Methods and systems for improved delivery via ultrasound

Abstract

Methods are provided for improving expression of a nucleic acid construct in a cell or organ of a subject using sonoporosis and optimization of the ultrasonic acoustic energy mechanical index.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 516,488, filed July 28, 2023, U.S. Provisional Patent Application No. 63 / 592,106, filed October 20, 2023, and U.S. Provisional Patent Application No. 63 / 656,376, filed June 5, 2024, each of which is incorporated herein by reference in its entirety and 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 suffer from significant drawbacks, such as low transfection rates and insufficient gene expression, which hinder the clinical development and commercialization of these methods. 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] In ultrasound therapy, the mechanical index (MI) is a unitless number that measures the power of an ultrasound beam and its potential to induce biological effects in tissue. Sonopore formation protocols for gene therapy products typically seek to maximize nucleic acid delivery to cells while minimizing the ultrasound energy applied to the cells, particularly by maintaining ultrasound application with a low mechanical index to improve the safety and tolerability of the cellular procedure. It has been demonstrated that applying ultrasound with a high mechanical index can induce tissue damage in some cases, resulting from uncontrolled cavitation, inflammatory responses, and vascular injury in the target tissue, often failing to achieve the goals of ultrasound therapy. This disclosure provides a method for optimizing nucleic acid payload delivery to cells using ultrasound protocols with an elevated mechanical index, where the elevated mechanical index ultrasound remains safe while significantly increasing nucleic acid payload delivery to target cells. As described herein, applying ultrasound using an alternating mechanical index protocol at an elevated mechanical index induces stable vibration and inertial cavitation of the acoustic agent applied to the subject and results in increased delivery of nucleic acid payload to target cells without significantly reducing the safety and tolerability of the procedure within the tissue.

[0004] The aspects disclosed herein provide a method for delivering a nucleic acid payload to target cells of a subject, the method comprising: administering a nucleic acid construct containing the nucleic acid payload to the subject; administering a acoustic agent to the subject; 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 1.3 and up to 2.9. The aspects disclosed herein provide a method for delivering a nucleic acid payload to target cells of a subject, the method comprising: administering a nucleic acid construct containing the nucleic acid payload to the subject; administering a acoustic agent to the subject; 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 of at least 2.0. This document discloses a method for delivering a nucleic acid payload to target cells of a subject, the method comprising: administering to the subject a nucleic acid construct containing the nucleic acid payload, wherein the nucleic acid construct is a microplasmid; administering to the subject a acoustic active agent; applying ultrasonic acoustic energy to the target cells at a first mechanical index (MI) of up to 0.4; and applying ultrasonic acoustic energy to the target cells at a second MI greater than 0.4 and up to 2.3. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI greater than 1.5 and up to 2.9. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI greater than 1.8 and up to 2.9. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI of at least 2.0. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI of at least 2.2. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI of at least 2.4. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI of at least 2.6. In some embodiments, ultrasonic acoustic energy is applied to the target cells at a second MI of at least 2.9. In some embodiments, ultrasonic energy is applied to target cells at a second MI greater than 2.2 and at most 2.9. In some embodiments, ultrasonic energy is applied to target cells at a second MI greater than 2.6 and at most 2.9. In some embodiments, an ultrasonic transducer applying the ultrasonic energy to the target cells is in continuous contact with the subject's tissue and continuously (1) applies ultrasonic 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 the expression cassette. In some embodiments, the nucleic acid construct is administered systemically. In some embodiments, the application of ultrasonic energy at the first MI and the application of ultrasonic energy at 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 application of ultrasonic energy at the first MI and the application of ultrasonic energy at the second MI are repeated 9 times. In some embodiments, the ultrasonic transducer is in continuous contact with the subject during the application of ultrasonic energy at the first MI and the second MI. 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, d. applying the ultrasonic energy includes applying the ultrasonic energy at the second MI using a pulse. In some embodiments, applying the ultrasonic energy at the second MI includes applying the ultrasonic energy at the second MI using a pulse with a duration of about 1 μs to about 500 µs. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 100 μs to about 3300 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 1 μs to about 3300 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 200 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of up to 200 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of up to 500 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 1 μs to about 200 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 1 μs to about 5 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 2.3 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of at least 2.3 µs with the second MI.In some embodiments, applying the ultrasonic energy at the first MI includes initially applying the ultrasonic energy at the first MI for about 2 s to about 30 s. In some embodiments, the method includes repeatedly applying the ultrasonic energy at the first MI and applying 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 acoustic agent 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 repeatedly applying 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. In some embodiments, the total duration of application of the ultrasonic energy in (c) and (d) ranges from about 1 s to about 60 m. In some embodiments, the total duration of the ultrasonic energy applied in (c) and (d) ranges from about 60 s to about 120 s. In some embodiments, the first MI ranges from about 0.05 to about 0.3. In some embodiments, the first MI ranges from about 0.09 to about 0.3. In some embodiments, the second MI ranges from about 1.0 to about 1.8. In some embodiments, the second MI ranges from about 1.4 to about 1.8. In some embodiments, the second MI ranges from about 1.4 to about 2.0. In some embodiments, the nucleic acid construct is a circular nucleic acid. In some embodiments, the nucleic acid construct is a microplasmid. In some embodiments, the microplasmid contains fewer than 500 base pairs and does not include an expression cassette. In some embodiments, the microplasmid does not contain an antibiotic resistance gene. In some embodiments, the microplasmid does not contain a bacterial genome. In some embodiments, the nucleic acid construct enhances the expression of non-endogenous genes. In some embodiments, the method induces the expression of the nucleic acid payload in the target cells within 20 hours of applying the ultrasonic energy. In some embodiments, the nucleic acid construct is used for gene enhancement, gene substitution, base editing, base knockdown, gene editing, gene knockdown, or gene deletion. In some embodiments, the nucleic acid construct is used to enhance in vivo stability. In some embodiments, the nucleic acid construct is administered at a dose of about 100 μg to about 200 μg. In some embodiments, the nucleic acid construct is administered at a dose of about 0.5 mg / kg to about 32 mg / kg.In some embodiments, approximately 2 × 10^13 to approximately 3 × 10^13 copies of the nucleic acid construct are administered to the subject. In some embodiments, the microplasmid contains a therapeutic transgene and / or regulatory element. In some embodiments, ultrasonic energy applied with the first MI induces stable vibrational cavitation of the acoustic agent. In some embodiments, ultrasonic energy applied with the first MI does not induce substantial destruction of the acoustic agent (e.g., bursting or inertial cavitation). In some embodiments, ultrasonic energy applied with the first MI does not induce substantial destruction of the acoustic agent in the vascular space and extravascular space, or induces stable vibrational cavitation of the acoustic agent in the vascular space and extravascular space. In some embodiments, ultrasonic energy applied with the second MI induces inertial cavitation of the acoustic agent to destroy the acoustic agent. In some embodiments, ultrasonic energy applied with the second MI induces inertial cavitation of the acoustic agent to destroy the acoustic agent in the vascular space and extravascular space. In some embodiments, the extravascular space includes interstitial space, subcutaneous space, intramuscular interosseous space, or lymphatic space. In some embodiments, the extravascular space includes extravascular tissue. In some embodiments, the extravascular tissue includes interstitial space, cytoplasmic space, subcutaneous tissue, lymphatic tissue, muscle, or a combination thereof. In some embodiments, the method does not cause substantial cellular damage to the target cells. In some embodiments, the method causes less than 1%, 5%, or 10% of the target cells to undergo apoptosis. In some embodiments, after (a)-(d), the following biomarkers of cell damage are not detected at apoptotic levels: ALT, AST, IL6, BCL2, or a combination thereof, and optionally, the target cells are located in the liver. In some embodiments, after (a)-(d), the following biomarkers of cell damage are not clinically elevated: ALT, AST, IL6, BCL2, or a combination thereof, and optionally, the target cells are located in the liver. In some implementations, ALT levels exceeding 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 U / L were not detected after (a)-(d). In some implementations, AST levels exceeding 225, 250, 275, or 300 U / L were not detected after (a)-(d). In some embodiments, IL6 levels exceeding 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, or 6 pg / mL were not detected after (a)-(d). In some embodiments, the target cells are located in the liver.In some embodiments, the target cells are located in the kidneys. In some embodiments, the target cells are located in the heart or skeletal muscle. In some embodiments, the target cells are located in the brain. In some embodiments, the target cells are located in the pancreas. In some embodiments, the target cells are located in a tumor or tumor cells. In some embodiments, the ultrasonic energy is applied with the first mechanical index to induce the formation of intercellular or interendothelial space. In some embodiments, the intercellular or interendothelial space ranges from about 10 nm to about 10 μm. In some embodiments, the method includes moving a nucleic acid construct from the intravenous space to the interstitial space. In some embodiments, the method includes moving a nucleic acid construct from the interstitial space to the intracellular space. In some embodiments, stable vibrational cavitation of the acoustic agent moves the nucleic acid construct from the intravenous space to the interstitial space. In some embodiments, inertial cavitation further moves the nucleic acid construct from the interstitial space to the intracellular space. In some embodiments, the ultrasonic energy is applied with the second mechanical index to induce the formation of pores in the cell membrane. In some embodiments, the pores in the cell membrane are formed in the range of about 10 nm to about 10 μm. In some embodiments, the nucleic acid payload comprises a transgene. In some embodiments, the transgene comprises a therapeutic transgene. In some embodiments, the transgene comprises a detectable biomarker. In some embodiments, the transgene comprises luciferase. In some embodiments, the nucleic acid construct comprises a promoter sequence containing CAG. In some embodiments, the nucleic acid construct comprises a promoter sequence containing ApoE. In some embodiments, the nucleic acid construct comprises a promoter sequence containing SERP. In some embodiments, the nucleic acid construct comprises a promoter sequence containing P3. In some embodiments, the method comprises inducing expression of the nucleic acid payload in the target cells. In some embodiments, inducing expression of the nucleic acid payload comprises inducing luciferase expression. In some embodiments, inducing expression of the nucleic acid payload comprises inducing a flux of 2, 3, 4, or 5 times that induced without repetitions (c) and (d). In some embodiments, inducing expression of the nucleic acid payload comprises inducing a flux of at least 10^6 p / s. In some embodiments, inducing the expression of the nucleic acid payload includes inducing a flux of about 10^6 p / s to about 10^9 p / s. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the production of RNA encoded by the payload. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the production of a protein encoded by the payload. In some embodiments, the acoustic agent is administered at a concentration of about 5 x 10^8 to about 1.2 x 10^9 microstructures / mL.In some embodiments, the acoustic active agent comprises sonazoid microvesicles. In some embodiments, the acoustic active agent comprises lipid-stabilized microstructures. In some embodiments, the acoustic active agent comprises phospholipid-stabilized microstructures. In some embodiments, the phospholipid-stabilized microstructure comprises a high-molecular-weight gas core or a perflutran core. In some embodiments, the acoustic active agent is administered at a concentration of about 10^9 microstructures / mL. In some embodiments, the acoustic active agent is administered at a concentration of about 0.1 to about 0.8 mL / kg. In some embodiments, the acoustic active agent is administered at a concentration of about 0.1 to about 20.0 mL / kg. In some embodiments, the acoustic active agent comprises protein-stabilized microstructures. In some embodiments, the acoustic active agent comprises optison microvesicles. In some embodiments, the acoustic active agent is administered at a concentration of about 5 x 10^8 to about 8 x 10^8 microstructures / mL. In some embodiments, the ultrasonic energy is applied at a distance of about 0.5 cm to about 20 cm from the target cell. In some embodiments, the nucleic acid construct and the acoustic active agent are administered together. In some embodiments, the nucleic acid construct and the acoustic active agent are mixed prior to co-administration. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent occurs continuously, in parallel, sequentially, or continuously. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent occurs continuously. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent occurs in parallel. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent occurs sequentially. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent occurs continuously. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent is by intravenous administration. In some embodiments, the administration of the nucleic acid construct and the acoustic active agent is by intramuscular, subcutaneous, interosseous, or postvesicular administration. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 3 hours of administering the payload. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 6 hours of administering the payload. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 12 hours of administering the payload. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression in cells in the liver. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression in cells in the kidney. In some embodiments, the method includes inducing the expression of the nucleic acid payload and maintaining the expression of the protein encoded by the nucleic acid payload for at least 1, 2, 3, 4, 5, 6, or 7 days.In some embodiments, the method includes inducing the expression of the nucleic acid payload and maintaining the expression of the protein encoded by the nucleic acid payload for at least 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, 31, or 32 days. In some embodiments, the method increases the persistence of the expression of the protein encoded by the nucleic acid payload. In some embodiments, the method includes increasing the expression of the nucleic acid payload by increasing the dose of the nucleic acid payload administered to the subject. In some embodiments, the method includes increasing the expression of the nucleic acid payload by linearly increasing the dose of the nucleic acid payload administered to the subject. In some embodiments, the method includes increasing the expression of the nucleic acid payload by administering at least 5, 50, 250, or 500 μg of the nucleic acid payload to the subject. In some embodiments, delivering the nucleic acid payload to the target cells of the subject increases or decreases gene expression in the target cells. In some embodiments, delivering the nucleic acid payload to the target cells results in a copy number of at least 0.15 of the nucleic acid payload per diploid genome. In some embodiments, delivering the nucleic acid payload to the target cells results in a copy number of at least 0.2 of the nucleic acid payload per diploid genome. In some embodiments, delivering the nucleic acid payload to the target cells results in a copy number of 0.15 to 0.3 of the nucleic acid payload per diploid genome. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding FVIII. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding FIX. 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 therapeutic transgene comprises a nucleic acid sequence encoding PKD1. In some embodiments, the therapeutic transgene contains a nucleic acid sequence encoding PKD2. In some embodiments, delivering the nucleic acid payload to the target cells of the subject increases or decreases gene expression in the target cells.

[0005] The aspects disclosed herein provide a kit comprising: a first container containing microbubbles for sono-induced pore formation; and a second container containing a microplasmid containing a transgene. In some embodiments, the microplasmid further comprises an expression cassette. In some embodiments, the first and second containers are configured to induce expression of the transgene in the target cells of a subject within 20 hours post-transfection. In some embodiments, the kit further includes instructions for operating ultrasound hardware and software parameters sufficient to destroy the sono-active agent. In some embodiments, the kit further includes instructions for administering the first and second containers.

[0006] Aspects disclosed herein provide a 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 1.3 and up to 2.9 (e.g., 1.3 < MI ≤ 2.9), wherein the subject has been administered a nucleic acid construct comprising a nucleic acid payload and a sonosensitizer, and wherein the nucleic acid construct is a microplasmid. In some embodiments, the ultrasound transducer that applies the ultrasonic acoustic energy to the target cells is in continuous contact with the tissue of the subject 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 microplasmid. 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 twice. 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 9 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 spaces or endothelial intercellular spaces. 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 with 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 with a duration of up to 200 μs.In some embodiments, applying the ultrasonic energy in the second MI comprises applying the ultrasonic energy in the second MI using a pulse for a duration of up to 500 µs. In some embodiments, applying the ultrasonic energy in the second MI comprises applying the ultrasonic energy in the second MI using a pulse for a duration of about 1 µs to about 200 µs. In some embodiments, applying the ultrasonic energy in the second MI comprises applying the ultrasonic energy in 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 in the first MI and applying the ultrasonic energy in the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy in the first MI for a duration sufficient to allow reperfusion of the acoustic agent in the tissue containing the target cells. In some embodiments, the repetition comprises applying the ultrasonic energy in the first MI for 1-30 seconds before repeatedly applying the ultrasonic energy in the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy in the first MI for 5-15 seconds before applying the ultrasonic energy in the second MI. In some embodiments, the repetition includes applying the ultrasonic energy at the first MI for 10 seconds prior to applying the ultrasonic energy at the second MI. A aspect disclosed herein provides a computer-readable medium configured to perform a method of applying ultrasonic energy to target cells of a subject, the method comprising: applying ultrasonic energy to the target cells at a first mechanical index (MI) of up to 0.4; and applying ultrasonic energy to the target cells at a second MI greater than 1.3 and up to 2.9, 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 sonoactive agent. In some embodiments, an ultrasonic transducer applying the ultrasonic energy to the target cells is in continuous contact with the subject's tissue and continuously (1) applies ultrasonic 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 energy at the first MI and the application of the ultrasonic energy at 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 at least 9 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 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 range of the second MI is 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 energy at the second MI includes applying the ultrasonic energy at the second MI using a pulse for a duration of from about 1 μs to about 500 µs. 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 up to 200 µs. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of up to 500 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 1 µs to about 200 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse for a duration of about 2.3 µs with the second MI. In some embodiments, the specification includes repeating the application of the ultrasonic energy with the first MI and the application of the ultrasonic energy with the second MI. In some embodiments, the repetition includes applying the ultrasonic energy with the first MI for a duration sufficient to allow reperfusion of the acoustic agent in the tissue containing the target cells. In some embodiments, the repetition includes applying the ultrasonic energy with the first MI for 1-30 seconds before repeating the application of the ultrasonic energy with the second MI. In some embodiments, the repetition includes applying the ultrasonic energy with the first MI for 5-15 seconds before applying the ultrasonic energy with 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. Attached Figure Description

[0007] The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the invention, and in the accompanying drawings: Figures 1A-1D The vector map of the exemplary nucleic acid construct used in this disclosure is shown.

[0008] Figure 2 The results of nucleic acid transfection and expression, as measured by the mean radiance of IVIS, are illustrated compared to the control, with fluorescence observed in mouse liver resulting from gene transfection and expression.

[0009] Figure 3A and Figure 3B The results of nucleic acid transfection and expression, as measured by the mean radiance of IVIS, are illustrated compared to the control, with fluorescence observed in mouse liver resulting from gene transfection and expression.

[0010] Figure 4 Examples of nucleic acid transfection and expression results measured by IVIS mean radiance using different high MI pulse bursts are illustrated, with fluorescence observed in mouse liver resulting from gene transfection and expression.

[0011] Figures 5A-5C Nucleic acid transfection and expression results were illustrated using different high MI pulse trains and IVIS mean radiance measurements, with fluorescence observed in mouse livers resulting from gene transfection and expression.

[0012] Figures 6A-6C Examples of nucleic acid transfection and expression results using different doses of luciferase nanoparticles (5 µg, 50 µg, 250 µg, or 500 µg) are illustrated, with IVIS mean radiance measurements showing fluorescence generated by gene transfection and expression observed in mouse liver.

[0013] Figure 7 The results of nucleic acid transfection and expression, measured by mean IVIS irradiance, are illustrated at 3, 6, 12, 18, 24, and 30 hours after delivery of luciferase microplasmids via sonopore formation in four different subjects.

[0014] Figures 8A-8C The levels of biomarkers in mice transfected using sonoporosis are illustrated.

[0015] Figure 9 The weight of the transfected mice is shown in the figure. The mice were weighed daily for one week after transfection.

[0016] Figure 10 The mean radiance of the fluorescent reporter factor is illustrated in the 500 μg dose cohort over 7 days.

[0017] Figure 11 Examples are shown of the levels of exogenous gene expression in hepatocytes after delivery of nucleic acid payloads via sonoforming pores using different vectors, as measured by quantitative polymerase chain reaction (qPCR).

[0018] Figure 12 An exemplary ultrasonic transducer system is illustrated, the computer processor having a computer-readable medium storing instructions for implementing the methods of this disclosure.

[0019] Figure 13 The mean fluorescence emissivity measurement after sonoporous gene therapy, which delivers nucleic acids encoding fluorescent reporter genes in multiple organ systems, is illustrated.

[0020] Figure 14 Data on the copy number (CN / DG) of each diploid genome in the kidneys (left) and liver (right) of NHP are shown.

[0021] Figures 15A-15C The mean fluorescence emissivity measurement after sonoporous gene therapy, which delivers nucleic acids encoding fluorescent reporter genes in multiple organ systems, is illustrated. Detailed Implementation

[0022] In ultrasound therapy, the mechanical index (MI) is a unitless number that measures the power of an ultrasound beam and its potential to induce biological effects in tissue. Sonopore formation protocols for gene therapy products typically seek to maximize nucleic acid delivery to cells while minimizing the ultrasound energy applied to the cells, particularly by maintaining ultrasound application with a low mechanical index to improve the safety and tolerability of the cellular procedure. It has been demonstrated that applying ultrasound with a high mechanical index can induce tissue damage in some cases, resulting from uncontrolled cavitation, inflammatory responses, and vascular injury in the target tissue, often failing to achieve the goals of ultrasound therapy. This disclosure provides a method for optimizing nucleic acid payload delivery to cells using ultrasound protocols with an elevated mechanical index, where the elevated mechanical index ultrasound remains safe while significantly increasing nucleic acid payload delivery to target cells. As described herein, applying ultrasound using an alternating mechanical index protocol at an elevated mechanical index induces stable vibration and inertial cavitation of the acoustic agent applied to the subject and results in increased delivery of nucleic acid payload to target cells without significantly reducing the safety and tolerability of the procedure within the tissue.

[0023] 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 formation (e.g., processes involving the application of ultrasonic acoustic energy to cells, tissues, or organs, such as to provide increased porosity in those cells, tissues, or organs). In one aspect, this disclosure provides methods for delivering nucleic acid payloads to target cells by optimizing parameters or protocols of the applied ultrasonic acoustic energy, including methods for increasing or decreasing gene expression in target cells by applying ultrasonic acoustic energy with alternating mechanical indices to induce stable vibrational cavitation and inertial cavitation of the acoustic active agent. In some cases, the nucleic acid payload is a microplasmid, and delivery with alternating mechanical indices to induce stable vibrational cavitation and inertial cavitation of the acoustic active agent enhances the delivery of the microplasmid to the target cells.

[0024] In some embodiments herein, methods are provided for transfecting nucleic acid constructs into target cells or tissues (e.g., target cells or tissues of a subject) by applying a first ultrasonic energy to a cell, tissue, or organ and a second ultrasonic energy to the same cell, tissue, or organ. In specific embodiments herein, methods are provided for transfecting nucleic acid constructs into target cells or tissues by applying a first ultrasonic energy having a first mechanical index (MI) and a second ultrasonic energy having a second mechanical index (MI). This disclosure provides a method for enhancing the transfection of nucleic acid constructs into target cells or tissues by applying alternating ultrasonic energy, which alternates between a first mechanical index (MI) and a second MI. The application of ultrasonic energy can be repeated several times during sonoforming pore formation to increase the efficiency of nucleic acid construct transfection and / or delivery.

[0025] The aspects disclosed herein provide a method for delivering a nucleic acid payload to target cells of a subject, comprising: administering a nucleic acid construct containing the nucleic acid payload to the subject; administering a acoustic active agent to the subject; 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 1.3 and up to 2.9. In some embodiments, ultrasonic acoustic energy is applied to the target cells with a second MI greater than 1.5 and up to 2.9. In some embodiments, ultrasonic acoustic energy is applied to the target cells with a second MI greater than 1.8 and up to 2.9. In some embodiments, ultrasonic acoustic energy is applied to the target cells with a second MI of at least 2.0. In some embodiments, ultrasonic acoustic energy is applied to the target cells with a second MI of at least 2.2. In some embodiments, ultrasonic energy is applied to the target cells at a second MI of at least 2.4. In some embodiments, ultrasonic energy is applied to the target cells at a second MI of at least 2.6. In some embodiments, ultrasonic energy is applied to the target cells at a second MI of at least 2.9. In some embodiments, ultrasonic energy is applied to the target cells at a second MI greater than 2.2 and at most 2.9. In some embodiments, ultrasonic energy is applied to the target cells at a second MI greater than 2.6 and at most 2.9.

[0026] In some embodiments, the method provided herein provides acoustic apertureing with two or more different ultrasonic acoustic energies (e.g., a first ultrasonic acoustic energy and a second ultrasonic acoustic energy having a first MI and a second MI, respectively). In some embodiments, the method provided herein provides a method in which ultrasonic acoustic energy is continuously applied (e.g., the ultrasonic acoustic energy transitions from a first ultrasonic acoustic energy to a second ultrasonic acoustic energy without a period of not applying ultrasonic acoustic energy). In some embodiments, transient (e.g., a third, fourth, etc.) ultrasonic acoustic energy is applied between the application of the first and second ultrasonic acoustic energy.

[0027] In some embodiments, the acoustic aperture forming process (e.g., a single cycle of applying a first ultrasonic energy, a second ultrasonic energy, and a second ultrasonic energy, or a series of cycles including multiple applications of the first ultrasonic energy and multiple applications of the second ultrasonic energy) can last for several seconds (e.g., 1-100 seconds) or longer, such as up to several minutes (e.g., 1-3 minutes). In specific embodiments, the acoustic aperture forming process lasts for 1-30 seconds. In some specific embodiments, the acoustic aperture forming process lasts for 5-100 seconds. In some embodiments, the acoustic aperture forming process lasts for at least 1 minute (e.g., 1-30 minutes).

[0028] In some embodiments, the first MI is a low MI (e.g., less than 0.4). In some embodiments, the second MI is a high MI (e.g., 0.4 or greater). In some embodiments, the first MI is a low MI (e.g., less than 0.4), and the second MI is a high MI (e.g., 0.4 or greater). In some embodiments, the second MI is a low MI (e.g., less than 0.4), and the first MI is a high MI (e.g., 0.4 or greater).

[0029] In some implementations, the low MI is <0.3. In a particular implementation, the low MI is <0.2. In a more specific implementation, the low MI is <0.1. In an even more specific implementation, the low MI is about 0.09. In an even more specific implementation, the low MI is about 0.04. In an even more specific implementation, the low MI is about 0.03.

[0030] In some implementation schemes, high MI > 0.5. In a specific embodiment, the high MI is 0.5 to 2.0 or between 0.5 and 2.0. In a more specific embodiment, the high MI is 0.5 to 1 or between 0.5 and 2.0. In some embodiments, the high MI is 1.5. In some embodiments, the high MI is 1.8. In some embodiments, the high MI is 2.0. In some embodiments, the high MI is greater than 0.4. In some embodiments, the high MI... >0.5. In a more specific embodiment, the high MI is 0.5 to 1 or between 0.5 and 2.0. In some embodiments, the high MI is 1.5. In some embodiments, the high MI is 1.8. In some embodiments, the high MI is 2.0. In some embodiments, ultrasonic energy is applied to the target cells with a second MI greater than 1.3 and at most 2.9. In some embodiments, ultrasonic energy is applied to the target cells with a second MI greater than 1.8 and at most 2.9. In some embodiments, ultrasonic energy is applied to the target cells with a second MI of at least 2.0. In some embodiments, ultrasonic energy is applied to the target cells with a second MI of at least 2.2. In some embodiments, ultrasonic energy is applied to the target cells with a second MI of at least 2.4. In some embodiments, ultrasonic energy is applied to the target cells with a second MI of at least 2.6. In some embodiments, ultrasonic energy is applied to the target cells with a second MI of at least 2.9. In some embodiments, ultrasonic energy is applied to the target cells with a second MI greater than 2.2 and at most 2.9. In some implementations, ultrasonic energy is applied to the target cells at a second MI greater than 2.6 and at most 2.9.

[0031] In some embodiments, any method provided herein (e.g., acoustic aperture treatment) involves the application of continuous ultrasonic acoustic energy (which may have different energy levels) alternating between low MI and high MI (e.g., alternating with the same, similar, or variable period). In some embodiments, a low MI (e.g., < 0.1) (e.g., first) ultrasonic energy (also referred to herein as low MI), and a set number of pulses (e.g., less than 30 seconds) of high MI (e.g., second) ultrasonic energy (also referred to herein as high MI). In some embodiments, the method provided herein includes, for example, applying multiple pulses of high MI (e.g., second) ultrasonic energy during an additional continuous application of low MI (e.g., first) ultrasonic energy. In specific embodiments, the number of high MI pulses is about 4 or more, such as up to about 12, or an unlimited number of pulses. In specific embodiments, the number of high MI pulses is 6-30. In even more specific embodiments, the number of high MI pulses is between 8, 9, 12, 15, or 18, or any number therebetween. In some embodiments, at least 8, 9, 12, 15, or 18 high MI pulses are applied to the subject between applications of low MI ultrasonic energy.

[0032] In some embodiments, high-MI ultrasonic energy is applied in the form of pulses. In specific embodiments, the pulse length is any suitable length, such as less than 30 seconds. In more specific embodiments, the pulse length is less than 15 seconds. In even more specific embodiments, the pulse length is less than 10 seconds. In even more specific embodiments, the pulse length is less than 5 seconds. In even more specific embodiments, the pulse length is less than 2 seconds. In even more specific embodiments, the pulse length is less than 1 second and / or may be greater than or equal to 1 microsecond. In some embodiments, the pulse length ranges from 100 microseconds to 300 microseconds. In some embodiments, the pulse length is at most about 200 microseconds. In some embodiments, the pulse length is at most about 500 microseconds. In some embodiments, the pulse length ranges from 1 microsecond to 500 microseconds.

[0033] In various embodiments, high-MI ultrasonic energy is provided first in time (e.g., sequentially first). In other embodiments, low-MI ultrasonic energy is provided second in time (e.g., sequentially second).

[0034] In some implementations, any method provided herein may also include administering a nucleic acid (e.g., any nucleic acid provided herein) to a subject (e.g., systemic administration, such as via infusion) (e.g., a subject to which ultrasound energy has been applied).

[0035] In some embodiments, any method provided herein also includes administering (e.g., systemic administration, such as via infusion) an acoustically active structure (e.g., any acoustically active structure or microbubble described herein) to a subject (e.g., a subject to which ultrasound energy has been applied).

[0036] In some embodiments, this document provides a method for delivering a nucleic acid payload into target cells (e.g., target cells of a tissue or organ) of a subject, the method comprising: (a) administering a nucleic acid construct containing the nucleic acid payload to the subject; (b) administering a sonoactive agent to the subject; and (c) administering a sonopore-forming treatment.

[0037] In some embodiments, the acoustic pore-forming process includes applying ultrasonic acoustic energy (e.g., ultrasonic acoustic energy having a certain mechanical index (MI)) to target cells (e.g., target cells of a subject's tissue or organ). In some embodiments, applying ultrasonic acoustic energy to the target cells includes applying a first ultrasonic acoustic energy to the target cells and applying a second ultrasonic acoustic energy to the target cells. In some embodiments, (e.g., the first or the second) ultrasonic acoustic energy has a first mechanical index (MI). In some embodiments, (e.g., the first or the second) ultrasonic acoustic energy has a second mechanical index (MI). In some embodiments, (e.g., the first or the second) MI is less than 0.4. In some embodiments (e.g., the other of the first or the second) MI is greater than 0.4 (e.g., and less than 2.0).

[0038] In a specific implementation plan, the first ultrasonic acoustic energy and the second ultrasonic acoustic energy are applied sequentially in a repeated manner.

[0039] In some embodiments, a first (high MI or low MI) ultrasound energy is applied before or after the application of any other reagent (such as nucleic acid and / or acoustically active structure). In some embodiments, the first ultrasound energy is applied after the application of the acoustically active structure to the subject. In some embodiments, the first ultrasound energy is applied after the application of nucleic acid to the subject. In some embodiments, the first ultrasound energy is applied after the application of both nucleic acid and acoustically active structure.

[0040] In some embodiments, the first ultrasonic energy is applied within 60 minutes of the application of the nucleic acid and / or acoustically active structure. In a specific embodiment, the first ultrasonic energy is applied within 30 minutes of the application of the nucleic acid and / or acoustically active structure. In a more specific embodiment, the first ultrasonic energy is applied within 5 minutes of the application of the nucleic acid and / or acoustically active structure. In an even more specific embodiment, the first ultrasonic energy is applied within 2 minutes of the application of the nucleic acid and / or acoustically active structure. In an even more specific embodiment, the first ultrasonic energy may be applied simultaneously with the application of the nucleic acid and / or acoustically active structure.

[0041] In a specific implementation, a first (e.g., high MI) ultrasonic energy is applied immediately after the application (e.g., infusion) of the acoustically active structure and / or nucleic acid, or some time after the application (e.g., infusion) of the acoustically active structure and / or nucleic acid.

[0042] In some embodiments, the first or second ultrasonic energy is ultrasonic energy (e.g., low MI) that causes stable cavitation (or stable vibrational cavitation) of the acoustically active structure and / or a change in the average diameter of the acoustically active structure (e.g., due to the inherent resonant properties of microbubbles) when applied to the cells, tissues, or organs of a subject.

[0043] In some embodiments, the first or second ultrasonic energy is ultrasonic energy (e.g., high MI) that causes inertial cavitation or collapse of acoustically active structures and / or disruption of cell membrane and / or vascular endothelial integrity when applied to the cells, tissues or organs of a subject.

[0044] In some embodiments, the first or second ultrasonic energy is ultrasonic energy that causes stable cavitation (or stable vibrational cavitation) and / or a change in mean diameter of an acoustically active structure when applied to the cells, tissues, or organs of the subject (e.g., low MI), and the other of the first or second ultrasonic energy is ultrasonic energy that causes inertial cavitation or collapse of an acoustically active structure and / or disruption of the integrity of the cell membrane and / or vascular endothelium when applied to the cells, tissues, or organs of the subject (e.g., high MI).

[0045] In some cases, disruption of the cell membrane allows target cells to become permeable to circulating agents such as nucleic acid constructs. In certain situations, such circulating agents can then enter target cells, tissues, or organs, such as in a more rapid manner (e.g., relative to the application of low-MI or high-MI ultrasound energy alone, or in the absence of ultrasound energy application).

[0046] In some embodiments, the method herein includes alternating between applied ultrasound energy and a first ultrasound energy having a first MI and a second ultrasound energy having a second MI. In some embodiments, the alternating ultrasound energy applied to the subject is repeated multiple times between the first MI and the second MI, such as to enhance gene transfection in target cells, tissues, or organs (e.g., relative to similar methods in which the first and / or second ultrasound energies are not used and / or not alternately applied and / or not repeatedly alternately applied).

[0047] In some implementations, applying ultrasonic acoustic energy includes applying ultrasonic acoustic energy at a frequency of about 1 MHz to about 10 MHz. In some embodiments, the applied ultrasonic acoustic energy includes frequencies from about 1 MHz to about 1.1 MHz, from about 1 MHz to about 1.5 MHz, from about 1 MHz to about 2 MHz, from about 1 MHz to about 3 MHz, from about 1 MHz to about 4 MHz, from about 1 MHz to about 5 MHz, from about 1 MHz to about 7 MHz, from about 1 MHz to about 8 MHz, from about 1 MHz to about 9 MHz, from about 1 MHz to about 9.3 MHz, from about 1 MHz to about 10 MHz, from about 1.1 MHz to about 1.5 MHz, from about 1.1 MHz to about 2 MHz, from about 1.1 MHz to about 3 MHz, from about 1.1 MHz to about 4 MHz, from about 1.1 MHz to about 5 MHz, from about 1.1 MHz to about 7 MHz, from about 1.1 MHz to about 8 MHz, from about 1.1 MHz to about 9 MHz, from about 1.1 MHz to about 9.3 MHz, from about 1.1 MHz to about 10 MHz, from about 1.5 MHz to about 2 MHz, and from about 1.5 MHz to about 1.5 MHz. MHz to 3 MHz, about 1.5 MHz to 4 MHz, about 1.5 MHz to 5 MHz, about 1.5 MHz to 7 MHz, about 1.5 MHz to 8 MHz, about 1.5 MHz to 9 MHz, about 1.5 MHz to 9.3 MHz, about 1.5 MHz to 10 MHz, about 2 MHz to 3 MHz, about 2 MHz to 4 MHz, about 2 MHz to 5 MHz, about 2 MHz to 7 MHz, about 2 MHz to 8 MHz, about 2 MHz to 9 MHz, about 2 MHz to 9.3 MHz, about 2 MHz to 10 MHz, about 3 MHz to 4 MHz, about 3 MHz to 5 MHz, about 3 MHz to 7 MHz, about 3 MHz to 8 MHz, about 3 MHz to 9 MHz, about 3 MHz to 9.3 MHz, about 3 MHz to 10 MHz, about 4 MHz to 5 MHz, about 4 MHz to 7 MHz MHz, approximately 4 MHz to approximately 8 MHz, approximately 4 MHz to approximately 9 MHz, approximately 4 MHz to approximately 9.3 MHz, approximately 4 MHz to approximately 10 MHz, approximately 5 MHz to approximately 7 MHz, approximately 5 MHz to approximately 8 MHz, approximately 5 MHz to approximately 9 MHz, approximately 5 MHz to approximately 9.3 MHz, approximately 5 MHz to approximately 10 MHz, approximately 7 MHz to approximately 8 MHz, approximately 7 MHz to approximately 9 MHz, approximately 7 MHz to approximately 9.3 MHz, approximately 7 MHz to approximately 10 MHz, approximately 8 MHz to approximately 9 MHz, approximately 8 MHz to approximately 9.The ultrasonic acoustic energy can be applied at frequencies ranging from 3 MHz, about 8 MHz to about 10 MHz, about 9 MHz to about 9.3 MHz, about 9 MHz to about 10 MHz, or about 9.3 MHz to about 10 MHz (inclusive). In some embodiments, applying ultrasonic acoustic energy includes applying ultrasonic acoustic energy at frequencies of about 1 MHz, about 1.1 MHz, about 1.5 MHz, about 2 MHz, about 3 MHz, about 4 MHz, about 5 MHz, about 7 MHz, about 8 MHz, about 9 MHz, about 9.3 MHz, or about 10 MHz. In some embodiments, applying ultrasonic acoustic energy includes applying ultrasonic acoustic energy at frequencies of at least about 1 MHz, at least about 1.1 MHz, at least about 1.5 MHz, at least about 2 MHz, at least about 3 MHz, at least about 4 MHz, at least about 5 MHz, at least about 7 MHz, at least about 8 MHz, at least about 9 MHz, or at least about 9.3 MHz. In some embodiments, applying ultrasonic acoustic energy includes applying ultrasonic acoustic energy at frequencies of up to about 1.1 MHz, up to about 1.5 MHz, up to about 2 MHz, up to about 3 MHz, up to about 4 MHz, up to about 5 MHz, up to about 7 MHz, up to about 8 MHz, up to about 9 MHz, up to about 9.3 MHz, or up to about 10 MHz. In some embodiments, the first ultrasonic acoustic energy and the second ultrasonic acoustic energy are applied at the same frequency. In some embodiments, the first ultrasonic acoustic energy applied at a first MI and the second ultrasonic acoustic energy applied at a second MI are applied at the same frequency. In some embodiments, the first ultrasonic acoustic energy and the second ultrasonic acoustic energy are applied at different frequencies. In some embodiments, the first ultrasonic acoustic energy applied at a first MI and the second ultrasonic acoustic energy applied at a second MI are applied at different frequencies.

[0048] 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.

[0049] 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.

[0050] 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. 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.

[0051] In some embodiments, the cells, tissues, or organs are those of the liver. In some embodiments, the cells, tissues, or organs are those of the kidney.

[0052] In some embodiments, the subjects of this study are mammals. In some embodiments, mammals, as non-limiting examples, are humans, rats, mice, monkeys, and other non-human primates.

[0053] In some embodiments, the parameters of the ultrasonic energy or the intermittent oscillation (MI) can be varied to induce and / or enhance transgene expression in the cells or organs of the subject. In one aspect, this document provides a method for transfection by alternating ultrasonic energy using a first MI and a second MI. In some embodiments, the first MI, which induces stable vibrational cavitation, is applied before the second MI, which induces inertial cavitation. In some embodiments, the ultrasonic energy using the first and second MIs is applied multiple times to increase transfection efficiency at the target cells. In some embodiments, during the application of sono-oscillation pore formation, the ultrasonic energy is continuously applied with the first MI, except when the ultrasonic energy is applied with the second MI. For example, ultrasonic energy is applied to the target cells with the first MI, then with the second MI, repeated 4 to 18 times. In some embodiments, ultrasonic energy is applied to the target cells with the first MI, then with the second MI, repeated an unlimited number of times. In one aspect, during this period, the ultrasonic energy of the first MI is continuously applied except when the ultrasonic energy of the second MI is applied.

[0054] In some embodiments, the first MI ranges from about 0.05 to about 0.4. In some embodiments, the first MI ranges from about 0.05 to about 0.3. In some embodiments, the first MI ranges from about 0.05 to about 0.4. In some embodiments, the first MI ranges from about 0.09 to about 0.3.

[0055] In some embodiments, the second MI ranges from about 0.5 to about 2.0. In some embodiments, the second MI ranges from greater than 1.4 to about 1.8. In some embodiments, the second MI ranges from greater than 1.4 to about 2.0. In some embodiments, the second MI ranges from about 1.5 to about 2.0.

[0056] 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 application of ultrasonic energy at the first MI and the application of ultrasonic energy at the second MI are repeated 8 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 9 times.

[0057] In some embodiments, applying ultrasonic energy includes applying ultrasonic energy c. or d. without stopping the application of ultrasonic energy c. or d. In some embodiments, applying ultrasonic energy includes applying ultrasonic energy c. in addition to applying ultrasonic energy d. In some embodiments, the ultrasonic probe applying ultrasonic energy is in constant contact with the surface of the subject's skin at the application site (e.g., abdomen, chest wall, skull, etc.). In some embodiments, the ultrasonic transducer applying the ultrasonic energy to the target cells is in continuous contact with the subject's tissue and continuously (1) applies ultrasonic energy to the subject or (2) receives reflected ultrasonic energy from the subject. In some embodiments, transient (e.g., third, fourth, etc.) ultrasonic energy is applied between the application of the first and second ultrasonic energy. In some embodiments, applying ultrasonic energy includes applying ultrasonic energy without considering an EKG gating signal that modulates the application of ultrasonic energy. In some embodiments, applying ultrasonic energy includes applying ultrasonic energy without turning off the power to the ultrasonic transducer. In some implementations, applying ultrasonic energy includes the ultrasonic transducer transmitting or receiving reflected ultrasonic energy for at least 95% of the time period during which the ultrasonic transducer is in continuous contact with the subject.

[0058] In some cases, a second MI (e.g., high MI) of ultrasonic acoustic energy is applied using pulses. In some cases, the pulses include applying ultrasonic acoustic energy in short pulses (e.g., microsecond-length pulses). In some cases, high MI is applied in pulses, causing inertial cavitation and disruption of acoustically active microstructures, resulting in disruption of cell membrane and vascular endothelial integrity, and transducing nucleic acid payloads into cells. In some cases, the pulses are applied for a duration of about 1 μs to about 200 μs. In some cases, the pulses are applied for a duration of about 1 μs to about 200 μs or longer.

[0059] In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI. In some cases, the duration of the applied second MI ranges from 0.1 μs to about 200 μs. In some cases, the duration of the applied second MI ranges from 1 μs to about 200 μs or longer. In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI for a duration of about 1 μs to about 200 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI for a duration of up to 200 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI for a duration of about 1 μs to about 500 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI for a duration of up to 500 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI for a duration of about 2.3 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy with a pulse at the second MI for a duration of at least 2.3 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy to the second MI using a pulse with a duration ranging from 1 to 500 µs. In some embodiments, (d) includes applying ultrasonic acoustic energy to the second MI using a pulse with a duration ranging from 0.1 to 500 µs.

[0060] In some cases, alternating ultrasound energy multiple times between the first and second MIs also allows for the reperfusion of the acoustic active agent and nucleic acid construct to the target cells, tissues, or organs after the acoustic active agent has been destroyed in or near the target cells, tissues, or organs.

[0061] In some implementations, the repeated application of ultrasonic energy between the first MI and the second MI includes applying ultrasonic energy (c) before applying ultrasonic energy (d) again, for a duration sufficient to allow the acoustic active agent to be reperfused in the tissue containing the target cells.

[0062] In some embodiments, the method includes applying ultrasonic energy (c) for 1-30 seconds before repeating (d) the application of ultrasonic energy. In some embodiments, the method includes applying ultrasonic energy (c) for 5-15 seconds before repeating (d) the application of ultrasonic energy. In some embodiments, the method includes applying ultrasonic energy (c) for 10 seconds before repeating (d) the application of ultrasonic energy.

[0063] In some cases, the duration of the first MI applied ranges from about 2 s to about 30 s. In some embodiments, (c) includes initially applying ultrasonic acoustic energy for about 2 s to about 30 s with the first MI.

[0064] In some embodiments, the total duration of repeating the application of ultrasonic energy at the first MI and the second MI ranges from about 1 s to about 60 m. In some embodiments, the total duration of repeating the application of ultrasonic energy at the first MI and the second MI ranges from about 60 s to about 120 s.

[0065] In some embodiments, applying ultrasonic energy in (c) induces stable vibrational cavitation of the acoustic active agent. In some embodiments, applying ultrasonic energy in (c) does not induce substantial destruction of the acoustic active agent. In some embodiments, applying ultrasonic energy in (c) does not induce substantial destruction of the acoustic active agent in the vascular system space and extravascular space, or induces stable vibrational cavitation of the acoustic active agent in the vascular system space and extravascular space.

[0066] In some embodiments, (c) the formation or endocytosis of intercellular or interendothelial space is induced. In some embodiments, the intercellular or interendothelial space is in the range of about 10 nm to about 10 μm. In some embodiments, the stable vibrational cavitation of the acoustic agent moves the nucleic acid construct from the intravenous space to the interstitial space or cytoplasm.

[0067] In some embodiments, ultrasonic energy is applied in (d) to induce inertial cavitation of the acoustic active agent, thereby destroying the acoustic active agent. In some embodiments, ultrasonic energy is applied in (d) to induce inertial cavitation of the acoustic active agent, thereby destroying the acoustic active agent in the vascular system space and the extravascular space. In some embodiments, the extravascular space includes interstitial space, subcutaneous space, intramuscular space, or lymphatic space. In some embodiments, the extravascular space includes extravascular tissue. In some embodiments, the extravascular tissue includes interstitial space, cytoplasmic space, subcutaneous tissue, lymphatic tissue, muscle, or a combination thereof.

[0068] In some embodiments, (d) the application of ultrasonic energy induces the formation of pores in the cell membrane. In some embodiments, the pores in the cell membrane range from about 10 nm to about 10 μm.

[0069] In some implementations, the administration of the acoustic active agent and the nucleic acid construct occurs simultaneously, wherein the acoustic active agent is mixed with a solution containing the nucleic acid construct before delivery to the subject. Such a mixture may contain 50% v / v of acoustic active agent (e.g., Optison acoustic active microbubbles) and 50% v / v of a solution containing the nucleic acid construct. Such mixtures may contain varying percentages of acoustic active agent, ranging from 5% to 90% v / v.

[0070] In some embodiments, the nucleic acid construct comprises a microplasmid backbone. As used herein, the term "microplasmid (mpDNA)" refers to a nucleic acid construct that is smaller in size than a conventional plasmid or pDNA (i.e., contains fewer base pairs (bp)). In some embodiments, the mpDNA construct comprises 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 comprises a backbone of less than 0.5 kb. 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 having a plasmid backbone of less than 500 bp and not containing an antibiotic resistance gene.

[0071] Microplasmid DNA nucleic acid 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 microplasmid 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 microplasmid does not contain an antibiotic resistance gene. In some embodiments, the microplasmid does not contain a bacterial genome. In some embodiments, the microplasmid contains a therapeutic transgene and / or regulatory element. In some embodiments, the microplasmid is a nanoplasmid. 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 implementations, the persistence of protein expression encoded by a nucleic acid payload can be increased relative to the expression of the same protein in another nucleic acid construct.

[0072] 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).

[0073] In some embodiments, the nucleic acid payload includes an expression cassette. In some embodiments, the expression cassette includes a transgene. In some embodiments, the nucleic acid payload includes a transgene (endogenous or non-endogenous). In some embodiments, the transgene includes a therapeutic transgene. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the expression of a therapeutic transgene. In some embodiments, the transgene includes a detectable biomarker. In some embodiments, the transgene includes luciferase. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the expression of luciferase.

[0074] In some embodiments, the nucleic acid payload includes regulatory elements, such as promoters (e.g., APOE-ATT). In some embodiments, the total amount (e.g., dose) of DNA administered to the subject for sonoporous formation purposes can range from 100 micrograms to 200 mg.

[0075] In some implementations, the therapeutic payload is a non-endogenous gene. In some implementations, the nucleic acid payload is constructed to perform gene enhancement, gene replacement, gene editing, gene knockdown, or gene deletion.

[0076] In some embodiments, the nucleic acid construct includes one or more regulatory elements, such as promoters, enhancers, ribosome binding sites, or transcription termination signals. Examples of promoters considered herein include, but are not limited to, the CMV promoter, UbC promoter, CAG promoter, EF-1α promoter, ApoE promoter, ApoE-AAT1 promoter, 3XSERP promoter, or P3-heterozygous promoter. In some embodiments, the nucleic acid construct includes a CAG-containing promoter sequence. In some embodiments, the nucleic acid construct includes an ApoE-containing promoter sequence. In some embodiments, the nucleic acid construct includes a SERP-containing promoter sequence. In some embodiments, the nucleic acid construct includes a P3-containing promoter sequence.

[0077] In some embodiments, inducing the expression of the nucleic acid payload includes inducing the production of RNA encoded by the payload. In some embodiments, inducing the expression of the nucleic acid payload includes inducing the production of a protein encoded by the payload.

[0078] In some embodiments, the payload 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). In some embodiments, the nucleic acid construct is designed for gene enhancement, gene substitution, base editing, base knockdown, gene editing, gene knockdown, or gene knockout. In some embodiments, delivering the nucleic acid payload to the target cells of the subject increases or decreases gene expression in the target cells.

[0079] In some embodiments, the payload 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 a 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).

[0080] The acoustic active agents considered herein (also referred to as acoustic active microstructures, acoustic microspheres, or “microbubbles”) include, but are not limited to, those used as contrast agents in ultrasound imaging. In some embodiments, the acoustic active agent comprises phospholipid-stabilized microstructures. In some embodiments, the phospholipid-stabilized microstructure comprises a high-molecular-weight gas core or a perflutran core. Examples of acoustic active agents 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 is LUMASON (Bracco) (sulfur hexafluoride lipid-type A microspheres). In some embodiments, the acoustic active agent is SonoVue (sulfur hexafluoride microbubbles). In some embodiments, the acoustic active agent comprises protein-stabilized microstructures. In some embodiments, the acoustic active agent is Optison microbubbles.

[0081] The acoustic active agent can be applied before, after, or simultaneously with the nucleic acid construct (or nucleic acid payload) (e.g., co-application). In some embodiments, the nucleic acid construct and the acoustic active agent are co-application. In some embodiments, the application of the nucleic acid construct and the acoustic active agent occurs continuously, in parallel, sequentially, or continuously. In some embodiments, the application of the nucleic acid construct and the acoustic active agent occurs continuously. In some embodiments, the application of the nucleic acid construct and the acoustic active agent occurs in parallel. In some embodiments, the application of the nucleic acid construct and the acoustic active agent occurs sequentially. In some embodiments, the application of the nucleic acid construct and the acoustic active agent occurs continuously.

[0082] In some embodiments, the nucleic acid construct is administered at a dose of about 0.5 mg / kg to about 500 mg / kg. In some embodiments, about 2 × 10^13 to about 3 × 10^13 copies of the nucleic acid construct are administered to the subject. In some embodiments, each nucleic acid construct contains a copy of a transgene.

[0083] As used herein, the concentration of microstructures per mL refers to the concentration of the acoustic active agent in the pharmaceutical composition prior to administration to a subject. In some embodiments, the acoustic active agent is administered at a concentration of about 5 x 10^8 to about 1.2 x 10^10 microstructures per mL. In some embodiments, the acoustic active agent 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 agent may be administered at a concentration of about 5 M (millions) to about 8 M microstructures per mL. In some embodiments, 1 x 10^9 phospholipid-stabilized acoustic active agent (e.g., Sonazoid) is administered. In some embodiments, the phospholipid-stabilized acoustic active agent (e.g., Sonazoid) has a diameter of 1-5 micrometers. In some embodiments, the acoustic active agent is administered at a concentration of about 0.1 to about 0.8 mg / kg. In some embodiments, the acoustic active agent is applied at a concentration of about 0.1 to about 1.0 mL / kg. In some embodiments, the acoustic active agent is applied at a concentration of about 10^9 microstructures / mL. In some embodiments, the acoustic active agent is applied at a concentration of at least 5 x 10^8 microstructures / mL. In some embodiments, the acoustic active agent is applied at a concentration of up to 1.2 x 10^10 microstructures / mL. In some embodiments, the acoustic active agent is applied at a concentration of 5 x 10^8 to 8 x 10^8 microstructures / mL.

[0084] In some embodiments, the nucleic acid construct and the acoustic active agent are mixed before co-administration. In some cases, the acoustic active agent is mixed with the nucleic acid construct before administration to the subject. In some cases, the acoustic active agent is mixed with the nucleic acid construct and additional buffers or reagents (such as saline or other biocompatible solutions with different electrostatic charges and surface chemical structures and ligands) before administration to the subject. For example, Optison acoustic active microstructures can be mixed with nanoparticles containing APOE-Fluc and saline and administered together.

[0085] In some embodiments, the administration of the nucleic acid construct and the sonoactive agent is performed via intravenous, subcutaneous, intramuscular, intra-arterial, interosseous, or direct organ puncture.

[0086] In some implementations, ultrasonic energy is applied to target cells, tissues, or organs after the nucleic acid construct and acoustic agent are administered.

[0087] Once the nucleic acid construct is within the target cells, expression of the nucleic acid payload is induced. In some embodiments, the nucleic acid payload comprises a luciferase. In some embodiments, inducing expression of the nucleic acid payload using the microplasmid construct includes inducing expression to induce an average radiance of at least 2 x 10^4 p / sec / cm^2 / sr. In some embodiments, inducing expression of the nucleic acid payload includes inducing an average radiance of approximately 2 x 10^4 p / sec / cm^2 / sr to approximately 5 x 10^5 p / sec / cm^2 / sr.

[0088] In some embodiments, inducing the expression of the nucleic acid payload includes inducing a throughput of at least 10^6 p / s. In some embodiments, inducing the expression of the nucleic acid payload includes inducing a throughput of about 10^6 p / s to about 10^9 p / s.

[0089] In some implementations, inducing the expression of the nucleic acid payload includes inducing an expression at a flux of 2, 3, 4, or 5 times that induced without repeated application of the ultrasonic energy at the first MI and the second MI.

[0090] In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 3 to approximately 12 hours after administration of the payload. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 3 hours after administration. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 6 hours after administration. In some embodiments, inducing the expression of the nucleic acid payload includes inducing expression within approximately 12 hours after administration.

[0091] Adverse effects on living cells or tissues may occur due to the application of ultrasound. In some embodiments, this disclosure provides a method for improving gene transfection using sonoforming pore formation by alternating ultrasound energy between a first MI and a second MI, without causing substantial DNA or cellular damage in target cells, tissues, or organs. In some embodiments, the method does not cause substantial cellular damage to target cells. In some embodiments, the method induces apoptosis in less than 1%, 5%, or 10% of target cells.

[0092] Cell damage can be detected using apoptosis biomarkers. 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 include interleukin-6 (IL6) or B-cell lymphoma 2 (BCL2 or BCL2 apoptosis regulator). In some embodiments, after delivery of the nucleic acid payload to the subject's target cells, the following biomarkers of cell damage are not detected at apoptotic levels: ALT, AST, IL6, BCL2, or combinations thereof. In some embodiments, after delivery of the nucleic acid payload to the subject's target cells, the following biomarkers of cell damage are not clinically elevated: ALT, AST, IL6, BCL2, or combinations thereof. In some embodiments, after delivery of the nucleic acid payload to the subject's target cells, the following biomarkers of cell damage are not detected at apoptotic levels: ALT, AST, IL6, BCL2, or combinations thereof, and optionally, said target cells are located in the liver. In some embodiments, after delivery of the nucleic acid payload to the subject's target cells, the following biomarkers of cell damage are not clinically elevated: ALT, AST, IL6, BCL2, or combinations thereof, and optionally, the target cells are located in the liver. In some embodiments, after delivery of the nucleic acid payload to the subject's target cells, the following biomarkers of cell damage are not clinically elevated: urinary creatinine levels, the ratio of urinary albumin to creatine, 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 embodiments, after delivery of the nucleic acid payload to the subject's target cells, the following biomarkers of cell damage are not clinically elevated: blood troponin levels, or creatinine phosphokinase, and optionally, the target cells are located in the heart or skeletal muscle.

[0093] The sonoporation process described herein can be used to induce the expression of nucleic acid payloads in liver or kidney cells.

[0094] 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.

[0095] 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.

[0096] 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. In some embodiments, the method includes (a) administering a nucleic acid construct containing a nucleic acid payload (e.g., a therapeutic transgene) to a subject; (b) administering a sonoactive agent to the subject; and (c) administering a sonopore-forming treatment. In some embodiments, the sonopore-forming treatment includes applying ultrasound energy to the liver at a first mechanical index (MI) of less than 0.4; (d) applying ultrasound energy to the liver at a second MI of greater than 0.4 and less than 2.0; in some embodiments, the method includes repeating the application of ultrasound energy at the first MI and the second MI multiple times. In some embodiments, the method includes systemic delivery of the nucleic acid payload and the sonoactive agent (e.g., via intravenous administration).

[0097] In some embodiments, provided herein is a method of treating a subject having hemophilia A, comprising administering to the subject a nucleic acid construct comprising a therapeutic transgene; administering to the subject an acoustic agent; 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).

[0098] In some embodiments, provided herein is a method of treating a subject having Wilson's disease, comprising administering to the subject a nucleic acid construct comprising a therapeutic transgene; administering to the subject an acoustic agent; 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). In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding ATP7B. In some embodiments, the nucleic acid construct and the acoustic agent are administered systemically (e.g., by intravenous administration).

[0099] In one aspect, using the methods described herein, the present disclosure provides methods of treating a subject having a kidney disorder. In some embodiments, the kidney disorder being treated is: Alport Syndrome or autosomal dominant polycystic kidney disease.

[0100] In some embodiments, the present disclosure provides methods of treating a subject having a kidney disorder with a therapeutic transgene. In some embodiments, the therapeutic transgene encodes one or more of COL4A3, COL4A4, COL4A5, PKD1, and / or PKD2.

[0101] In some embodiments, the present disclosure provides methods of treating a subject having a kidney disorder with a therapeutic transgene. In some embodiments, the kidney disorder is Alport Syndrome and the therapeutic transgene encodes one or more of COL4A3, COL4A4, and / or COL4A5. In some embodiments, the kidney disorder is autosomal dominant polycystic kidney disease and the therapeutic transgene encodes one or more of PKD1 and / or PKD2. In some embodiments, the method comprises (a) administering to the subject a nucleic acid construct comprising a nucleic acid payload; (b) administering to the subject an acoustic agent; and (c) administering a sonoporation treatment. In some embodiments, the sonoporation treatment comprises applying ultrasonic acoustic energy to the kidney at a first mechanical index (MI) less than 0.4; (d) applying ultrasonic acoustic energy to the kidney at a second MI greater than 0.4 and less than 2.0.

[0102] In some embodiments, provided herein is a method of treating a subject having Alport syndrome, comprising administering to the subject a nucleic acid construct comprising a therapeutic transgene; administering to the subject a sonosensitizer; 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). 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 construct and the sonosensitizer are administered systemically (e.g., by intravenous administration).

[0103] In some embodiments, provided herein is a method of treating a subject having autosomal polycystic kidney disease, comprising administering to the subject a nucleic acid construct comprising a therapeutic transgene; administering to the subject a sonosensitizer; 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). In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding PKD1. In some embodiments, the therapeutic transgene comprises a nucleic acid sequence encoding PKD2. In some embodiments, the nucleic acid construct and the sonosensitizer are administered systemically (e.g., by intravenous administration).

[0104] On the other hand, the present disclosure provides a kit for performing the methods described herein. In some embodiments, the kit comprises: (a) a first container comprising microbubbles for sonoporation; and (b) a second container comprising a minicircle plasmid containing a transgene and a mixing chamber (reservoir, syringe, Y-port, etc.).

[0105] In some embodiments, the minicircle plasmid further comprises an expression cassette. As used herein, an expression cassette comprises a nucleic acid sequence encoding a nucleic acid payload, e.g., an expression cassette containing a transgene. The expression cassette further comprises regulatory elements such as a promoter, an enhancer, a ribosome binding site, or a transcription termination signal.

[0106] In some embodiments, the first container and the second container are configured to induce expression of the transgene in the target cells of the subject within 20 hours after transfection.

[0107] In some embodiments, the method further includes inducing the expression of the nucleic acid payload and maintaining the expression of the protein encoded by the nucleic acid payload for at least 1, 2, 3, 4, 5, 6, or 7 days after administering the nucleic acid construct, the acoustic active agent, and applying ultrasonic energy to the target cells at low MI and high MI.

[0108] In some embodiments, the method further includes increasing the expression of the nucleic acid payload by increasing the dose of the nucleic acid payload administered to the subject. In some embodiments, the method further includes increasing the expression of the nucleic acid payload by increasing the dose of the nucleic acid payload administered to the subject in a linear manner. In some embodiments, the method further includes increasing the expression of the nucleic acid payload by administering at least 5, 50, 250, or 500 μg of the nucleic acid payload to the subject.

[0109] In some implementations, ALT levels exceeding 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 U / L were not detected after the nucleic acid payload was delivered to the subject's target cells. In some implementations, AST levels exceeding 225, 250, 275, or 300 U / L were not detected after the nucleic acid payload was delivered to the subject's target cells. In some implementations, IL6 levels exceeding 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, or 6 pg / mL were not detected after the nucleic acid payload was delivered to the subject's target cells.

[0110] In some embodiments, the kit also includes software and hardware instructions for the safe and effective operation of an ultrasonic machine sufficient to disrupt the acoustic active agent to induce a sonopore-forming process, which includes, but is not limited to, disrupting microstructures, inducing inertia and stabilizing cavitation, promoting endocytosis and interendothelial space formation, and microfluidics on the cell surface, thereby increasing the transfection of nucleic acid payloads into cells. In some embodiments, the instructions describe a method for improving gene transfection using sonopore-forming by applying alternating ultrasonic energy between a first MI and then a second MI. In some embodiments, the kit also includes instructions for applying the first and second containers.

[0111] This disclosure provides an ultrasound system including a computer system programmed to implement the methods of this disclosure. Figure 12 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 for communicating with one or more other systems (e.g., network adapters), and peripheral devices such as caches, other memories, data storage, and / or electronic display adapters. Memory, storage units, interfaces, and peripheral devices communicate with the CPU via a communication bus (solid line) such as the motherboard. Storage units can be data storage units (or databases) for storing data. The computer system can be operatively coupled to a computer network (“network”) via a communication interface. A network can 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 can include one or more computer servers that can enable distributed computing, such as cloud computing. In some cases, the network can be a peer-to-peer network, enabling devices coupled to the computer system to act as clients or servers.

[0112] 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 sonosensitizer, 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 or endothelial 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 with a duration of 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 with a duration of up to 200 μs.In some embodiments, applying the ultrasonic energy in the second MI comprises applying the ultrasonic energy in the second MI using a pulse for a duration of up to 500 µs. In some embodiments, applying the ultrasonic energy in the second MI comprises applying the ultrasonic energy in the second MI using a pulse for a duration of about 1 µs to about 200 µs. In some embodiments, applying the ultrasonic energy in the second MI comprises applying the ultrasonic energy in 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 in the first MI and applying the ultrasonic energy in the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy in the first MI for a duration sufficient to allow reperfusion of the acoustic agent in the tissue containing the target cells. In some embodiments, the repetition comprises applying the ultrasonic energy in the first MI for 1-30 seconds before repeatedly applying the ultrasonic energy in the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy in the first MI for 5-15 seconds before applying the ultrasonic energy in 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.

[0113] 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) an acoustic active agent. In some embodiments, an ultrasound 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 energy with the second MI includes applying the ultrasonic energy with a pulse of duration from about 1 μs to about 500 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse of duration from the second MI for a duration of up to 200 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse of duration from the second MI for a duration of up to 500 µs with the second MI. In some embodiments, applying the ultrasonic energy with the second MI includes applying the ultrasonic energy with a pulse of duration from the second MI for a duration of about 1 μs to about 200 µs with the second MI.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 approximately 2.3 µs. In some embodiments, the specification includes repeatedly applying the ultrasonic energy at the first MI and 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 acoustic agent 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 prior to 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 prior to applying the ultrasonic energy at the second MI. In some embodiments, the repetition comprises applying the ultrasonic energy at the first MI for 10 seconds prior to applying the ultrasonic energy at the second MI.

[0114] 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.

[0115] 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).

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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®.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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 Active Server 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®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

[0134] 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.

[0135] 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.

[0136] Suitable mobile application development environments are available from several sources. As non-limiting examples, commercially available development environments 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, webOSSDK, and Windows® Mobile SDK.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] The terms “determine,” “measure,” “evaluate,” “assess,” “determine,” and “analyze” are often used interchangeably in this document to refer to forms of measurement. These terms include determining the presence of an element (e.g., detecting). These terms may include quantitative, qualitative, or a combination of quantitative and qualitative determinations. Assessments may be relative or absolute. “Detecting the presence of…” may include determining the quantity of something present, in addition to determining whether something is present or not, depending on the context.

[0143] The terms “subject,” “individual,” or “patient” are often used interchangeably in this document. A subject can be a mammal. A mammal can be a human. A subject may be diagnosed or suspected of being at high risk for a disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk for the disease.

[0144] The term "in vivo" is used to describe events that occur inside a subject's body.

[0145] The term "ex vivo" is used to describe events that occur outside the subject's body. Measurements are not performed on the subject in vitro. Instead, measurements are performed on samples separated from the subject. An example of an ex vivo measurement of a sample is an "in vitro" measurement.

[0146] The term "in vitro" is used to describe events that occur in a container used to contain laboratory reagents, separating them from the biological source from which the material is obtained. In vitro assays can cover cell-based assays, in which live or dead cells are used. In vitro assays can also cover cell-free assays, in which intact cells are not used.

[0147] As used herein, the terms “treatment” or “treating” are used to refer to a drug or other intervention intended to achieve a beneficial or desired outcome in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits and / or preventative benefits. A therapeutic benefit may refer to the eradication or improvement of symptoms or the underlying condition being treated. Similarly, a therapeutic benefit may be achieved by eradicating or improving one or more physical symptoms associated with an underlying condition, resulting in an improvement observed in the subject, although the subject may still have the underlying condition. Preventative effects include delaying, preventing, or eliminating the onset of a disease or symptom, delaying or eliminating the onset of symptoms of a disease or symptom, slowing, stopping, or reversing the progression of a disease or symptom, or any combination thereof. For preventative benefits, a subject at risk of developing a particular disease or a subject reporting one or more physical symptoms of a disease may receive treatment, even if the disease has not yet been diagnosed.

[0148] As used herein, the terms “ultrasound,” “ultrasound energy,” “ultrasound acoustic energy,” “ultrasound power,” and “ultrasound acoustic energy” are used interchangeably.

[0149] The term "IVIS" refers to an in vivo imaging system.

[0150] Example The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0151] Example 1: Generation of microparticles for sono-induced pore formation.

[0152] In this experiment, microplasmids for transfection were generated. In short, microplasmid vector backbones, such as nanoplasmids, were used. The nanoplasmids were generated / purchased by Aldeveron (Fargo, SD). Wild-type firefly luciferase was used as the reporter gene in this experiment and was located below the promoter sequence. Nucleic acid constructs included: CAG-Fluc, ApoE-AAT-Fluc, 3xSERP-Enh-TTR-Fluc, and P3-heterozygous-Fluc. A map of the nanoplasmid vectors is shown in Figure 1.

[0153] Example 2: Optimization of gene therapy expression and persistence in rat liver.

[0154] This experiment evaluated the transfection and expression of reporter gene luciferase in rat liver.

[0155] Experimental conditions and procedures: Twenty Sprague Dawley rats were studied. All animals were anesthetized with 2% isoflurane and their abdomens were shaved and treated with a depilatory agent. The injection solution consisted of 1 mL Optison and 250 µL of a nucleic acid payload containing DNA (1.125 mg) of one of the following nanoparticles: a nanoparticle containing the promoter sequence ApoE-AAT, CAG, 3xSERP, or P3; and each nanoparticle containing luciferase (e.g., the nanoparticle generated in Example 1). The nucleic acid payload was diluted with 750 µL of PBS, with an estimated dead zone of approximately 75 μL. The solution was infused intravenously via the tail vein over 70 seconds. The acoustic contact agent (Aqua gel) was applied directly to the abdominal surface and onto the skin surface of the rat's upper abdomen. Acoustic parameters included the following: • Low MI operates at an MI of 0.09.

[0156] • High MI mode operates at MI 1.4.

[0157] The ultrasound was delivered at a frequency of 9.3 MHz.

[0158] The treatment procedure is as follows: • While administering the infusion via the tail vein, place the ultrasound transducer on the abdomen and initiate low-MI ultrasound imaging of the liver (0.09) for 20 seconds.

[0159] • After 21 seconds, a high MI pulse of 1.4 is applied, with a pulse duration of 0.98 μs.

[0160] • After the high MI pulse, continue with low MI imaging (0.09), and apply the high MI pulse every 10 seconds for 8 times (total 90 seconds).

[0161] In vivo bioluminescence imaging (IVIS) was performed at 24, 48, 72, and 144 hours. Bioluminescence values ​​are reported as mean radiance (photons / sec / cm²). 2 / Sphericity).

[0162] result: like Figure 2 As shown, groups A and B are control groups. Groups C, D, E, and F (N=4 per group) received nucleic acid payloads containing DNA in the following order: CAG-Fluc, ApoE-AAT-Fluc, 3xSERP-Enh-TTR-Fluc, and P3-hybrid-Fluc. The mean radiance within IVIS is expressed as (photons / second / cm²). 2 / sphericity) records. As previously mentioned, control animals did not exhibit bioluminescence, and groups D and E revealed stable mean radiance at 144 hours, with increased variability noted in groups C and F.

[0163] Figure 2 This study depicts quantitative results of nucleic acid transfection and expression from an in vivo imaging system (IVIS) using bioluminescent imaging (BLI) of rat livers with nucleic acid payloads comprising CAG-Fluc, ApoE-AAT-Fluc, 3xSERP-Enh-TTR-Fluc, and P3-hybrid-Fluc.

[0164] Example 3: Optimization of gene therapy expression and persistence in mouse liver - Study I.

[0165] In this experiment, the kinetics of transfection and expression of the reporter gene luciferase in mouse liver were investigated.

[0166] Experimental conditions and procedures: Twelve C57BL / 6 mice were studied. All animals were anesthetized with 2% isoflurane and their abdomens were shaved and treated with a depilatory agent. The ApoE-ATT / luciferase nanoparticles produced in Example 1 were used. The injection solution contained a total injection volume of 240 μL (157.5 µg of ApoE-AAT with luciferase nanoparticles), with 95 μL of PBS and 120 μL of Optison in 35 μL, and an estimated dead zone of approximately 50 μL. This total volume was infused intravenously via the tail vein and an external ultrasound transducer over 70 seconds. An acoustic contact agent (Aqua gel) was applied directly to the abdominal surface, and ultrasound energy was applied to the upper abdominal skin surface of the rats. Acoustic parameters included the following: • Low MI operates at an MI of 0.09 or 0.3.

[0167] • High MI mode operates at MI 1.5.

[0168] The ultrasound was delivered at a frequency of 9.3 MHz.

[0169] The treatment procedure is as follows: • Low-MI imaging of the liver (0.09) was initiated within the first 20 seconds after tail vein infusion.

[0170] • After 21 seconds, a high MI pulse of 1.5 is applied, with a pulse duration of 2.28 μs.

[0171] • After high MI mode, continue with low MI imaging (0.09), and perform high MI every 10 seconds, 9 times (total 100 seconds). Other studies included variations in the aforementioned acoustic parameters, with a low MI of 0.09 in some mice and a low MI of 0.3 in others.

[0172] Liver-based protein dynamics were characterized using IVIS following sono-induced pore formation, with measurements initiated at 3 hours and recorded sequentially at 6, 12, 18, 24, 30, 48, and 72 hours.

[0173] result: like Figure 3A As shown, the background control group did not reveal any recorded bioluminescence. Based on timing kinetic data, an initial IVIS scan performed at 3 hours revealed the presence of luciferase signaling. Bioluminescence signal levels increased at 6, 12, 18, and 24 hours, with a peak signal observed at 30 hours in two animals treated with low MI (0.09) (all other conditions were constant). Bioluminescence signals were significantly lower in animals treated with reduced pulse numbers (N=4 vs. N=9), or with low MI increased by 0.3, or with insufficient tail vein injection. Bioluminescence imaging results are shown in… Figure 3B middle.

[0174] Figure 3A and Figure 3B The results of nucleic acid transfection and expression (kinetic study) from IVIS using BLI from mouse livers of Study I with a nucleic acid payload containing ApoE-AAT-Fluc are depicted. Figure 3A A graph showing the average emissivity measured compared to a control is presented. Figure 3B The same in vivo imaging system (IVIS) using bioluminescence imaging (BLI) is shown. Figure 3BThe following observations were made: No fluorescence was observed in Figures A-C; blue / green fluorescence was observed in mouse liver in Figure D; no fluorescence was observed in Figures E-G; blue / green fluorescence was observed in mouse liver in Figure H, with an increase in blue / green fluorescence compared to Figure D; a small amount of blue fluorescence was observed in mouse liver in Figure I; observable blue / green fluorescence was observed in mouse liver in Figures J-L, with an increase in blue / green fluorescence compared to Figure H; no fluorescence was observed in Figures M-T; blue / green fluorescence was observed in mouse liver in Figure U; and blue / green fluorescence was observed in mouse liver in Figures V-W. Figure X - Green / red fluorescence is present in mouse liver in Figure Y; blue / green fluorescence is present in mouse liver in Figure Z, with an increase in blue / green fluorescence compared to Figure A in Figure V; green / red fluorescence is present in mouse liver in Figures A1-C1, with an increase in red / green fluorescence compared to Figures W-Y in Figures A1-C1; blue / green fluorescence is present in mouse liver in Figure D1; green / red fluorescence is present in mouse liver in Figures E1-G1; no fluorescence is observed in Figures H1, K1, or N1; blue / green fluorescence is present in mouse liver in Figures I1 and L1, with an increase in blue / green fluorescence compared to Figure I1 in Figure L1; red / green fluorescence is present in Figures J1 and M1, with fluorescence in Figure M1 compared to Figure J1; and red / green fluorescence is present in Figures P1 and M1, with fluorescence in Figure P1 compared to Figure M1. Blue fluorescence indicates approximately 10 × 10^5 p / s / cm. 2 The fluorescence intensity of / sr; blue / green fluorescence indicates approximately 20*10^6 p / s / cm. 2 The fluorescence intensity of / sr; green fluorescence indicates approximately 30*10^6 p / s / cm. 2 Fluorescence intensity of / sr; yellow fluorescence indicates approximately 40*10^6 p / s / cm. 2 The fluorescence intensity of / sr; and the red fluorescence indicator of approximately 50*10^6 p / s / cm. 2 / sr fluorescence intensity. Increased fluorescence indicates a higher degree of luciferase expression. Example 4: Optimization of gene therapy expression and persistence in mouse liver - Study II.

[0175] In this experiment, the transfection and expression of the reporter gene luciferase in mouse liver were investigated.

[0176] Experimental conditions and procedures: Twelve BalbC mice were studied. All animals were anesthetized with 2% isoflurane and their abdomens were shaved and treated with a depilatory agent. The nanoparticles produced in Example 1 were used. The injection solution consisted of a total injection volume of 300 µL (157.5 µg of ApoE-AAT with luciferase nanoparticles, 115 µL of PBS and 150 µL of Optison in 35 µL, with an estimated dead zone of approximately 50 µL). This total volume was infused intravenously via the tail vein and an external ultrasound transducer over 3 seconds. The acoustic contact agent (Aqua gel) was applied directly to the abdominal surface, and the transducer was applied to the skin surface of the upper abdomen of the rats. Acoustic parameters included the following: • Low MI operates at an MI of 0.05-0.07.

[0177] • High MI mode operates at an MI of 0.8.

[0178] The ultrasound was delivered at a frequency of 9.3 MHz.

[0179] The treatment procedure is as follows: • During tail vein infusion, high-MI pulses are administered at 8-second intervals with a repetition rate of 4, 9, or 18 sequences (32, 72, or 144 seconds, respectively), with a pulse duration of 2.28 μs.

[0180] • Low MI imaging was maintained at (0.05–0.07) throughout the treatment and applied between high MI pulses.

[0181] IVIS was performed at 24, 48, and 72 hours.

[0182] result: like Figure 4 As shown, the background does not reveal bioluminescence. After receiving 9 pulses at 8-second intervals (e.g., ... Figure 4 The most stable bioluminescence results were recorded in the animals shown; while animals receiving 4 or 18 pulse sequences at 8-second intervals did not reveal a stable bioluminescence pattern at 72 hours, indicating a significant decrease in luciferase expression at 72 hours. Animals receiving 9 pulses experienced a stable illumination response, indicating that luciferase expression was maintained.

[0183] Example 5: Expression and persistence of gene therapy in mouse kidneys.

[0184] In this experiment, the efficacy and durability of gene therapy following sonoporosis in mouse kidneys were investigated.

[0185] Experimental conditions and procedures: Eight BalbC mice were studied. All animals were anesthetized with 2% isoflurane and their abdomens and lower backs were shaved and treated with a depilatory agent. The nanoparticle CAG produced in Example 1 was used. The injection solution consisted of a total injection volume of 300 µL (157.5 µg of CAG with luciferase nanoparticles, 115 µL of PBS and 150 µL of Optison in 35 µL, with an estimated dead zone of approximately 50 µL). This total volume was infused intravenously via the tail vein and an external ultrasound transducer over 3 seconds.

[0186] The acoustic contact agent Aqua gel was applied directly to the left lateral ventral surface, and the transducer was applied to the skin surface of the left upper abdomen, focusing on the left renal region. Imaging allowed for clear acoustic visualization of the left kidney. All treated animals received DNA (CAG). Acoustic parameters included the following: • Low MI operates at an MI of 0.05-0.07.

[0187] • High MI mode operates at 0.8 MI with a pulse duration of approximately 2 microseconds.

[0188] The ultrasound was delivered at a frequency of 9.3 MHz.

[0189] The treatment procedure is as follows: • During tail vein infusion, high-MI pulses were initially administered every 3 seconds, repeated for 10 pulse sequences (27 seconds in total). Low-MI imaging remained at (0.05–0.07) throughout the treatment.

[0190] In vivo bioluminescence imaging (IVIS) was performed at 17 and 36 hours.

[0191] result: like Figure 5A As shown, bioluminescence was recorded in two of the four animals (two mice on the left) 17 hours after sonoscopic pore formation, particularly in the left region. Both animals underwent sonoscopic pore formation targeting the left kidney and were imaged in the short-axis plane; while the two mice on the right of the image were imaged in the long-axis plane. Figure 5B The results showed that reporter gene expression was still observed 36 hours after sono-induced pore formation. Figure 5C As shown, no bioluminescence was observed in the left region of the control animals. Table 1 below shows the quantitative results of this experiment.

[0192] Figures 5A-5C IVIS imaging results of mouse kidneys after CAG-Fluc treatment at 17 and 36 hours post-treatment were depicted. Figure 5A The image shows blue fluorescence in the leftmost part of the subject's kidney. Figure 5AThe blue / green fluorescence in the second image from the far left of the subject's kidney is shown. Figure 5A The third image from the far left of the subject's kidney showed no fluorescence. Figure 5A The third image from the far left of the subject's kidney showed no fluorescence. Figure 5B The image showing the rightmost kidney of the subject reveals blue / green fluorescence. Figure 5B The blue / green fluorescence in the second image from the far left of the subject's kidney is shown. Figure 5B The third image from the far left of the subject's kidney showed no fluorescence. Figure 5B The image showing the rightmost kidney of the subject has no fluorescence. Blue fluorescence indicates approximately 10 × 10^5 p / s / cm. 2 The fluorescence intensity of / sr; blue / green fluorescence indicates approximately 20*10^6 p / s / cm. 2 The fluorescence intensity of / sr; green fluorescence indicates approximately 30*10^6 p / s / cm. 2 Fluorescence intensity of / sr; yellow fluorescence indicates approximately 40*10^6 p / s / cm. 2 The fluorescence intensity of / sr; and the red fluorescence indicator of approximately 50*10^6 p / s / cm. 2 / sr fluorescence intensity. Increased fluorescence indicates a higher degree of luciferase expression. Figure 5C IVIS imaging results of the kidneys from control mice were depicted. Figure 5C The image showing the leftmost kidney of the subject showed no fluorescence. Figure 5C The second image from the far left of the subject's kidney shows no fluorescence. Figure 5C The third image from the far left of the subject's kidney showed no fluorescence. Figure 5C The image showing the rightmost kidney of the subject has no fluorescence. Blue fluorescence indicates approximately 10 × 10^5 p / s / cm. 2 The fluorescence intensity of / sr; blue / green fluorescence indicates approximately 20*10^6 p / s / cm. 2 The fluorescence intensity of / sr; green fluorescence indicates approximately 30*10^6 p / s / cm. 2 Fluorescence intensity of / sr; yellow fluorescence indicates approximately 40*10^6 p / s / cm. 2 The fluorescence intensity of / sr; and the red fluorescence indicator of approximately 50*10^6 p / s / cm. 2 / sr fluorescence intensity. Increased fluorescence indicates a higher degree of luciferase expression.

[0193] Table 1: Quantification of IVIS readings.

[0194] Overall, the data shows that the alternating MI protocol can deliver nucleic acid payloads containing nanoparticle constructs with luciferase to the kidneys.

[0195] Table 2 below provides a summary of the parameters used in Examples 2-5.

[0196] Table 2: Summary of parameters used in Examples 2-5.

[0197] Example 6: Dose-responsive delivery in mouse liver.

[0198] In this experiment, the effect of transgenic dosage on expression in mouse liver was investigated. The injection solution comprised 5 µg, 50 µg, 250 µg, or 500 µg luciferase nanoparticles. Sonopore formation was performed according to the methods described herein. Gene expression was analyzed by IVIS.

[0199] The same experimental procedure was performed on mouse livers as described in the previous examples. Ultrasound was applied at 1.3 MHz, low MI of 0.1–0.4, and high MI of 1.4.

[0200] like Figure 6A As shown, a higher mean emissivity was observed with increasing nanoparticle dose. Furthermore, a linear relationship was observed between mean emissivity and the abundance of nanoparticles in the blood. Figure 6B The original IVIS image of the mouse is shown in... Figure 6C middle.

[0201] Figures 6A-6C The IVIS results of mouse livers after receiving 5 μg, 50 μg, 250 μg, or 500 μg of luciferase nanoparticles were depicted. Figure 6A The average radioactivity (p / s / cm) for each nanoparticle dose tested is shown. 2 / sr). Figure 6B The average radioactivity is shown based on the relative DNA abundance in the blood. Figure 6C Exemplary raw IVIS images of each nanoparticle dose tested are shown. Blue fluorescence indicates a fluorescence intensity of approximately 0.5 × 10^7 p / s / cm. 2 / sr; the blue / green indicator fluorescence intensity is approximately 1*10^7 p / s / cm. 2 / sr; the green fluorescence indicator shows a fluorescence intensity of approximately 1.5*10^7 p / s / cm. 2 / sr; Green / yellow fluorescence indicates fluorescence intensity of approximately 2*10^7 p / s / cm. 2 / sr; the orange fluorescence indicates a fluorescence intensity of approximately 2.5*10^7 p / s / cm.2 / sr; The red fluorescence indicates a fluorescence intensity of approximately 3*10^7 p / s / cm. 2 / sr. Increased fluorescence indicates a higher level of luciferase expression. Figure 6C The leftmost image shows that the nucleic acid construct treated with 0 μg showed no fluorescence; Figure 6C The leftmost image shows no fluorescence in the nucleic acid construct treated with 0 μg; the second image from the left shows blue fluorescence in the nucleic acid construct treated with 5 μg; the third image from the left (middle image) shows a large area of ​​blue / blue-green fluorescence in the nucleic acid construct treated with 50 μg; the second image from the right shows green fluorescence surrounded by blue fluorescence in the nucleic acid construct treated with 250 μg; and the right image shows a nucleic acid construct treated with 500 μg, where green-yellow fluorescence is surrounded by blue fluorescence, with observable red fluorescence in the center.

[0202] Example 7: Kinetics and persistence of transgene expression in mouse liver In this experiment, as described in Example 3, the kinetics of transgene expression were examined after sonoformation of luciferase nanoparticles into mouse livers.

[0203] Transgenic expression was measured by IVIS at 3, 6, 12, 18, 24, and 30 hours post-delivery. Figure 7 As shown, transgene expression can be detected for the first time 3 hours after delivery, indicating the rapid dynamics of DNA delivery to the cell nucleus.

[0204] Figure 7 IVIS results of mouse livers at 3, 6, 12, 18, 24, and 30 hours after delivery of luciferase nanoparticles via sonopore formation in four different animals were depicted. Blue fluorescence indicates a fluorescence intensity of approximately 1 × 10^5 p / s / cm. 2 / sr; the blue / green indicator fluorescence intensity is approximately 2*10^5 p / s / cm. 2 / sr; the green fluorescence indicator shows a fluorescence intensity of approximately 3*10^5 p / s / cm. 2 / sr; the yellow fluorescence indicates a fluorescence intensity of approximately 4*10^5 p / s / cm. 2 / sr; The red fluorescence indicates a fluorescence intensity of approximately 5*10^5 p / s / cm. 2 / sr. Increased fluorescence indicates a higher level of luciferase expression. Figure 7 No fluorescence was observed at AC, DF, and U; blue fluorescence was observed at D, H, and VX; blue-green fluorescence was observed at M, N, Q, and U; and yellow-red fluorescence was observed at O, P, RT, and VX.

[0205] Furthermore, it was observed that expression remained at a consistent level for 7 days, such as Figure 10 As shown.

[0206] Example 8: Safety evaluation after treatment In this experiment, various safety endpoints were evaluated after transgenes were delivered to the liver via sonopore formation.

[0207] Evaluate blood levels of ALT, AST, and IL-6 one day after delivery. Figures 8A-8C As shown, no increase in ALT or AST activity was observed. Furthermore, based on previously published values, the measured ALT and AST levels were within the normal range. In BALB / c mice, normal ALT activity ranges from 40–170 U / L (female) or 41–131 U / L (female), and normal AST activity ranges from 67–381 U / L (female) or 55–381 U / L (male). Similarly, no increase in blood IL6 levels was observed. Figure 8C ).

[0208] Figure 8A The ALT activity (U / L) in mouse blood transfected with a specified nanoparticle dose is shown. Figure 8B The AST activity (U / L) in mouse blood transfected with a specified nanoparticle dose is shown. Figure 8C The concentration (pg / mL) of IL6 in mouse blood transfected with a specified nanoparticle dose is shown.

[0209] One week after delivery of the transgene under different promoter controls, the weight of the treated animals was also examined. Figure 9 As shown, no weight loss was observed, and the weight of the treated animals followed a similar trend to that of the control animals.

[0210] In summary, these data confirm the safety of ultrasound-mediated transgene delivery to the liver.

[0211] Example 9: Expression levels of exogenous DNA delivered via sonopore formation of hepatocytes using different vectors In this experiment, quantitative polymerase chain reaction (qPCR) was used to measure the copy number of each diploid genome of exogenous genes delivered via sonopores using different expression vectors in hepatocytes.

[0212] Laboratory animals and protocols Encoding downstream of the CAG promoter via acoustic perforation Firefly luciferase ( FlucThe gene vector was delivered to the livers of six groups of mice, each group containing four Rag2 mice. Prior to sonopore formation, 100 μg of DNA was administered to each mouse via a jugular vein catheter. Different vectors were used in each group to deliver the firefly luciferase (Fluc) gene. The vectors used in each group were as follows: Group 1, plasmid (pUC57-CAG-Fluc); Group 2, nanoparticle plasmid (NTC9385R\(3xCpG)-CAG2.0 Fluc-CpG-free BGH pA); Group 3, linear DNA (db312-001 TpUC CAG2.0-Fluc-CpG-free bGHpA_pUC57); Group 4, GenCircle (GC-CAG-Fluc); Group 5, MiniCircle (MC-CAG-Fluc); Group 6, negative control (no vector delivered).

[0213] One month after DNA delivery, livers were harvested from mice in all groups, and two liver samples from each animal were analyzed. Genomic DNA (gDNA) was isolated from the liver samples using the QIAGEN AllPrep kit. Samples were processed and isolated within a MystairePrep Station enclosure. The DNA was diluted in TE buffer so that 50 ng of DNA was included in each qPCR reaction. qPCR reactions were run using TaqPath ProAmp MasterMix with Flux5 p / ps.

[0214] Standard curves were generated using constructs serially diluted from 1:100,000 to 1:1,000,000 in primary gDNA from the following six groups of samples: (1) pUC57-CAG-Fluc (pUC57, 4.596 mg / ml, 6364 bp); (2) NTC9385R\(3xCpG)-CAG2.0 Fluc-CpG-free BGH pA (nanoplasmid, 4.93 mg / ml, 4092 bp); (3) db312-001 TpUC CAG2.0-Fluc-CpG-free bGHpA_pUC57 (linear, 3.958 mg / ml, 4224 bp); (4) GC-CAG-Fluc (GenCircle, 5.0 mg / ml, 4083 bp); (5) MC-CAG-Fluc (microrings, 5.0 mg / ml, 3699 bp); (6) untreated samples, copy number calculated based on the average of parameters from the above standard curves. result Using qPCR, the number of each diploid genome in hepatocytes was measured. Fluc Genetically modified copy number. Figure 11For each of the two samples, the mean transgenic copy number in mice across the six groups is provided. The bar height indicates the mean transgenic copy number in each group, and the dots indicate the sample measurement for each animal. Fluc The abundance was highest in group 2, where nanoparticle vectors were used. Using the sonopore-forming treatment protocol described herein, copies per diploid genome (CN / DG) were observed with nanoparticle vectors. Fluc A significant increase in abundance was observed, and this nanoplasmid vector was superior to many other vectors, including the standard plasmid (pUC57), a linear DNA form with blocked ends, a modified plasmid DNA sequence (microcircle) with prokaryotic DNA sequences removed, and a modified small circular double-stranded DNA vector (GenCircle) with a vector backbone of approximately 430 bp and the removal of antibiotic resistance genes. In some cases, compared with other vector formats, the abundance measured in Group 2 was significantly higher. Fluc The abundance was 2 to 10 times.

[0215] Example 10: Induction of nucleic acid payloads in non-human primates via DNA transfection using a high-MI ultrasound protocol strong expression Laboratory animals and protocols In this embodiment, the delivery of a nucleic acid payload encoding a green fluorescent protein (EGFP) reporter gene to multiple organ systems in a non-human primate was performed. Three experimental animals, each a male cynomolgus monkey, were used. Two cynomolgus monkeys (NHP01 and NHP02) were co-administered a microplasmid construct (Nanoplasmid™, Aldevron, SD) and a mixture of acoustically active microstructures along with delivered ultrasound (US) energy. The third cynomolgus monkey was naive and did not receive any intravenous injection of microvesicles or plasmids, nor was it subjected to external ultrasound at any time. The nucleic acid payloads used are summarized below.

[0216] Table 3. Summary of nucleic acid payloads.

[0217] Prior to the start of the experimental period, an IV catheter was inserted into the subject's saphenous vein. A dose of sonoactive microstructure and DNA solution was prepared first, following the label instructions: the microstructure was removed from the 4C reservoir and rolled between the fingers for 20 seconds; the protective plastic and aluminum capping was removed from the Optison® vial; a 25G needle was inserted through the rubber gasket of the Optison® vial to provide a pressure relief orifice; and 15 mL of Optison® (5 vials) was drawn into a syringe using a 1.5-inch 18G needle (including needle dead zone in the calculation). Using the same needle and syringe, 4 mL of DNA payload (5 mg DNA / mL solution) was drawn into the syringe to combine the DNA and Optison®. The Optison® microbubbles and DNA payload were mixed in the syringe until homogeneous by rolling the syringe between the fingers. The DNA + Optison® solution was withdrawn from the needle dead zone. Five syringes were prepared, each containing 15 mL of Optison® and 4 mL of DNA solution. The DNA+Optison® mixture is delivered intravenously in multiple 1 mL boluses at a rate of 1 mL / 1 min. The total delivery time is 15–20 minutes.

[0218] While administering a mixture of 19 mL microbubbles and nucleic acid payload via the saphenous vein, ultrasound energy was delivered to the subject's kidneys (e.g., unilateral or bilateral), liver, and quadriceps using an M5Sc probe positioned perpendicular to the subject's skin. The frequency of the applied ultrasound energy was 2.07–2.90 MHz, depending on the selected contrast imaging frequency label (Pen, Gen, Res), the depth was set to 7–8 cm, and the scaling was set to 0. Ultrasound was continuously delivered, alternating between a low mechanical index (MI) value of 0.07 and a high MI value of 1.5, without stopping the application of ultrasound energy at any point during treatment. Nine high MI ultrasound flashes of 1.5 were delivered at 5–20 second intervals between each set of high MI flashes, and the application of nine pulses was repeated three times. The duration of the high MI pulse was approximately 2.28 microseconds. The ultrasound probes and parameters used are summarized below.

[0219] Table 4. Summary of Ultrasound Parameters result Imaging of target organs and tissues of interest to measure the fluorescence radiation signal generated by EGFP. Figure 13Quantitative results of fluorescence emissivity measurements in organs of interest from non-human primates are presented. The kidneys of NHP01 and NHP02 were imaged. Fluorescence was observed on the entire surface (transverse and longitudinal cross-sections) of the kidneys of both NHP01 and NHP02. Fluorescence was also observed at the peripheral border of the liver, the leftmost portion of the heart, and along the periphery of skeletal muscle. Figure 13 The intensity of fluorescent radiation in these organs was quantified.

[0220] Figure 13 Quantitative results showed that the average radiation rate in the liver was approximately 5*10^6 p / s / cm. 2 The average emissivity in skeletal muscle is approximately 7*10^6 p / s / cm. 2 / sr, and the average radiation rate in the kidneys is approximately 8*10^7 p / s / cm. 2 / sr.

[0221] Analysis of transgene copy number (CN / DG) for each diploid genome in the treated organs was consistent with fluorescence radiation data, which tended to show high levels of gene expression in the kidneys, quite different from those in the liver. Figure 14 The comparison of CN / DG of EGFP in the kidney and liver is shown. In subject 9275, the CN / DG of EGFP was observed to be approximately 0.25 in the liver and approximately 2.5 in the kidney; and in subject 9286, the CN / DG of EGFP was observed to be approximately 0.5 in the liver and approximately 5.0 in the kidney. The additional CN / DG of EGFP observed in the liver and kidney compared to... Figure 13 The optical fluorescence data shown are consistent.

[0222] Example 11: Sonication with increased high MI values ​​in the kidneys and livers of a non-human primate model Hole treatment The data provided in this embodiment show that increasing the mechanical index of high MI pulses directly improves gene delivery and expression in sonoporosis treatment in non-human primate models.

[0223] Laboratory animals and protocols In this embodiment, the delivery of a nucleic acid payload encoding a fluorescent reporter gene to multiple organ systems in a non-human primate was performed. Three experimental animals, each a male cynomolgus monkey, were used. Two cynomolgus monkeys (NHP01 and NHP02) were co-administered a microplasmid construct (Nanoplasmid™, Aldevron, SD) and a mixture of acoustically active microstructures along with delivered ultrasound (US) energy. The nucleic acid payloads used and experimental conditions are summarized below.

[0224] Table 5: Summary of Nucleic Acid Payloads Prior to the start of the experimental period, an IV catheter was inserted into the subject's saphenous vein. A specific dose of sonoactive microstructures and DNA solution was prepared by first preparing the sonoactive agent as indicated on the label.

[0225] For Optison®, remove the microstructure from the 4C reservoir 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 relief port; and use a 1.5-inch 18G needle to draw the dose of Optison® into the syringe. Using the same needle and syringe, draw the dose of the DNA payload into the syringe to combine the DNA and Optison®. Mix the Optison® microbubbles and DNA payload in the syringe until the solution is homogeneous by rolling the syringe between your fingers. Withdraw the DNA + Optison® solution from the needle dead zone. Prepare multiple syringes, each containing the DNA + Optison® mixture, with a microbubble solution to DNA solution volume ratio of approximately 4:1. Administer the DNA + Optison® mixture via multiple 1mL boluses intravenously at a rate of 1 mL / 1 min. The total delivery time is approximately 30 minutes.

[0226] For Sonazoid®, prepare a dose of the sonoactive microstructure and DNA solution by first preparing the sonoactive agent as indicated on the label: Remove the injection powder from the manufacturer's packaging by twisting the top of the ampoule, insert the syringe directly into the ampoule without using a cannula, add phosphate-buffered saline from the syringe to the vial and manually shake for one minute to ensure the product is homogeneous, withdraw the product into the syringe and re-inject it into the vial, shake the vial to reconstitute the product immediately before injection, and withdraw it into the syringe for injection. Using the same needle and syringe, draw the dose of DNA payload into the syringe to combine the DNA and Sonozoid® sonoactive agent, and mix the Sonozoid® sonoactive agent and DNA payload in the syringe until the solution is homogeneous by rolling the syringe between your fingers. Withdraw the DNA + Sonozoid® sonoactive agent solution from the needle dead zone. Then replace the 18G needle with a 25G blunt needle for injection into the IV catheter. The DNA + Sonozoid® mixture is delivered intravenously in multiple 1 mL boluses at a rate of 1 mL / 1 min. The total delivery time is approximately 25 minutes.

[0227] While administering a mixture of microbubbles and nucleic acid payloads via the saphenous vein, ultrasound energy was delivered to the subject's kidneys (e.g., unilateral or bilateral) or liver region using an ultrasound probe positioned perpendicular to the subject's skin. The listed probes, used in conjunction with the GE LOGIQ system, were employed to deliver the ultrasound energy. The applied ultrasound energy was delivered at a frequency of 2.07–2.90 MHz, a depth of 7–8 cm, and a scaling of 0. Ultrasound was delivered continuously, alternating between listed low mechanical index (MI) and high MI values, without interrupting the ultrasound probe's contact with the subject's skin or the application of ultrasound energy at any point during treatment. High MI ultrasound flashes of five (5) times at listed values ​​were delivered to the subject at intervals, with low MI applied between high MI applications, and the ultrasound probe was moved to a new position approximately every 8 seconds. The high MI pulse duration was approximately 2.28 microseconds.

[0228] result Imaging of target organs and tissues of interest was performed to measure the fluorescence radiation signals generated by EGFP and TdTom. Figures 15A-15C Quantitative results of fluorescence emissivity measurements in the kidneys and livers of each subject are shown and cited below. It was observed that, under the same nucleic acid payload dose and the same ultrasound parameters, the mean fluorescence emissivity measurements in the kidneys (with approximately 3 times the blood perfusion of the liver) were approximately 2 to 6 times that of the liver.

[0229] When this data was compared with Example 9, which used standard low / high mechanical index values ​​of 0.07 and 1.5 to deliver the EGFP reporter gene in the same animal model, it was observed that ultrasound with increased mechanical index values ​​of 0.3 and 2.7–2.9 had beneficial technical effects, with subjects treated with increased low / high mechanical index values ​​of 0.3 and 2.7–2.9 exhibiting fluorescence approximately 10-fold greater than that observed in animals treated with standard low / high mechanical index values ​​of 0.07 and 1.5. This data suggests that, under otherwise similar treatment conditions, the increased mechanical index is a parameter directly associated with increased gene delivery and / or expression when sonopore treatment is applied.

[0230] Table 6: Summary of Results While preferred embodiments of the invention have been shown and described herein, such embodiments are provided by way of example only and will be apparent to those skilled in the art. Various modifications, variations, and substitutions will now be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. This means that the following claims define the scope of the invention, and the methods and structures within the scope of these claims and their equivalents are therefore covered.

Claims

1. A method for delivering a nucleic acid payload to target cells of a subject, comprising: a. Administering the nucleic acid construct containing the nucleic acid payload to the subject; b. Administer the sonoactive agent to the subject; c. Apply ultrasonic energy to the target cells with a first mechanical index (MI) of up to 0.4; and d. Apply ultrasonic energy to the target cells at a second MI greater than 1.3 and at most 2.

9.

2. A method for delivering a nucleic acid payload to target cells of a subject, comprising: a. Administering the nucleic acid construct containing the nucleic acid payload to the subject; b. Administer the sonoactive agent to the subject; c. Apply ultrasonic energy to the target cells with a first mechanical index (MI) of up to 0.4; and d. Apply ultrasonic energy to the target cells at a second MI of at least 2.

0.

3. A method for delivering a nucleic acid payload to target cells of a subject, comprising: a. Administering to the subject a nucleic acid construct containing the nucleic acid payload, wherein the nucleic acid construct is a microplasmid; b. Administer the sonoactive agent to the subject; c. Apply ultrasonic energy to the target cells with a first mechanical index (MI) of up to 0.4; and d. Apply ultrasonic energy to the target cells at a second MI greater than 0.4 and at most 2.

3.

4. The method of any one of claims 1 or 2, wherein the ultrasonic energy is applied to the target cells at a second MI greater than 1.5 and at most 2.

9.

5. The method of any one of claims 1 or 2, wherein the ultrasonic energy is applied to the target cells at a second MI greater than 1.8 and at most 2.

9.

6. The method of any one of claims 1 or 3, wherein the ultrasonic energy is applied to the target cells at a second MI of at least 2.

0.

7. The method of any one of claims 1-3, wherein the ultrasonic energy is applied to the target cell at a second MI of at least 2.

2.

8. The method of any one of claims 1 or 2, wherein the ultrasonic energy is applied to the target cells at a second MI of at least 2.

4.

9. The method of any one of claims 1 or 23, wherein the ultrasonic energy is applied to the target cells at a second MI of at least 2.

6.

10. The method of any one of claims 1 or 2, wherein the ultrasonic energy is applied to the target cells at a second MI of at least 2.

9.

11. The method of any one of claims 1 or 2, wherein the ultrasonic energy is applied to the target cells at a second MI greater than 2.2 and at most 2.

9.

12. The method of any one of claims 1 or 2, wherein the ultrasonic energy is applied to the target cells at a second MI greater than 2.6 and at most 2.

9.

13. The method of any of the preceding claims, wherein the ultrasound transducer that applies the ultrasound energy to the target cells is in continuous contact with the tissue of the subject and continuously (1) applies the ultrasound energy to the subject or (2) receives reflected ultrasound energy from the subject.

14. The method of any of the preceding claims, wherein the nucleic acid construct is a microplasmid, wherein the length of the microplasmid is less than or equal to 500 base pairs and does not include an expression cassette.

15. The method of any of the preceding claims, wherein the nucleic acid construct is administered systemically.

16. The method as claimed in any of the preceding claims, wherein 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.

17. The method as claimed in any of the preceding claims, wherein 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 4 to 18 times.

18. The method as claimed in any of the preceding claims, wherein 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 6 to 12 times.

19. The method as claimed in any of the preceding claims, wherein 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 8 to 10 times.

20. The method as claimed in any of the preceding claims, wherein 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 9 times.

21. The method as claimed in any of the preceding claims, wherein 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 9 times.

22. The method of any of the preceding claims, wherein the ultrasonic transducer remains in continuous contact with the subject during the application of the ultrasonic energy at the first MI and the application of the ultrasonic energy at the second MI.

23. The method of any of the preceding claims, wherein the ultrasonic transducer transmits ultrasonic acoustic energy or receives reflected ultrasonic acoustic energy for at least 95% of the time period during which the ultrasonic transducer is in continuous contact with the subject.

24. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse.

25. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of about 1 μs to about 500 μs.

26. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of about 100 μs to about 3300 μs.

27. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of about 200 μs.

28. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of up to 200 μs.

29. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of up to 500 μs.

30. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of about 1 μs to about 200 μs.

31. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with a pulse for a duration of about 1 μs to about 5 μs with the second MI.

32. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of about 2.3 µs.

33. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse for a duration of at least 2.3 µs.

34. The method of any of the preceding claims, wherein applying the ultrasonic acoustic energy with the first MI comprises initially applying the ultrasonic acoustic energy with the first MI for about 2 s to about 30 s.

35. The method as claimed in any of the preceding claims, further comprising repeatedly applying the ultrasonic acoustic energy at the first MI and at the second MI.

36. The method of claim 35, wherein the repetition comprises applying the ultrasonic energy at the first MI for a duration sufficient to allow reperfusion of the acoustic agent in the tissue containing the target cells.

37. The method of claim 35, wherein the repetition comprises applying the ultrasonic energy for 1-30 seconds at the first MI before repetition of applying the ultrasonic energy at the second MI.

38. The method of claim 35, wherein the repetition comprises applying the ultrasonic energy for 5-15 seconds at the first MI before applying the ultrasonic energy at the second MI.

39. The method as claimed in any of the preceding claims, wherein the repetition comprises applying the ultrasonic energy for 10 seconds at the first MI before applying the ultrasonic energy at the second MI.

40. The method as claimed in any of the preceding claims, wherein the total duration of the application of the ultrasonic energy in (c) and the ultrasonic energy in (d) ranges from about 1 s to about 60 m.

41. The method as claimed in any of the preceding claims, wherein the total duration of the application of the ultrasonic energy in (c) and the ultrasonic energy in (d) ranges from about 60 s to about 120 s.

42. The method as claimed in any of the preceding claims, wherein the range of the first MI is from about 0.05 to about 0.

3.

43. The method as claimed in any of the preceding claims, wherein the range of the first MI is from about 0.09 to about 0.

3.

44. The method as described in any of the preceding claims, wherein the range of the second MI is from about 1.0 to about 1.

8.

45. The method as claimed in any of the preceding claims, wherein the range of the second MI is from about 1.4 to about 1.

8.

46. ​​The method as claimed in any of the preceding claims, wherein the range of the second MI is from about 1.4 to about 2.

0.

47. The method of any of the preceding claims, wherein the microplasmid does not contain an antibiotic resistance gene.

48. The method of any of the preceding claims, wherein the microplasmid does not contain a bacterial genome.

49. The method of any of the preceding claims, wherein the nucleic acid construct enhances the expression of non-endogenous genes within the microplasmid.

50. The method of any of the preceding claims, wherein the method induces the expression of the nucleic acid payload in the target cells within 20 hours of applying the ultrasonic acoustic energy.

51. The method of any of the preceding claims, wherein the nucleic acid construct is used for gene enhancement, gene replacement, base editing, base knockdown, gene editing, gene knockdown, or gene removal.

52. The method of any of the preceding claims, wherein the nucleic acid construct is configured to enhance in vivo stability.

53. The method of any of the preceding claims, wherein the nucleic acid construct is administered at a dose of about 100 μg to about 200 μg.

54. The method of any of the preceding claims, wherein the nucleic acid construct is administered at a dose of about 0.5 mg / kg to about 32 mg / kg.

55. The method as claimed in any of the preceding claims, wherein about 2 × 10^13 to about 3 × 10^13 copies of the nucleic acid construct are administered to the subject.

56. The method of any of the preceding claims, wherein the microplasmid comprises a therapeutic transgene and / or a regulatory element.

57. The method of any of the preceding claims, wherein the acoustic agent is a microbubble.

58. The method of any of the preceding claims, wherein the ultrasonic energy applied by the first MI induces stable vibrational cavitation of the acoustic active agent.

59. The method of any of the preceding claims, wherein applying ultrasonic acoustic energy with the first MI does not induce substantial damage to the acoustic active agent (e.g., bursting or inertial cavitation).

60. The method of any of the preceding claims, wherein applying ultrasonic energy with the first MI does not induce substantial destruction of the acoustic agent in the vascular system space and the extravascular space, or induces stable vibrational cavitation of the acoustic agent in the vascular system space and the extravascular space.

61. The method of any of the preceding claims, wherein the ultrasonic energy applied by the second MI induces inertial cavitation of the acoustic active agent to destroy the acoustic active agent.

62. The method of any of the preceding claims, wherein the ultrasonic energy applied by the second MI induces inertial cavitation of the acoustic agent to disrupt the acoustic agent in the vascular space and the extravascular space.

63. The method of any of the preceding claims, wherein the extravascular space includes interstitial space, subcutaneous space, intramuscular interosseous space or lymphatic space.

64. The method of any of the preceding claims, wherein the extravascular space comprises extravascular tissue.

65. The method of any of the preceding claims, wherein the extravascular tissue comprises interstitial space, cytoplasmic space, subcutaneous tissue, lymphatic tissue, muscle, or a combination thereof.

66. The method as described in any of the preceding claims, wherein the method does not cause substantial cellular damage to the target cells.

67. The method of any of the preceding claims, wherein the method causes less than 1%, 5% or 10% of the target cells to undergo apoptosis.

68. The method of any of the preceding claims, wherein after the nucleic acid payload is delivered to the target cells of the subject, no of the following biomarkers of cell damage at the apoptosis level are detected: ALT, AST, IL6, BCL2 or combinations thereof, and optionally wherein the target cells are located in the liver.

69. The method of any of the preceding claims, wherein after the nucleic acid payload is delivered to the target cells of the subject, the following biomarkers of cell damage are not clinically elevated: ALT, AST, IL6, BCL2, or combinations thereof, and optionally wherein the target cells are located in the liver.

70. The method of any of the preceding claims, wherein after delivering the nucleic acid payload to the target cells of the subject, no ALT level exceeding 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 U / L is detected.

71. The method of any of the preceding claims, wherein no level of AST exceeding 225, 250, 275 or 300 U / L is detected after the nucleic acid payload is delivered to the target cells of the subject.

72. The method of any of the preceding claims, wherein after delivering the nucleic acid payload to the target cells of the subject, no level of IL6 exceeding 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, or 6 pg / mL is detected.

73. The method of any of the preceding claims, wherein the target cells are located in the liver.

74. The method of any of the preceding claims, wherein the target cells are located in the kidney.

75. The method of any of the preceding claims, wherein the target cells are located in the heart or skeletal muscle.

76. The method of any of the preceding claims, wherein the target cells are located in the brain.

77. The method of any of the preceding claims, wherein the target cells are located in the pancreas.

78. The method of any of the preceding claims, wherein the target cells are located in a tumor or are tumor cells.

79. The method of any of the preceding claims, wherein the ultrasonic energy is applied with the first mechanical index to induce the formation of intercellular or interendothelial spaces.

80. The method of any of the preceding claims, wherein the intercellular space or the interendothelial space is in the range of about 10 nm to about 10 μm.

81. The method as described in any of the preceding claims, further comprising moving the nucleic acid construct from the intravenous space into the interstitial space.

82. The method as described in any of the preceding claims, further comprising moving the nucleic acid construct from the interstitial space to the intracellular space.

83. The method of any of the preceding claims, wherein the stable vibrational cavitation of the acoustic active agent moves the nucleic acid construct from the intravenous space into the interstitial space.

84. The method of any of the preceding claims, wherein the inertial cavitation further moves the nucleic acid construct from the interstitial space into the intracellular space.

85. The method of any of the preceding claims, wherein the ultrasonic energy is applied with the second mechanical index to induce the formation of pores in the membrane of the cell.

86. The method of any of the preceding claims, wherein the pores in the membrane of the cell are formed in the range of about 10 nm to about 10 μm.

87. The method of any of the preceding claims, wherein the nucleic acid payload comprises a transgene.

88. The method of any of the preceding claims, wherein the genetic modification comprises a therapeutic genetic modification.

89. The method of any of the preceding claims, wherein the transgene comprises a detectable biomarker.

90. The method of any of the preceding claims, wherein the transgene comprises luciferase.

91. The method of any of the preceding claims, wherein the nucleic acid construct comprises a promoter sequence containing CAG.

92. The method of any of the preceding claims, wherein the nucleic acid construct comprises a promoter sequence containing ApoE.

93. The method of any of the preceding claims, wherein the nucleic acid construct comprises a promoter sequence containing SERP.

94. The method of any of the preceding claims, wherein the nucleic acid construct comprises a promoter sequence containing P3.

95. The method of any of the preceding claims, further comprising inducing the expression of the nucleic acid payload in the target cells.

96. The method of claim 95, wherein inducing the expression of the nucleic acid payload comprises inducing the expression of luciferase.

97. The method of claim 95, wherein inducing the expression of the nucleic acid payload comprises inducing a throughput of at least 10^6 p / s.

98. The method of claim 95, wherein inducing the expression of the nucleic acid payload comprises inducing a throughput of about 10^6 p / s to about 10^9 p / s.

99. The method of claim 95, wherein inducing the expression of the nucleic acid payload comprises inducing an expression at a flux of 2, 3, 4, or 5 times that induced without repeated application of the ultrasonic energy at the first MI and the ultrasonic energy at the second MI.

100. The method of claim 95, wherein inducing the expression of the nucleic acid payload comprises inducing the production of RNA encoded by the payload.

101. The method of claim 95, wherein inducing the expression of the nucleic acid payload comprises inducing the production of a protein encoded by the payload.

102. The method of any of the preceding claims, wherein the acoustic agent is applied at a concentration of about 5 x 10^8 to about 1.2 x 10^9 microstructures / mL.

103. The method of any of the preceding claims, wherein the acoustic agent comprises a lipid-stabilized microstructure.

104. The method of any of the preceding claims, wherein the acoustic agent comprises a phospholipid-stabilized microstructure.

105. The method of claim 104, wherein the acoustic agent comprises Sonazoid microbubbles.

106. The method of any of the preceding claims, wherein the phospholipid-stabilized microstructure comprises a high molecular weight gas core or a perflutran core.

107. The method of claim 106, wherein the acoustic agent is applied at a concentration of about 10^9 microstructures / mL.

108. The method of any of the preceding claims, wherein the acoustic agent is applied at a concentration of about 0.1 to about 20.0 mL / kg.

109. The method of any of the preceding claims, wherein the acoustic agent is applied at a concentration of about 0.1 to about 0.8 mg / kg.

110. The method of any of the preceding claims, wherein the acoustic agent comprises a protein-stabilized microstructure.

111. The method of claim 110, wherein the acoustic active agent comprises optison microbubbles.

112. The method of claim 110, wherein the acoustic agent is applied at a concentration of about 5 x 10^8 to about 8 x 10^8 microstructures / mL.

113. The method of any of the preceding claims, wherein the ultrasonic energy is applied at a distance of about 0.5 cm to about 20 cm from the target cell.

114. The method of any of the preceding claims, wherein the nucleic acid construct and the acoustic active agent are administered together.

115. The method of claim 114, wherein the nucleic acid construct and the acoustic active agent are mixed prior to co-application.

116. The method of any of the preceding claims, wherein the application of the nucleic acid construct and the acoustic active agent occurs continuously, in parallel, sequentially, or persistently.

117. The method of any of the preceding claims, wherein the application of the nucleic acid construct and the acoustic active agent occurs continuously.

118. The method as described in any of the preceding claims, wherein the application of the nucleic acid construct and the acoustic active agent occurs in parallel.

119. The method as described in any of the preceding claims, wherein the application of the nucleic acid construct and the acoustic active agent occurs sequentially.

120. The method of any of the preceding claims, wherein the application of the nucleic acid construct and the acoustic active agent occurs continuously.

121. The method of any of the preceding claims, wherein the administration of the nucleic acid construct and the acoustic active agent is by intravenous administration.

122. The method of any of the preceding claims, wherein the administration of the nucleic acid construct and the sonoactive agent is by intramuscular, subcutaneous, interosseous, or post-vesicle administration.

123. The method as described in any of the preceding claims, wherein inducing the expression of the nucleic acid payload comprises inducing expression within approximately 3 hours of administering the payload.

124. The method as claimed in any of the preceding claims, wherein inducing the expression of the nucleic acid payload comprises inducing expression within approximately 6 hours of administering the payload.

125. The method of any of the preceding claims, wherein inducing the expression of the nucleic acid payload comprises inducing expression within approximately 12 hours of administering the payload.

126. The method of any of the preceding claims, wherein inducing the expression of the nucleic acid payload comprises inducing expression in cells in the liver.

127. The method of any of the preceding claims, wherein inducing the expression of the nucleic acid payload comprises inducing expression in cells of the kidney.

128. The method as claimed in any of the preceding claims, further comprising inducing the expression of the nucleic acid payload and maintaining the expression of the protein encoded by the nucleic acid payload for at least 1, 2, 3, 4, 5, 6 or 7 days.

129. The method as claimed in any of the preceding claims, further comprising inducing the expression of the nucleic acid payload and maintaining the expression of the protein encoded by the nucleic acid payload for at least 1, 2, 3, 4, 5, 6 or 7 days.

130. The method as claimed in any of the preceding claims, wherein the method increases the persistence of expression of the protein encoded by the nucleic acid payload.

131. The method of any of the preceding claims, further comprising increasing the expression of the nucleic acid payload by increasing the dose of the nucleic acid payload applied to the subject.

132. The method of any of the preceding claims, further comprising increasing the expression of the nucleic acid payload by linearly increasing the dose of the nucleic acid payload applied to the subject.

133. The method as claimed in any of the preceding claims, further comprising increasing the expression of the nucleic acid payload by administering at least 5, 50, 250, or 500 μg of the nucleic acid payload to the subject.

134. The method of any of the preceding claims, wherein delivering the nucleic acid payload to the target cells of the subject increases or decreases gene expression in the target cells.

135. The method of any of the preceding claims, wherein delivering the nucleic acid payload to the target cell causes the copy number of the nucleic acid payload in each diploid genome to be at least 0.

15.

136. The method of any of the preceding claims, wherein delivering the nucleic acid payload to the target cell causes the copy number of the nucleic acid payload in each diploid genome to be at least 0.

2.

137. The method of any of the preceding claims, wherein delivering the nucleic acid payload to the target cell results in a copy number of the nucleic acid payload of 0.15 to 0.3 per diploid genome.

138. A reagent kit comprising: a. A first container containing microbubbles for sonoforming pore formation; and b. A second container containing microplasmids containing genetically modified organisms.

139. The kit of claim 138, wherein the microplasmid further comprises an expression cassette.

140. The kit of claim 138, wherein the first container and the second container are configured to induce expression of the transgene in the target cells of the subject within 20 hours after transfection.

141. The kit of claim 138, further comprising instructions for operating ultrasonic hardware and software parameters sufficient to destroy the acoustic active agent.

142. The kit of claim 138, further comprising instructions for administering the first container and the second container.

143. A system comprising: An ultrasonic transducer configured to apply ultrasonic acoustic energy to a subject with multiple mechanical indices; A computer system, including a computer processor and a computer-readable medium, wherein the computer system is configured to perform a method of applying ultrasonic acoustic energy to target cells of the subject, the method comprising: a. Apply ultrasonic energy to the target cells with a first mechanical index (MI) of up to 0.4; as well as b. Apply ultrasonic energy to the target cells at a second MI greater than 1.3 and at most 2.

9. The subjects were given (1) a nucleic acid construct containing a nucleic acid payload and (2) a sonoactive agent.

144. The system of claim 143, wherein the ultrasonic transducer applying the ultrasonic energy to the target cells is in continuous contact with the tissue of the subject and continuously (1) applies the ultrasonic energy to the subject or (2) receives reflected ultrasonic energy from the subject.

145. The system of claim 143, wherein the length of the nucleic acid construct is less than or equal to 500 base pairs, excluding the expression cassette.

146. The system of claim 143, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated at least twice.

147. The system of claim 143, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated 4 to 18 times.

148. The system of claim 143, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated 6 to 12 times.

149. The system of claim 143, wherein 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 8 to 10 times.

150. The system of claim 143, wherein the second MI ranges from about 1.4 to about 2.

0.

151. The system of claim 143, wherein the ultrasonic energy applied with the second mechanical index induces the formation of pores in the membrane of the cell.

152. The system of claim 143, wherein the ultrasonic energy applied with the first mechanical index induces the formation of intercellular or interendothelial space.

153. The system of claim 143, wherein the ultrasonic transducer transmits ultrasonic acoustic energy or receives reflected ultrasonic acoustic energy for at least 95% of the time period during which the ultrasonic transducer is in continuous contact with the subject.

154. The system of claim 143, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse.

155. The system of claim 143, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with a pulse for a duration of about 1 μs to about 500 μs with the second MI.

156. The system of claim 143, wherein applying the ultrasonic acoustic energy at the second MI comprises applying the ultrasonic acoustic energy at the second MI using a pulse for a duration of up to 200 μs.

157. The system of claim 143, wherein applying the ultrasonic acoustic energy at the second MI comprises applying the ultrasonic acoustic energy at the second MI using a pulse for a duration of up to 500 μs.

158. The system of claim 143, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with a pulse for a duration of about 1 μs to about 200 µs with the second MI.

159. The system of claim 143, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with a pulse for a duration of about 2.3 µs with the second MI.

160. The system of claim 143, further comprising repeatedly applying the ultrasonic acoustic energy at the first MI and at the second MI.

161. The system of claim 160, wherein the repetition comprises the duration for which the ultrasonic energy is applied at the first MI for a duration sufficient to allow reperfusion of the acoustic agent in the tissue containing the target cells.

162. The system of claim 160, wherein the repetition comprises applying the ultrasonic energy for 1-30 seconds at the first MI before repetition at the second MI.

163. The system of claim 160, wherein the repetition comprises applying the ultrasonic energy for 5-15 seconds at the first MI before applying the ultrasonic energy at the second MI.

164. The system of claim 160, wherein the repetition comprises applying the ultrasonic energy for 10 seconds at the first MI before applying the ultrasonic energy at the second MI.

165. A computer-readable medium configured to perform a method of applying ultrasonic acoustic energy to target cells of a subject, the method comprising: a. Apply ultrasonic energy to the target cells with a first mechanical index (MI) of up to 0.4; as well as b. Apply ultrasonic energy to the target cells at a second MI greater than 1.3 and at most 2.

9. The subjects were given (1) a nucleic acid construct containing a nucleic acid payload and (2) a sonoactive agent.

166. The computer-readable medium of claim 165, wherein the ultrasonic transducer that applies the ultrasonic energy to the target cells is in continuous contact with the tissue of the subject and continuously (1) applies the ultrasonic energy to the subject or (2) receives reflected ultrasonic energy from the subject.

167. The computer-readable medium of claim 165, wherein the length of the nucleic acid construct is less than or equal to 500 base pairs, excluding the expression cassette.

168. The computer-readable medium of claim 165, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated at least twice.

169. The computer-readable medium of claim 165, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated 4 to 18 times.

170. The computer-readable medium of claim 165, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated 6 to 12 times.

171. The computer-readable medium of claim 165, wherein the application of the ultrasonic acoustic energy at the first MI and the application of the ultrasonic acoustic energy at the second MI are repeated 8 to 10 times.

172. The computer-readable medium of claim 165, wherein the second MI ranges from about 1.4 to about 2.

0.

173. The computer-readable medium of claim 165, wherein the ultrasonic energy applied with the second mechanical index induces the formation of pores in the membrane of the cell.

174. The computer-readable medium of claim 165, wherein the ultrasonic energy applied with the first mechanical index induces the formation of intercellular or interendothelial space.

175. The computer-readable medium of claim 165, wherein the ultrasonic transducer transmits ultrasonic acoustic energy or receives reflected ultrasonic acoustic energy for at least 95% of the time period during which the ultrasonic transducer is in continuous contact with the subject.

176. The computer-readable medium of claim 165, wherein applying the ultrasonic acoustic energy with the second MI comprises applying the ultrasonic acoustic energy with the second MI using a pulse.

177. The computer-readable medium of claim 165, wherein applying the ultrasonic acoustic energy in the second MI comprises applying the ultrasonic acoustic energy in the second MI using a pulse with a duration of about 1 μs to about 500 µs.

178. The computer-readable medium of claim 165, wherein applying the ultrasonic acoustic energy at the second MI comprises applying the ultrasonic acoustic energy at the second MI using a pulse for a duration of up to 200 μs.

179. The computer-readable medium of claim 165, wherein applying the ultrasonic acoustic energy at the second MI comprises applying the ultrasonic acoustic energy at the second MI using a pulse for a duration of up to 500 μs.

180. The computer-readable medium of claim 165, wherein applying the ultrasonic acoustic energy in the second MI comprises applying the ultrasonic acoustic energy in the second MI using a pulse with a duration of about 1 μs to about 200 µs.

181. The computer-readable medium of claim 165, wherein applying the ultrasonic acoustic energy at the second MI comprises applying the ultrasonic acoustic energy at the second MI using a pulse for a duration of about 2.3 µs.

182. The computer-readable medium of claim 165, further comprising repeatedly applying the ultrasonic acoustic energy at the first MI and at the second MI.

183. The computer-readable medium of claim 182, wherein the repetition comprises the duration for which the ultrasonic acoustic energy is applied at the first MI for a duration sufficient to allow reperfusion of the acoustic agent in the tissue containing the target cells.

184. The computer-readable medium of claim 182, wherein the repetition comprises applying the ultrasonic energy for 1-30 seconds at the first MI before repetition at the second MI.

185. The computer-readable medium of claim 182, wherein the repetition comprises applying the ultrasonic energy for 5-15 seconds in the first MI before applying the ultrasonic energy in the second MI.

186. The computer-readable medium of claim 182, wherein the repetition comprises applying the ultrasonic energy for 10 seconds at the first MI before applying the ultrasonic energy at the second MI.

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