weeks. Throughout the study, expression levels appeared to correlate with increasing nucleic acid dose, with the highest mean expression levels observed in the 250 μg nucleic acid group lasting 14 weeks, and the lowest expression levels observed in mice receiving 5 μg nucleic acid. Figure 22 ).

[0288] Example 19: Long-term persistence of administration in a rat liver model using multiple injections and multiple ultrasound localizations Laboratory animals and protocols There were two experimental groups, each consisting of four RAG2 knockout mice. Prior to the experiment, a jugular vein catheter (JVC) was implanted into each mouse, through which the acoustically active microstructure and nucleic acid construct were administered.

[0289] In this experiment, mice underwent a single sono-induced pore-forming process, which included three injections of microplasmid DNA (mpDNA) and the application of sono-active microstructures during the treatment. The mpDNA construct used in this experiment was the Nanoplasmid™ construct. Ultrasonic (US) energy was delivered percutaneously to transfect the target organ, the liver. Group 1 received three injections of 33 μg DNA, delivering a total DNA payload of 100 μg during the treatment, and Group 2 received three injections of 76 μg DNA, delivering a total DNA payload of 228 μg during the treatment. The volume ratio of the DNA payload solution to the sono-active microstructure solution used was 1:4.

[0290] Prepare a dose of sonoactive microstructure and DNA solution by first preparing the sonoactive microstructure according to the label instructions: Remove the microstructure from the 4C storage and roll it between your palms for 20 seconds; remove the protective plastic and aluminum capping from the Optison vial; insert a 25G needle through the rubber gasket of the Optison vial to provide a pressure outlet; and draw 12 mL of Optison into the syringe using a 1.5-inch 18G needle (the dead space of the needle is included in the calculation). Using the same needle and syringe, draw 228 μg or 100 μg of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the IV catheter.

[0291] Each procedure consists of three bolus injections of 33 μg DNA, delivering a total of 100 μg, or three bolus injections of 76 μg DNA payload, delivering a total of 228 μg DNA, along with a mixture of acoustically active microstructures in PBS, delivered via a jugular catheter with each injection. The microplasmid DNA construct contains nucleic acid encoding a luciferase gene coupled to the ApoE promoter. Acoustically active microstructures Optison™ microbubbles were delivered to mice at a 1:4 volume ratio (mpDNA solution: acoustically active microstructure mixture, including needle dead space dispersed in the mixture).

[0292] Following the administration of the first bolus of microbubbles and nucleic acid payload, in these experiments, ultrasound energy was delivered to the liver region of mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the liver at a low mechanical index (MI) value of 0.07 using B-mode ultrasound imaging. The depth setting was set to 2 cm, and the zoom setting was 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) value of 0.07 and a high MI value of 0.8. Nine flashes of high MI ultrasound at 0.8 were delivered, with a 4-second interval between each flash, and the administration of the nine pulses was repeated three times. The high MI pulse duration was approximately 0.82 microseconds. After the administration of the second bolus of microbubbles and nucleic acid payload, the ultrasound probe was moved to a second location on the liver, and ultrasound energy was delivered to this second liver location. After the administration of the third bolus of microbubbles and nucleic acid payload, the ultrasound probe was moved to a third location on the liver, and ultrasound energy was delivered to this third liver location.

[0293] result Following the first procedure, IVIS fluorescence radiance imaging was performed on all groups at 24, 48, and 72 hours, and then repeated weekly for 52 weeks. Figure 20 All mice in each group underwent IVIS fluorescence imaging at each time point. Large dots along the bars represent individual mouse readings, and the bars represent group averages. The left bar represents the 228 μg group, and the right bar represents the 100 μg group. Mice were observed to exhibit stable gene expression levels over 52 weeks when treated with a multiple bolus and multiple ultrasound localization protocol.

[0294] Example 20: Long-term persistence of administration in a rat liver model using multiple injections and multiple ultrasound localizations Laboratory animals and protocols This study involved an experimental group of four BALB / c mice. Mice were administered multiple boluses of DNA payloads and acoustically active microstructures via ultrasound administration to multiple organ sites, and were followed for 52 weeks to assess persistent gene expression. Prior to the experiment, a jugular vein catheter (JVC) was implanted in each mouse through which the acoustically active microstructures and nucleic acid constructs were administered.

[0295] To prepare a dose of sonoactive microstructure and DNA solution, first follow the label instructions: remove from 4C storage and roll between your fingers for 20 seconds; remove the protective plastic and aluminum cover from the Optison vial; insert a 25G needle through the rubber gasket to provide a pressure outlet; and draw 225 uL of Optison into the syringe using a 1.5-inch 18G needle (the needle dead space (approximately 50 microliters (uL) is included in the calculation)). Using the same needle and syringe, draw 75 uL of DNA payload into the syringe to combine the DNA and Optison. Mix the Optison microbubbles and DNA payload in the syringe by rolling the syringe between your fingers until the solution is homogenized. Withdraw the DNA + Optison solution from the needle dead space. Then replace the 18G needle with a 25G blunt needle for injection into the patient's JVC.

[0296] Each treatment session consisted of one dose of mpDNA and acoustically active microstructure mixture in PBS, totaling 150 μL, delivered via a jugular vein catheter. The microplasmid DNA construct contained nucleic acid encoding a luciferase gene coupled to the CAG promoter. Acoustically active microstructure Optison™ microbubbles were delivered to mice at a 1:4 volume ratio (mpDNA solution: acoustically active microstructure mixture, including needle dead space dispersed in the mixture).

[0297] Mice were administered three bolus injections of approximately 33 μg of DNA payload and acoustically active microstructures, totaling approximately 100 μg of DNA per treatment. Following the administration of the microbubbles and nucleic acid payload, in these experiments, ultrasound energy was delivered to the kidney region of the mice using an L6-24 probe positioned perpendicular to the mouse location to localize a lateral view of the kidney using B-mode ultrasound imaging at a low mechanical index (MI) of 0.07. The depth setting was set to 2 cm, and the zoom setting to 0. Ultrasound was delivered continuously, alternating between a low mechanical index (MI) of 0.07 and a high MI of 0.8, without interruption of ultrasound energy application at any point during the treatment. Nine flashes of high MI ultrasound at 0.8 were delivered, with 4-second intervals between each flash, and this nine-pulse delivery was repeated three times. The high MI pulse duration was approximately 0.82 microseconds. Subsequently, bolus injections of acoustically active microstructures and DNA solution were administered approximately every 30 seconds. During the second bolus injection, the ultrasound probe was moved from a cross-sectional view to a longitudinal view. When administering the second injection, move the ultrasound probe from the transverse section view back to the cross section view.

[0298] result Mice were imaged using IVIS fluorescence imaging at 1-week intervals for a total of 52 weeks. IVIS fluorescence irradiance measurements in all groups indicated that the luciferase genetic payload was stably expressed in mouse kidneys for 52 weeks. Figure 21 ).

[0299] Implementation plan with numbering 1. A method for delivering a nucleic acid payload to target cells of a subject, comprising: The treatment process is provided to the subject, wherein the treatment process includes: a. Administer the nucleic acid payload to the subject; b. Administering multiple microbubbles to the subject; and c. Applying ultrasound energy to the vicinity of the target cells of the subject; and Repeat the treatment at least once within 6 hours but within 10 days after starting the first treatment session.

[0300] 2. A method for delivering a nucleic acid payload to target cells of a subject, comprising: The treatment process is provided to the subject, wherein the treatment process includes: a. Administer the nucleic acid payload to the subject; b. Administering multiple microbubbles to the subject; and c. Applying ultrasound energy to the vicinity of the target cells of the subject; and Repeat the treatment process at least once, more than 21 days after the start of the first treatment course.

[0301] 3. A method for delivering a nucleic acid payload to target cells of a subject, comprising: The treatment process is provided to the subject, wherein the trea...

Claims

1. A method for delivering a nucleic acid payload to target cells of a subject, the method comprising: The first treatment procedure is administered to the subject, the first treatment procedure comprising: A certain amount of a first therapeutic composition is administered to the subject, the first therapeutic composition comprising: i) the nucleic acid payload, and ii) a plurality of acoustic agents; Apply ultrasound energy to target cells at a first location in the target tissue of the subject; and Applying ultrasound energy to target cells at a second location in the target tissue of the subject, wherein the first location and the second location are different; and Following the first treatment procedure, a second treatment procedure is administered to the subject, the second treatment procedure comprising: A certain amount of a second therapeutic composition is administered to the subject, the second therapeutic composition comprising: i) the nucleic acid payload and ii) a plurality of acoustic active agents; Apply ultrasound energy to target cells at a third location in the target tissue of the subject; and Ultrasonic energy is applied to target cells at a fourth location in the target tissue of the subject, wherein the third location and the fourth location are different.

2. The method of claim 1, further comprising: Following the second treatment procedure, a subsequent treatment procedure is administered to the subject, the subsequent treatment procedure comprising: A certain amount of the first therapeutic composition or the second therapeutic composition is administered to the subject, wherein the first therapeutic composition or the second therapeutic composition comprises: i) the nucleic acid payload and ii) the multiple acoustic active agents; Apply ultrasound energy to target cells at a fifth location in the target tissue of the subject; and Ultrasonic energy is applied to target cells at a sixth location in the target tissue of the subject, wherein the fifth location and the sixth location are different.

3. The method as described in any of the preceding claims, further comprising, during the first treatment, applying ultrasonic energy to target cells at a subsequent location in the target tissue of the subject after applying ultrasonic energy to the second location.

4. The method as claimed in any of the preceding claims, further comprising, during the second treatment, applying ultrasonic energy to target cells at subsequent locations in the target tissue of the subject after applying ultrasonic energy to the fourth location.

5. The method of claim 2, further comprising, during the subsequent treatment, applying ultrasound energy to target cells at subsequent locations in the target tissue of the subject after applying ultrasound energy to the sixth location.

6. The method as claimed in any of the preceding claims, wherein the second treatment procedure occurs more than 6 hours but within 10 days after the first treatment procedure.

7. The method as described in any of the preceding claims, wherein the second treatment process occurs more than 21 days after the first treatment process.

8. The method of any of the preceding claims, wherein the administration of the first therapeutic composition and / or the second therapeutic composition is performed via a peripheral vein intravenously.

9. The method of any of the preceding claims, wherein the nucleic acid payload comprises a therapeutic transgene of length greater than 4.7 kbp and / or an expression cassette of length greater than 4.7 kbp.

10. The method of claim 9, wherein the therapeutic transgenic material comprises FVIII, COL4A5, or PKD1, GLP-1, INS, Reg3g, MafA, PDX-1, NUEROG3, NGN3, DRYK, DYRK1A, DYRK1B, factor VIII, factor IX, PKD2, COL4A3, COL4A4, Klotho, Smad7, TGF-β, SLC7A1, SLC3A9, UMOD, REN, HNF1B, MUC1, SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, NPHS1, NPHS2, CTNS, or combinations thereof.

11. The method of any of the preceding claims, wherein administering the amount of the first therapeutic composition comprises administering a first dose of the first therapeutic composition and a second dose of the first therapeutic composition to the subject during the first treatment.

12. The method of claim 11, wherein during or after the administration of the second dose of the first therapeutic composition, ultrasonic energy is applied to the second location in the target tissue of the subject.

13. The method of claim 11, wherein administering the amount of the first therapeutic composition during the first treatment includes administering at least a third dose of the first therapeutic composition.

14. The method of claim 13, further comprising, during or after the administration of the third dose, applying ultrasonic energy to the subsequent location of the subject during the first treatment.

15. The method of any of the preceding claims, wherein administering the amount of the second therapeutic composition during the second treatment comprises administering a first dose of the second therapeutic composition and a second dose of the second therapeutic composition to the subject.

16. The method of claim 15, wherein during the second treatment period, during or after the administration of the second dose of the second therapeutic composition, ultrasonic energy is applied to the third location in the target tissue of the subject.

17. The method of claim 15, wherein administering the amount of the second therapeutic composition during the second treatment includes administering at least a third dose of the second therapeutic composition.

18. The method of claim 17, further comprising, during or after the administration of the third dose of the second therapeutic composition, applying ultrasonic energy to the subsequent location of the subject during the second treatment process.

19. The method of any one of claims 11 to 18, wherein each dose of the first therapeutic composition or the second therapeutic composition is administered by intravenous injection.

20. The method of claim 19, wherein the intravenous injection is administered over discrete time periods.

21. The method of claim 20, wherein the discrete time period does not exceed 60 seconds, 120 seconds, or 180 seconds.

22. The method as claimed in any of the preceding claims, wherein the first location in the target tissue and the third location in the target tissue are the same location.

23. The method of any one of claims 1-21, wherein the first location in the target tissue and the third location in the target tissue are different.

24. The method of claim 3, wherein during the first treatment, the first location in the target tissue and the subsequent location in the target tissue are the same location.

25. The method of claim 3, wherein during the first treatment, the first location in the target tissue and the subsequent location in the target tissue are different.

26. The method as claimed in any of the preceding claims, wherein the second location in the target tissue and the fourth location in the target tissue are the same location.

27. The method of any one of claims 1-25, wherein the second location in the target tissue and the fourth location in the target tissue are different.

28. The method of claim 4, wherein during the second treatment period, the subsequent location in the target tissue and the target tissue are the same location during the second treatment period.

29. The method of claim 4, wherein during the second treatment, the second location in the target tissue and the subsequent location in the target tissue are different.

30. The method of claim 2, wherein the fifth position is the same position as any one of the first to fourth positions.

31. The method of claim 2, wherein the sixth position is the same position as any one of the first to fourth positions.

32. The method of any of the preceding claims, wherein the ultrasonic energy is applied at a minimum MI of 1.6, 2.1 or 2.

3.

33. The method of any of the preceding claims, wherein the acoustic agent comprises a protein-stabilized shell, a lipid-stabilized shell, a perfluoropropane gas core, an SF6 gas core, or a combination thereof.

34. The method of any of the preceding claims, wherein the ultrasonic energy is applied transdermally.

35. The method of any of the preceding claims, wherein the target cells and / or the target tissue are in the liver.

36. The method of claim 35, wherein the first position is the first lobe of the liver, and the second position or the subsequent position is the second lobe or a subsequent lobe of the liver.

37. The method of claim 36, wherein the first lobe of the liver is a right lobe, wherein the second lobe is a left lobe, and wherein the subsequent lobe is one or both of a caudate lobe or a quadrate lobe.

38. The method of claim 35, wherein the nucleic acid payload comprises a therapeutic transgene, wherein the target cell is a hepatocyte, and wherein at least 50% of the cells expressing the therapeutic transgene in the liver are hepatocytes.

39. The method of any one of claims 1 to 34, wherein the target cells and / or the target tissue are in the kidney.

40. The method of claim 39, wherein the first location is in a first region of the kidney, and the second location is in a second region of the kidney.

41. The method of claim 39, wherein the expression of the nucleic acid payload is induced in multiple cell types of the kidney.

42. The method of claim 39, wherein the transgene encoded by the nucleic acid payload is expressed in cells in all regions of the kidney.

43. The method of claim 39, wherein the transgene encoded by the nucleic acid payload is expressed in cells across four non-overlapping spatial regions of the kidney, the four non-overlapping spatial regions of the kidney defining the entire kidney.

44. The method of any of the preceding claims, wherein the distance between the first location in the target tissue and the second location or the subsequent location in the target tissue is at least 25% of the maximum distance of the long axis of the organ containing the target tissue.

45. The method of any of the preceding claims, wherein the distance between the third position in the target tissue and the fourth or subsequent position in the target tissue is at least 25% of the maximum distance of the long axis of the organ containing the target tissue.

46. ​​The method of any of the preceding claims, wherein the distance between the first location in the target tissue and the second location or the subsequent location in the target tissue is at least 1 cm, 2 cm or 3 cm.

47. The method as claimed in any of the preceding claims, wherein the distance between the third position in the target tissue and the fourth or subsequent position in the target tissue is at least 1 cm, 2 cm or 3 cm.

48. The method of any of the preceding claims, wherein the distance between the first location in the target tissue and the second location or the subsequent location in the target tissue is at least the diameter of the focused ultrasound beam.

49. The method of any of the preceding claims, wherein the distance between the third position in the target tissue and the fourth or subsequent position in the target tissue is at least the diameter of the focused ultrasound beam.

50. The method of any of the preceding claims, wherein the ultrasound energy is applied at the second or subsequent location in the target tissue to increase microvascular perfusion of the target tissue.

51. The method as described in any of the preceding claims, wherein the target tissue exhibits a cystic lesion.

52. The method of claim 51, wherein the delivery and / or expression of the nucleic acid payload to the target cells in the target tissue presenting the cystic lesion is increased.

53. The method as claimed in any of the preceding claims, wherein the first therapeutic composition and the second therapeutic composition are the same therapeutic composition.

54. The method of any of the preceding claims, wherein the first therapeutic composition and the second therapeutic composition comprise different doses of the nucleic acid payload.

55. The method of any of the preceding claims, wherein the first therapeutic composition and the second therapeutic composition comprise different doses of a sonoactive agent, or different sonoactive agents.

56. The method of any of the preceding claims, wherein applying ultrasound energy to the first and second locations of the subject comprises moving an ultrasound probe through the skin surface of the subject from the first location to the second location.

57. The method of any of the preceding claims, wherein applying the second treatment process increases the delivery or expression of the nucleic acid payload by at least 25%, 50%, 75%, 100%, 250%, 300%, 350%, 400%, 450%, or 500% compared to a method comprising applying the first treatment process without applying the second treatment process.

58. The method of claim 2, wherein, compared with a method comprising applying the first treatment process without applying the second treatment process, or compared with a method comprising applying the first treatment process and the second treatment process without applying the subsequent treatment process, applying the subsequent treatment process increases the delivery or expression of the nucleic acid payload by at least 100%, 250%, 300%, 350%, 400%, 450%, or 500%.

59. The method as claimed in any of the preceding claims, wherein the first position and the second position are adjacent positions of the target tissue.

60. The method of claim 2, wherein the second position and the subsequent position are adjacent positions of the target tissue.

61. The method as claimed in any of the preceding claims, wherein the third position and the fourth position are adjacent positions of the target tissue.

62. The method of claim 3, wherein the fourth position and the subsequent position are adjacent positions of the target tissue.

63. The method of claim 2, wherein the fifth position or the sixth position is adjacent to any one of the first position to the fourth position.

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