Methods and compositions for delivery of therapeutic agents via ultrasound

Abstract

Provided are nucleic acid delivery vectors configured to drive expression in multiple cell types, and methods of use. In some cases, the nucleic acid delivery vectors disclosed herein can prevent or reduce an immune response to a transgene expressed from a nucleic acid delivery vector, and which maintains expression of the transgene at a therapeutic level suitable for treating genetic disorders. One of the multiple cell types may be an LSEC.

Description

METHODS AND COMPOSITIONS FOR DELIVERY OF THERAPEUTIC AGENTS VIA ULTRASOUNDCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 709,962 filed 21-Oct-2024, and U.S. Provisional Patent Application No. 63 / 678,027 filed 31- Jul-2024, each of which is incorporated herein by reference in its entirety and for all purposes.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 62668-735601. XML, created July 25, 2025, which is 195 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Gene therapy, in which a functional copy of a gene is delivered (e.g., transfected) into a cell, has been proposed as a method of treating genetic diseases. However, methods of gene therapy using ultrasound or sonoporation may suffer from significant shortcomings such as low transfection rates, insufficient gene expression, and significant variability between subjects and methodologies, all which have limited the clinical development and commercialization. There remains a need for improved methods of administering gene therapy that can transfect a gene to a cell in an organ or a tissue of a subject in a safe, effective, and predictable manner.SUMMARY

[0004] While various ultrasound-based techniques have been developed for increasing delivery of exogenous payloads to cells, they often fail to achieve therapeutically relevant levels of gene expression when delivering nucleic acids, and fail to maintain therapeutically relevant levels of gene expression for extended periods. One barrier to achieving durable and therapeutic effect with ultrasound guided gene therapies is achieving and maintaining expression of nucleic acids to multiple target cell types within an organ, as ultrasound-based techniques are generally not cell specific, and most expression cassettes are not optimized to induce expression in multiple cell types. Another barrier to achieving durable and therapeutic effect with ultrasoundguided gene therapies and with gene therapy techniques more generally is immunogenicity to expression products of exogenous transgenes of the gene therapy, which in some cases can completely eliminate any detectable expression production of the transgene, preventing an effective therapy. Disclosed herein nucleic acid delivery vectors configured to induce expression of a nucleic acid in multiple cell types and methods of use in methods of ultrasound mediated delivery and sonoporation. Also disclosed herein are nucleic acid delivery vectors which prevent or reduce an immune response to a transgene expressed from a nucleic acid delivery vector, and which maintains expression of the transgene at a therapeutic level suitable for treating genetic disorders. Utilizing the nucleic acid delivery vectors, compositions, and methods disclosed herein, increased levels of gene expression for extended periods of time can be achieved.

[0005] Aspects disclosed herein provide a nucleic acid delivery vector preventing or reducing an immune response to a transgene, comprising: a first expression cassette encoding the transgene that selectively drives expression in a first cell type; a second expression cassette encoding the transgene that selectively drives expression in a second cell type, wherein expression of the transgene from the second cell type prevents or reduces the immune response to the transgene, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type, or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Aspects disclosed herein provide a nucleic acid delivery vector treating a genetic disorder, comprising: a first expression cassette encoding the transgene that selectively drives expression in a first cell type; a second expression cassette encoding the transgene that selectively drives expression in a second cell type, wherein the nucleic acid delivery vector maintains expression of the transgene at a therapeutic level suitable for treating the genetic disorder, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type, or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Aspects disclosed herein provide a nucleic acid delivery vector, comprising: a first expression cassette comprising a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in a first cell type; a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein: the nucleic acid delivery vector is at least 15 kb in length; the first cell types and second cell type are different; the second cell type is a liver sinusoidal epithelial cell (LSEC); the second promoter selectively drives expression of the transgene in the LSECs; the second expression cassette driving expression of the transgene from the LSECs prevents or reduces an immune response to an expression productof the transgene as measured at least a 5-fold reduction in antibody titers for an antibody binding the expression product in which the reduction in antibody titers is maintained for a period of at least 26 days, thereby maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Aspects disclosed herein provide a nucleic acid delivery vector comprising: a first expression cassette comprising: a first nucleic acid coding sequence encoding a first copy of a transgene; a first promoter sequence operably linked to the first nucleic acid coding sequence; a second expression cassette comprising: a second nucleic acid coding sequence encoding a second copy of the transgene; a second promoter sequence operably linked to the second nucleic acid coding sequence, wherein the nucleic acid delivery vector is at least 10.5 kb in length, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector. Aspects disclosed herein provide a nucleic acid delivery vector comprising: a first expression cassette comprising: a first nucleic acid coding sequence encoding a first copy of a transgene; a first promoter sequence operably linked to the first nucleic acid coding sequence; a second expression cassette comprising: a second nucleic acid coding sequence encoding a second copy of the transgene; a second promoter sequence operably linked to the second nucleic acid coding sequence, wherein the nucleic acid delivery vector is at least 10.5 kb in length, wherein the nucleic acid delivery vector lacks viral genomic coding sequences. Aspects disclosed herein provide a nucleic acid delivery vector configured to induce expression of a transgene in multiple cell types, the nucleic acid delivery vector comprising: a first expression cassette comprising: a first nucleic acid coding sequence encoding a first copy of the transgene; a first promoter sequence operably linked to the first nucleic acid coding sequence; a second expression cassette comprising: a second nucleic acid coding sequence encoding a second copy of the transgene; a second promoter sequence operably linked to the second nucleic acid coding sequence, wherein the first expression cassette is configured to induce expression of the transgene in a first cell type, and wherein the second expression cassette is configured to induce expression of the transgene in a second cell type. Aspects disclosed herein provide a kit comprising a nucleic acid delivery vector as disclosed herein, and a sonoactive agent. Aspects disclosed herein provide a kit comprising a nucleic acid delivery vector as disclosed herein, a sonoactive agent, and instructions for administering to a subject the sonoactive agent and ultrasound energy. Aspects disclosed herein provide a nucleic acid delivery vector for expressing a transgene in multiple cell types, comprising: a first expression cassettecomprising a first nucleic acid sequence encoding the transgene operable linked to a first promoter that selectively drives expression in a first cell type; a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein the first and second cell types are different.

[0006] In some embodiments, the first expression cassette comprises a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in the first cell type, and the second expression cassette comprises a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in the second cell type. In some embodiments, the nucleic acid delivery vector maintains expression of the transgene at a therapeutic level as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. In some embodiments, expression of the transgene from the first cell type prevents or reduces the immune response to the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. In some embodiments, the immune response is an adaptive immune response. In some embodiments, the immune response is an antibody immune response to an expression product of the transgene. In some embodiments, the nucleic acid delivery vector tolerizes an immune system of a subject to an expression product of the transgene. In some embodiments, an amount of antibodies binding an expression product of the transgene are reduced. In some embodiments, the amount of antibodies binding an expression product of the transgene as measured by antibody titers are reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is measured in comparison to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is maintained for a period of at least 7, 14, 21, 26, or 30 days following administration of the nucleic acid delivery vector. In some embodiments, an amount of antibodies binding an expression product of the transgene are within a range of plus or minus 10, 20, 30, 40 or 50 % of an amount of antibodies binding the transgene present in a naive subject not administered the nucleic acid delivery vector as measured by antibody titers. In some embodiments, of antibodies binding an expression product of the transgene as measured by antibody titers is maintained within the range for a period of at least 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 75, or 90 days following administration of the nucleic aciddelivery vector. In some embodiments, the second cell type is a liver cell. In some embodiments, the first cell type is not a liver cell. In some embodiments, the second promoter sequence selectively drives expression in LSECs. In some embodiments, the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55. In some embodiments, at least 20% of a total transfected cell population expressing the transgene are liver sinusoidal endothelial cells. In some embodiments, the first expression cassette is upstream or positioned 5’ of the second expression cassette. In some embodiments, the first expression cassette is downstream or positioned 3’ of the second expression cassette. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector. In some embodiments, the nucleic acid delivery vector lacks viral genomic coding sequences, or combinations thereof. In some embodiments, the first expression cassette is configured to induce preferentially expression of the transgene in a first cell type, and wherein the second expression cassette is configured to preferentially induce expression of the transgene in a second cell type. In some embodiments, the first copy of the transgene is expressed relative to the second copy of the transgene, at a higher level in the first cell type and the second copy of the transgene is, relative to the first copy of the transgene, expressed at a higher level in the second cell type. In some embodiments, the first promoter sequence and the second promoter sequence comprise different nucleic acid sequences. In some embodiments, the first expression cassette and the second expression cassette do not comprise any nucleic acid sequences which comprise 15 or more same nucleotides in sequence. In some embodiments, the vector is configured to induce expression of the transgene in different cell types. In some embodiments, the first expression cassette and second expression cassette are configured to induce expression of the transgene in different cell types. In some embodiments, the first expression cassette is configured to express of the first nucleic acid coding sequence in a first cell type, and wherein the second expression cassette is configured to express of the second nucleic acid coding sequence in a second cell type. In some embodiments, the first cell type is a hepatocyte. In some embodiments, the second cell type is a liver sinusoidal endothelial cell. In some embodiments, the first promoter sequence comprises an APOE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9. In some embodiments, the second promoter sequence comprises a F8 promoter sequence. In some embodiments, the transgene is FVIII. In some embodiments, the first nucleic acid sequence encoding the transgene comprises a first nucleic acid coding sequence, wherein the second nucleic acid sequence encoding the transgene comprises a second nucleic acid coding sequence.In some embodiments, at least 20% of a total transfected cell population expressing the FVIII transgene are liver sinusoidal endothelial cells.

[0007] In some embodiments, the nucleic acid delivery vector is at least 12.5 or 15 kb in length. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence are codon diversified. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence comprise different nucleic acid sequences but encode the same transgene. In some embodiments, the nucleic acid delivery vector is single stranded. In some embodiments, the nucleic acid delivery vector is double stranded. In some embodiments, the first expression cassette and the second expression cassette are both in a sense orientation the nucleic acid delivery vector. In some embodiments, the first expression cassette in the sense orientation in the nucleic acid delivery vector, and wherein the second expression cassette is in an anti-sense orientation in the nucleic acid delivery vector. In some embodiments, the first expression cassette further comprises an intron sequence. In some embodiments, the second expression cassette further comprises an intron sequence. In some embodiments, the intron sequence comprises a hemoglobin subunit gamma intron (hBGi) sequence. In some embodiments, the intron sequence in the first expression cassette and the intron sequence in the second expression cassette comprise different nucleic acid sequences. In some embodiments, the first expression cassette further comprises a posttranscriptional regulatory element. In some embodiments, the second expression cassette further comprises a posttranscriptional regulatory element. In some embodiments, the posttranscriptional regulatory element in the second expression cassette and the posttranscriptional regulatory element in the first expression cassette comprise different nucleic acid sequences. In some embodiments, the posttranscriptional regulatory element comprises a woodchuck hepatitis posttranscriptional regulatory element. In some embodiments, the posttranscriptional regulatory element comprises a polyadenylation signal. In some embodiments, the posttranscriptional regulatory element comprises a polyadenylation signal coupled downstream to a woodchuck hepatitis posttranscriptional regulatory element. In some embodiments, the nucleic acid delivery vector further comprises a nuclear targeting sequence. In some embodiments, nuclear targeting sequence is positioned downstream of the first expression cassette and upstream of the second expression cassette. In some embodiments, nuclear targeting sequence is positioned downstream of the second expression cassette. In some embodiments, the nucleic acid delivery vector increases expression of the transgene when administered to a subject as compared to a nucleic acid delivery vector comprising either the first expression cassette or the second expression cassette. In some embodiments, the nucleic acid delivery vector is a non-viral vector. In some embodiments, thenucleic acid delivery vector is a DNA vector. In some embodiments, the nucleic acid delivery vector is not comprised within a viral capsid. In some embodiments, the nucleic acid delivery vector is configured to be administered to a subject as unencapsulated DNA. In some embodiments, the nucleic acid delivery vector comprises any one of SEQ ID NO: 1-2, 4-6, 9, 16, 24, 32, 33, or 39. In some embodiments, the nucleic acid delivery vector comprises any one of SEQ ID NO: 47 or 49. In some embodiments, the nucleic acid delivery vector comprises SEQ ID NO: 47 and 49. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45 over a minimum alignment length of at least 6500, 7000, or 7500 nucleotides. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the nucleic acid delivery vector comprises SEQ ID NO: 45. In some embodiments, the first coding sequence or the second coding sequence encodes an amino acid sequence of SEQ ID NO. 24. Aspects disclosed herein provide a method of delivering a nucleic acid to a subject comprising administering to the subject the nucleic acid delivery vector of any one of the preceding embodiment. Aspects disclosed herein provide a method of treating a bleeding disorder in a subject in need thereof comprising administering to the subject the nucleic acid delivery vector of any one of the preceding embodiment wherein the transgene is FVIII. In some embodiments, the bleeding disorder is Hemophilia A. In some embodiments, nucleic acid delivery vector further comprises a third expression cassette comprising a third nucleic acid coding sequence encoding a third copy of a transgene. In some embodiments, the third expression cassette comprises a third promoter sequence operably linked to the third nucleic acid coding sequence. In some embodiments, nucleic acid delivery vector further comprises a fourth expression cassette comprising: a fourth nucleic acid coding sequence encoding a fourth copy of the transgene. In some embodiments, the fourth expression cassette comprises a fourth promoter sequence operably linked to the fourth nucleic acid coding sequence. Aspects disclosed herein provide a use of the nucleic acid delivery vector any one of the preceding embodiments for treating a genetic disorder in a subject in need thereof, comprising administering the nucleic acid delivery vector to the subject, thereby inducing expression of the transgene and treating the genetic disorder.

[0008] Aspects disclosed herein provide a method of preventing or reducing an immune response to an expression product of a transgene in a subject: administering to the subject a nucleic acid delivery vector inducing expression of the transgene in a first cell type and a second cell type in the subject, thereby reducing the immune response to the expression product of the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in asingle cell type. Aspects disclosed herein provide a method of treating a genetic disorder in a subject in need thereof, comprising: administering to the subject a nucleic acid delivery vector inducing expression of a transgene in a first cell type and a second cell type in the subject, thereby maintaining a therapeutic level of an expression product of the transgene in the subject and treating the genetic disorder. Aspects disclosed herein provide a method of inducing expression of a transgene in a subject comprising: administering to the subject a sonoactive agent; administering to the subject a nucleic acid delivery vector comprising; a first expression cassette comprising a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in a first cell type; a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein: the first cell types and second cell type are different and the second cell type is a liver sinusoidal epithelial cell (LSEC), the second promoter selectively drives expression of the transgene in the LSECs, and the nucleic acid delivery vector is at least 15 kb in length; applying ultrasound energy to tissue(s) of the subject comprising the first cell type and the second cell type, thereby inducing expression of the transgene in the first cell type and the second cell type, wherein the second expression cassette driving expression of the transgene from the LSECs prevents or reduces an immune response to an expression product of the transgene as measured at least a 5- fold reduction in antibody titers for an antibody binding the expression product in which the reduction in antibody titers is maintained for a period of at least 26 days, thereby maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Aspects disclosed herein provide a method of delivering a nucleic acid a first cell type and a second cell type in a subject using a nucleic acid delivery vector, the method comprising: administering to the subject a nucleic acid delivery vector comprising a first nucleic acid coding sequence encoding a first copy of a transgene and a second nucleic acid coding sequence encoding a second copy of a transgene; and administering to the subject a sonoactive agent and ultrasound energy, thereby inducing expression of the transgene in the first cell type and the second cell type. Aspects disclosed herein provide method of treating a bleeding disorder in a subject comprising administering to the subject a nucleic acid delivery vector inducing expression of a transgene encoding a human clotting factor in both a first cell type and a second cell type in the subject, thereby maintaining a therapeutic level of the human clotting factor in a plasma of the subject.

[0009] In some embodiments, expression of the transgene in both the first cell type and the second cell type maintains a therapeutic level of an expression product of the transgene in the subject and treats a genetic disorder. In some embodiments, inducing expression of the transgene in the first cell type and the second cell type in the subject reduces an immune response to the expression product of the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. In some embodiments, the immune response is an adaptive immune response. In some embodiments, the immune response is an antibody immune response to an expression product of the transgene. In some embodiments, expression of the transgene in the second cell type tolerizes an immune system of a subject to an expression product of the transgene. In some embodiments, an amount of antibodies binding an expression product of the transgene are reduced. In some embodiments, the amount of antibodies binding an expression product of the transgene as measured by antibody titers are reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is measured in comparison to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is maintained for a period of at least 7, 14, 21, 26, or 30 days following administration of the nucleic acid delivery vector In some embodiments, an amount of antibodies binding an expression product of the transgene are within a range of plus or minus 10, 20, 30, 40 or 50 % of an amount of antibodies binding the transgene present in a naive subject not administered the nucleic acid delivery vector as measured by antibody titers In some embodiments, of antibodies binding an expression product of the transgene as measured by antibody titers is maintained within the range for a period of at least 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 75, or 90 days following administration of the nucleic acid delivery vector. In some embodiments, the second cell type is a liver cell. In some embodiments, the first cell type is not a liver cell. In some embodiments, the second promoter sequence selectively drives expression in LSECs. In some embodiments, the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55. In some embodiments, the first promoter sequence comprises an ApoE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9. In some embodiments, the method further includes comprising administering to the subject a second dose of the sonoactive agent and the nucleic acid delivery vector, and applying ultrasound a second time. In some embodiments, the method further includes administering to the subject a third dose of the sonoactive agent and the nucleic acid deliveryvector, and applying ultrasound energy a third time. In some embodiments, the second dose is administered at least 24 hours after the initial administration of the sonoactive agent and the nucleic acid delivery vector, and initial application of the ultrasound energy. In some embodiments, the third dose is administered at least 24 hours after the second administration of the sonoactive agent and the nucleic acid delivery vector, and second application of the ultrasound energy. In some embodiments, the nucleic acid delivery vector comprises a first expression cassette comprising: the first nucleic acid coding sequence encoding the first copy of a transgene; and a first promoter sequence operably linked to the first nucleic acid coding sequence. In some embodiments, the nucleic acid delivery vector comprises a second expression cassette comprising: the second nucleic acid coding sequence encoding the first copy of a transgene; and a second promoter sequence operably linked to the first nucleic acid coding sequence. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector. In some embodiments, the nucleic acid delivery vector is at least 5, 7.5, 10, 12.5 or 15 kb in length kb in length. In some embodiments, the nucleic acid delivery vector lacks viral genomic coding sequences. In some embodiments, the nucleic acid delivery vector wherein the first expression cassette and the second expression cassette do not comprise any nucleic acid sequences which comprise 15 or more same nucleotides in sequence. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence are codon diversified. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence comprise different nucleic acid sequences but encode the same transgene. In some embodiments, the nucleic acid delivery vector is single stranded. In some embodiments, the nucleic acid delivery vector is double stranded. In some embodiments, the first expression cassette and the second expression cassette are both in a sense orientation the nucleic acid delivery vector. In some embodiments, the first expression cassette is in the sense orientation in the nucleic acid delivery vector, and wherein the second expression cassette is in an anti-sense orientation in the nucleic acid delivery vector. In some embodiments, the sonoactive agent comprises a microbubble or nanobubble filled with a perfluorinated gas. In some embodiments, the sonoactive agent comprises protein stabilized microstructures. In some embodiments, the sonoactive agent comprises a C3F8 gas encapsulated within a protein stabilized shell. In some embodiments, the sonoactive agent comprises lipid stabilized microstructures. In some embodiments, the sonoactive agent comprises an SF6 gas encapsulated within a lipid stabilized shell. In some embodiments, the sonoactive agent comprises a C4F10 gas encapsulated within a lipid stabilized shell. In some embodiments, ultrasound energy is applied at an MI of at least 0.8,1.3, 1.8, 1.9, or 2.2. In some embodiments, administering to the subject ultrasound energy comprises applying an acoustic radiation force. In some embodiments, the first copy of the transgene is expressed in the first cell type, and wherein the second copy of the transgene is expressed in the second cell type. In some embodiments, the first cell type is a hepatocyte. In some embodiments, the second cell type is a liver sinusoidal endothelial cell. Th In some embodiments, the first promoter sequence comprises an APOE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9. In some embodiments, the second promoter sequence comprises an F8 promoter sequence. In some embodiments, the transgene is FVIII. In some embodiments, the delivery to the first cell type and the second cell type occurs simultaneously. In some embodiments, the expression in the first cell type and the second cell type occurs simultaneously. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45 over a minimum alignment length of at least 6500, 7000, or 7500 nucleotides. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the nucleic acid delivery vector comprises SEQ ID NO: 45. In some embodiments, the first coding sequence or the second coding sequence encodes an amino acid sequence of SEQ ID NO. 24. In some embodiments, the nucleic acid delivery vector is any nucleic acid delivery vector disclosed herein. In some embodiments, nucleic acid delivery vector further comprises a third expression cassette comprising a third nucleic acid coding sequence encoding a third copy of a transgene. In some embodiments, the third expression cassette comprises a third promoter sequence operably linked to the third nucleic acid coding sequence. In some embodiments, nucleic acid delivery vector further comprises a fourth expression cassette comprising: a fourth nucleic acid coding sequence encoding a fourth copy of the transgene. In some embodiments, the fourth expression cassette comprises a fourth promoter sequence operably linked to the fourth nucleic acid coding sequence. In some embodiments, the nucleic acid delivery vector comprises a first nucleic acid coding sequence encoding a first copy of a transgene and a second nucleic acid coding sequence encoding a second copy of the transgene. In some embodiments, the transgene comprises hFVIII, hFIX, or von-Willebrand factor, or Von Willebrand disease. In some embodiments, the bleeding disorder comprises hemophilia A, hemophilia B. In some embodiments, the transgene comprises hFVIII, wherein the bleeding disorder comprises hemophilia A, and wherein an average level of the human clotting factor in the plasma does not fall below thirty percent (30%) of a peak hFVIII plasma level induced by administration of the nucleic acid delivery vector. In some embodiments, the average level of the human clotting factor is assessed over one or more 30-day periods, and the peak fFVIII plasma level is a maximum discreet value within said one ormore 3O-day periods, each following administration of the nucleic acid delivery vector. In some embodiments, the transgene comprises hFVIII, wherein the bleeding disorder comprises hemophilia A, and wherein an average level of the human clotting factor in the plasma does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state hFVIII plasma level induced by administration of the nucleic acid delivery vector. In some embodiments, the average level of the human clotting factor and the average steady state hFVIII plasma level are assessed over one or more 30-day periods following administration of the nucleic acid delivery vector. In some embodiments, the therapeutic level of the human clotting factor is maintained for at least 30, 60, 70, 80, 90,100, 110 or 120 days following administration of the nucleic acid delivery vector. In some embodiments, at least 20% of a total population of cells expressing the transgene are liver sinusoidal endothelial cells. In some embodiments, an average expression level of the transgene does not fall below thirty percent (30%) of an expression level of the transgene induced by administration of the nucleic acid delivery vector. In some embodiments, the average expression level of the transgene and the peak average expression level of the transgene over one or more 30-day periods, and a peak expression level is a maximum discreet value within said one or more 30-day periods, each following administration of the nucleic acid delivery vector. In some embodiments, an average expression level of the transgene does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state expression level of the transgene induced by administration of the nucleic acid delivery vector. In some embodiments, the average expression level of the transgene and the average steady state expression level of the transgene are assessed over one or more 30-day periods following administration of the nucleic acid delivery vector. In some embodiments, expression of the transgene is maintained for at least 30, 60, 70, 80, 90,100, 110 or 120 days following administration of the nucleic acid delivery vector. In some embodiments, expression level of the transgene is determined by mRNA production, a reporter gene, or transgene protein production. Aspects disclosed herein provide a use of a nucleic acid delivery vector for treating a genetic disorder in a subject in need thereof, comprising administering the nucleic acid delivery vector to the subject in accordance with the method of any one of the preceding embodiments , thereby inducing expression of the transgene and treating the genetic disorder.

[0010] Aspects disclosed herein provide a nucleic acid having a sequence of any one of SEQ ID NO: 1-55. Aspects disclosed herein provide a nucleic acid having a sequence of SEQ ID NO: 9 or 50. In some embodiments, the sequence is a promoter sequence. In some embodiments, the sequence is SEQ ID NO: 50, wherein the sequence selectively drives gene expression in LSECs.INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0013] FIG. 1 illustrates protein expression levels in a murine model of sonoporation resulting from administration of a lower dose of the nucleic acid delivery vectors disclosed herein;

[0014] FIG. 2 illustrates protein expression levels in a murine model of sonoporation resulting from administration of a larger dose the nucleic acid delivery vectors disclosed herein;

[0015] FIG. 3 illustrates protein expression levels in a murine model of sonoporation resulting from administration of a larger dose nucleic acid delivery vectors disclosed herein over an extended period following treatment;

[0016] FIG. 4A illustrates the protein expression levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein;

[0017] FIG. 4B illustrates the protein expression levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein;

[0018] FIG. 5 illustrates an exemplary nucleic acid delivery vector as disclosed herein;

[0019] FIG. 6 illustrates data showing the identity and percentage of cells transfected by the nucleic acid delivery vectors disclosed herein;

[0020] FIG. 7A illustrates protein expression levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein;

[0021] FIG 7B illustrates protein expression levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein;

[0022] FIG. 8A illustrates protein expression levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein;

[0023] FIG. 8B illustrates protein expression levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein;

[0024] FIG. 9 illustrates antibody titer levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein; and

[0025] FIG. 10 illustrates antibody titer levels in a murine model of sonoporation resulting from administration of a dose nucleic acid delivery vectors disclosed herein.DETAILED DESCRIPTION

[0026] While various ultrasound-based techniques have been developed for increasing delivery of exogenous payloads to cells, they often fail to achieve therapeutically relevant levels of gene expression when delivering nucleic acids, and fail to maintain therapeutically relevant levels of gene expression for extended periods. One barrier to achieving durable and therapeutic effect with ultrasound guided gene therapies is achieving and maintaining expression of nucleic acids to multiple target cell types within an organ, as ultrasound-based techniques are generally not cell specific, and most expression cassettes are not optimized to induce expression in multiple cell types. Another barrier to achieving durable and therapeutic effect with ultrasound guided gene therapies and with gene therapy techniques more generally is immunogenicity to expression products of exogenous transgenes of the gene therapy, which in some cases can completely eliminate any detectable expression production of the transgene, preventing an effective therapy. Disclosed herein nucleic acid delivery vectors configured to induce expression of a nucleic acid in multiple cell types and methods of use in methods of ultrasound mediated delivery and sonoporation. Also disclosed herein are nucleic acid delivery vectors which prevent or reduce an immune response to a transgene expressed from a nucleic acid delivery vector, and which maintains expression of the transgene at a therapeutic level suitable for treating genetic disorders. Utilizing the nucleic acid delivery vectors, compositions, and methods disclosed herein, increased levels of gene expression for extended periods of time can be achieved.

[0027] Gene therapy delivery platforms, for example, viral vectors or lipid nanoparticles, are often specific to a particular organ and cell type. Further, many such platforms are also constrained by payload size, which limits the ability to design larger and more sophisticated payloads. By using nucleic acid delivery vectors configured to induce expression of a nucleic acid in multiple cell types, it is possible to transfect multiple cell types simultaneously in a single sonoporation treatment using only a single nucleic acid delivery vector. Moreover, it isalso possible to simultaneously drive expression of the therapeutic protein in multiple cell types within an organ simultaneously, increasing the amount of therapeutic protein produced, the stability of the resulting gene expression, as well as the durability of the gene expression. In some cases, bicistronic nucleic acid delivery vectors with a first expression cassette encoding the transgene that selectively drives expression in a first cell type, and a second expression cassette encoding the transgene that selectively drives expression in a second cell type, can prevent or reduce an immune response to a transgene expressed from a nucleic acid delivery vector, and which maintains expression of the transgene at a therapeutic level suitable for treating genetic disorders. By expressing the transgene from second cell type, for example a liver cell type such as the liver sinusoidal endothelial cells (LSECs), the immune system of a subject can be tolerized to the expression product of the transgene, and an adaptive immune response which would otherwise prevent ongoing expression of the transgene can be reduced or prevented. As is disclosed herein, a nucleic acid delivery vector comprising multiple expression cassettes optimized for targeting multiple cell types provided as a continuous nucleic acid construct can these provide beneficial technical effects, and can be reliably and successfully transfected to target cells using ultrasound-based techniques, including sonoporation.I. COMPOSITIONS

[0028] Immunogenicity remains a challenges in gene therapy which limiting the therapeutic effect of successfully transfected nucleic acid products. For example, when therapeutic expression products of an exogenous transgene are perceived as foreign, triggering pathogenic B- and T-cell responses that erode efficacy and prevent ongoing transgene expression. In some cases, for instance when the patient lacks endogenous expression, which is common in many monogenic disorders, the first appearance of the protein can prime naive CD4+T cells, which in turn activate B cells to class-switch and secrete high-affinity, neutralizing IgG antibodies that result in protein clearance. Further, peptides derived from the newly synthesized can be presented by MHC I complexes of transduced cells, and where the epitopes are sufficiently divergent from the host’s tolerized repertoire, cytotoxic CD8+T lymphocytes can be activated, leading to targeted lysis of transfected cells, and loss of therapeutic benefit. Such adaptive mechanisms necessitate improvements to gene therapy vectors and methods of use to provide for ongoing therapeutic benefit of successfully delivered gene therapies.

[0029] Aspects disclosed herein provide a nucleic acid delivery vector preventing or reducing an immune response to a transgene, comprising: a first expression cassette encoding the transgene that selectively drives expression in a first cell type; a second expression cassette encoding the transgene that selectively drives expression in a second cell type, whereinexpression of the transgene from the second cell type prevents or reduces the immune response to the transgene, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type, or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Aspects disclosed herein provide a nucleic acid delivery vector treating a genetic disorder, comprising: a first expression cassette encoding the transgene that selectively drives expression in a first cell type; a second expression cassette encoding the transgene that selectively drives expression in a second cell type, wherein the nucleic acid delivery vector maintains expression of the transgene at a therapeutic level suitable for treating the genetic disorder, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type, or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Aspects disclosed herein provide a nucleic acid delivery vector, comprising: a first expression cassette comprising a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in a first cell type; a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein: the nucleic acid delivery vector is at least 15 kb in length; the first cell types and second cell type are different; the second cell type is a liver sinusoidal epithelial cell (LSEC); the second promoter selectively drives expression of the transgene in the LSECs; the second expression cassette driving expression of the transgene from the LSECs prevents or reduces an immune response to an expression product of the transgene as measured at least a 5-fold reduction in antibody titers for an antibody binding the expression product in which the reduction in antibody titers is maintained for a period of at least 26 days, thereby maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. Without being bound to a particular theory, expressing the transgene in liver sinusoidal endothelial cells (LSECs) may exploits their intrinsically tolerogenic antigen-presentation pathway, which preferentially induces regulatory T cells and deletes reactive effector T cells, and prevents neutralizing antibody formation and cytotoxic T-cell responses against the therapeutic protein, enabling durable and higher systemic levels of expression without the need for broad immunosuppression.

[0030] In some embodiments, the first expression cassette comprises a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in the first cell type, and the second expression cassette comprises a second nucleicacid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in the second cell type. In some embodiments, the nucleic acid delivery vector maintains expression of the transgene at a therapeutic level as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. In some embodiments, expression of the transgene from the first cell type prevents or reduces the immune response to the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. In some embodiments, the immune response is an adaptive immune response. In some embodiments, the immune response is an antibody immune response to an expression product of the transgene. In some embodiments, the nucleic acid delivery vector tolerizes an immune system of a subject to an expression product of the transgene. In some embodiments, an amount of antibodies binding an expression product of the transgene are reduced. In some embodiments, the amount of antibodies binding an expression product of the transgene as measured by antibody titers are reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is measured in comparison to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is maintained for a period of at least 7, 14, 21, 26, or 30 days following administration of the nucleic acid delivery vector. In some embodiments, an amount of antibodies binding an expression product of the transgene are within a range of plus or minus 10, 20, 30, 40 or 50 % of an amount of antibodies binding the transgene present in a naive subject not administered the nucleic acid delivery vector as measured by antibody titers. In some embodiments, of antibodies binding an expression product of the transgene as measured by antibody titers is maintained within the range for a period of at least 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 75, or 90 days following administration of the nucleic acid delivery vector. In some embodiments, the second cell type is a liver cell. In some embodiments, the first cell type is not a liver cell. In some embodiments, the second promoter sequence selectively drives expression in LSECs. In some embodiments, at least 20% of a total transfected cell population expressing the transgene are liver sinusoidal endothelial cells. In some embodiments, the first expression cassette is upstream or positioned 5’ of the second expression cassette. In some embodiments, the first expression cassette is downstream or positioned 3’ of the second expression cassette. In some cases, the second promoter sequence selectively driving expression in LSECs provides a beneficial technical effect of preventing or reducing an immuneresponse to a transgene expressed from a nucleic acid delivery vector, and maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder. In some embodiments, the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55. In some cases, the second promoter sequence of SEQ ID NO: 50 selectively driving expression in LSECs provides a beneficial technical effect of preventing or reducing an immune response to a transgene expressed from a nucleic acid delivery vector, and maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder.

[0031] Aspects disclosed herein provide a nucleic acid delivery vector comprising: a first expression cassette comprising: a first nucleic acid coding sequence encoding a first copy of a transgene; a first promoter sequence operably linked to the first nucleic acid coding sequence; a second expression cassette comprising: a second nucleic acid coding sequence encoding a second copy of the transgene; a second promoter sequence operably linked to the second nucleic acid coding sequence, wherein the nucleic acid delivery vector is at least 10.5 kb in length, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector. Aspects disclosed herein provide a nucleic acid delivery vector comprising: a first expression cassette comprising: a first nucleic acid coding sequence encoding a first copy of a transgene; a first promoter sequence operably linked to the first nucleic acid coding sequence; a second expression cassette comprising: a second nucleic acid coding sequence encoding a second copy of the transgene; a second promoter sequence operably linked to the second nucleic acid coding sequence, wherein the nucleic acid delivery vector is at least 10.5 kb in length, wherein the nucleic acid delivery vector lacks viral genomic coding sequences. Aspects disclosed herein provide a nucleic acid delivery vector configured to induce expression of a transgene in multiple cell types, the nucleic acid delivery vector comprising: a first expression cassette comprising: a first nucleic acid coding sequence encoding a first copy of the transgene; a first promoter sequence operably linked to the first nucleic acid coding sequence; a second expression cassette comprising: a second nucleic acid coding sequence encoding a second copy of the transgene; a second promoter sequence operably linked to the second nucleic acid coding sequence, wherein the first expression cassette is configured to preferentially induce expression of the transgene in a first cell type, and wherein the second expression cassette is configured to preferentially induce expression of the transgene in a second cell type. In some embodiments, the first copy of the transgene is expressed relative to the second copy of the transgene, at a higher level in the first cell type and the second copy of the transgene is, relative to the first copy of the transgene, expressed at a higher level in the second cell type. In some embodiments, the second cell type isan LSEC. In some cases, the second cell type being an LSEC provides a beneficial technical effect of preventing or reducing an immune response to a transgene expressed from a nucleic acid delivery vector, and maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder. In some cases, the second promoter sequence of SEQ ID NO: 50 selectively driving expression in LSECs provides a beneficial technical effect of preventing or reducing an immune response to a transgene expressed from a nucleic acid delivery vector, and maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder.

[0032] In some embodiments, the nucleic acid delivery vector is at least 12.5 or 15 kb in length. In some cases, the nucleic acid constructs disclosed herein are configured for delivery using ultrasound and sonoactive agents and achieve high levels of transfection notwithstanding a large payload size of at least 12.5 or 15 kb. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector. In some cases, the nucleic acid constructs disclosed herein are configured for delivery using ultrasound and sonoactive agents and achieve high levels of transfection notwithstanding very long coding sequences collectively comprise at least 50, 60, 65, 70, or 75% of total nucleotides in the nucleic acid delivery vector. In some embodiments, the nucleic acid delivery vector lacks viral genomic coding sequences, or combinations thereof. In some embodiments, the first expression cassette is configured to preferentially induce expression of the transgene in a first cell type, and wherein the second expression cassette is configured to preferentially induce expression of the transgene in a second cell type. In some embodiments, the first copy of the transgene is expressed relative to the second copy of the transgene, at a higher level in the first cell type and the second copy of the transgene is, relative to the first copy of the transgene, expressed at a higher level in the second cell type. In some embodiments, the first promoter sequence and the second promoter sequence comprise different nucleic acid sequences. In some embodiments, the first expression cassette and the second expression cassette do not comprise any nucleic acid sequences which comprise 15 or more same nucleotides in sequence. In some cases, the lack of repeated coding sequence or codon diversified sequences among the expression cassettes provides a beneficial technical effect of driving expression in different cell types. In some embodiments, the vector is configured to induce expression of the transgene in different cell types. In some embodiments, the first expression cassette and second expression cassette are configured to induce expression of the transgene in different cell types. In some embodiments, the first expression cassette is configured to express of the first nucleic acid coding sequence in a first cell type, and whereinthe second expression cassette is configured to express of the second nucleic acid coding sequence in a second cell type. In some embodiments, the first cell type is a hepatocyte. In some embodiments, the second cell type is a liver sinusoidal endothelial cell. In some embodiments, the first promoter sequence comprises an ApoE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9. In some embodiments, the second promoter sequence comprises an F8 promoter sequence. In some embodiments, the transgene is FVIII. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence are codon diversified. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence comprise different nucleic acid sequences but encode the same transgene. In some embodiments, the nucleic acid delivery vector is single stranded. In some embodiments, the nucleic acid delivery vector is double stranded. In some embodiments, the first expression cassette and the second expression cassette are both in a sense orientation the nucleic acid delivery vector. In some embodiments, the first expression cassette is in the sense orientation in the nucleic acid delivery vector, and wherein the second expression cassette is in an anti-sense orientation in the nucleic acid delivery vector. In some embodiments, the first expression cassette further comprises an intron sequence. In some embodiments, the second expression cassette further comprises an intron sequence. In some embodiments, the intron sequence comprises a hemoglobin subunit gamma intron (hBGi) sequence. In some embodiments, the intron sequence in the first expression cassette and the intron sequence in the second expression cassette comprise different nucleic acid sequences. In some embodiments, the first expression cassette further comprises a posttranscriptional regulatory element. In some embodiments, the second expression cassette further comprises a posttranscriptional regulatory element. In some embodiments, the posttranscriptional regulatory element in the second expression cassette and the posttranscriptional regulatory element in the first expression cassette comprise different nucleic acid sequences. In some embodiments, the posttranscriptional regulatory element comprises a woodchuck hepatitis posttranscriptional regulatory element. In some embodiments, the posttranscriptional regulatory element comprises a polyadenylation signal. In some embodiments, the posttranscriptional regulatory element comprises a polyadenylation signal coupled downstream to a woodchuck hepatitis posttranscriptional regulatory element. In some embodiments, the nucleic acid delivery vector further comprises a nuclear targeting sequence. In some embodiments, nuclear targeting sequence is positioned downstream of the first expression cassette and upstream of the second expression cassette. In some embodiments, nuclear targeting sequence is positioned downstream of the second expression cassette. In some embodiments, the nucleic acid delivery vector increases expression of the transgene when administered to a subjectas compared to a nucleic acid delivery vector comprising either the first expression cassette or the second expression cassette. In some embodiments, the nucleic acid delivery vector is a non- viral vector. In some embodiments, the nucleic acid delivery vector is a DNA vector. In some embodiments, the nucleic acid delivery vector is not comprised within a viral capsid. In some embodiments, the nucleic acid delivery vector is configured to be administered to a subject as unencapsulated DNA. In some embodiments, the nucleic acid delivery vector comprises any one of SEQ ID NO: 1-2, 4-6, 9, 16, 24, 32, 33, or 39. In some embodiments, the nucleic acid delivery vector comprises any one of SEQ ID NO: 47 or 49. In some embodiments, the nucleic acid delivery vector comprises SEQ ID NO: 47 and 49. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45 over a minimum alignment length of at least 6500, 7000, or 7500 nucleotides. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the nucleic acid delivery vector comprises SEQ ID NO: 45. In some embodiments, the first coding sequence or the second coding sequence encodes an amino acid sequence of SEQ ID NO. 24. Aspects disclosed herein provide a method of delivering a nucleic acid to a subject comprising administering to the subject the nucleic acid delivery vector of any one of the preceding embodiment. Aspects disclosed herein provide a method of treating a bleeding disorder in a subject in need thereof comprising administering to the subject the nucleic acid delivery vector of any one of the preceding embodiment wherein the transgene is FVIII. In some embodiments, the bleeding disorder is Hemophilia. In some embodiments, nucleic acid delivery vector further comprises a third expression cassette comprising a third nucleic acid coding sequence encoding a third copy of a transgene. In some embodiments, the third expression cassette comprises a third promoter sequence operably linked to the third nucleic acid coding sequence. In some embodiments, nucleic acid delivery vector further comprises a fourth expression cassette comprising: a fourth nucleic acid coding sequence encoding a fourth copy of the transgene. In some embodiments, the fourth expression cassette comprises a fourth promoter sequence operably linked to the second nucleic acid coding sequence.

[0033] Aspects disclosed herein provide a nucleic acid delivery vector for expressing a transgene in multiple cell types, comprising: a first expression cassette comprising a first nucleic acid encoding the transgene operable linked to a first promoter that selectively drives expression in a first cell type; a second expression cassette comprising a second nucleic acid encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein the first and second cell types are different. In some embodiments, the first nucleic acid encoding the transgene comprises a first nucleic acid codingsequence, wherein the second nucleic acid encoding the transgene comprises a second nucleic acid coding sequence. In some embodiments, at least 20% of a total transfected cell population expressing the FVIII transgene are liver sinusoidal endothelial cells. Without being bound to a particular theory, the nucleic acid delivery vectors disclosed herein which are configured to express the therapeutic transgene in multiple cell types can provide for more stable and durable gene expression due to immune tolerization of the therapeutic transgene being expressed in multiple cell types in the target organ. In some cases, inducing expression of the therapeutic transgene from the hepatocytes and the LSECs can lead to a reduced immune response to the therapeutic transgene as compared to expression for only or predominantly a single cell type, and provides a beneficial technical effect of improved gene expression, including, for example, more stable gene expression and more durable gene expression.II. METHODS

[0034] Aspects disclosed herein provide a use of the nucleic acid delivery vector any one of the preceding embodiments for treating a genetic disorder in a subject in need thereof, comprising administering the nucleic acid delivery vector to the subject, thereby inducing expression of the transgene and treating the genetic disorder. Aspects disclosed herein provide a method of preventing or reducing an immune response to an expression product of a transgene in a subject: administering to the subject a nucleic acid delivery vector inducing expression of the transgene in a first cell type and a second cell type in the subject, thereby reducing the immune response to the expression product of the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. Aspects disclosed herein provide a method of treating a genetic disorder in a subject in need thereof, comprising: administering to the subject a nucleic acid delivery vector inducing expression of a transgene in a first cell type and a second cell type in the subject, thereby maintaining a therapeutic level of an expression product of the transgene in the subject and treating the genetic disorder. Aspects disclosed herein provide a method of inducing expression of a transgene in a subject comprising: administering to the subject a sonoactive agent; administering to the subject a nucleic acid delivery vector comprising; a first expression cassette comprising a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in a first cell type; a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein: the first cell types and second cell type are different and the second cell type is a liver sinusoidal epithelial cell (LSEC), the second promoter selectively drives expression of the transgene in the LSECs, and the nucleic acid delivery vectoris at least 15 kb in length; applying ultrasound energy to tissue(s) of the subject comprising the first cell type and the second cell type, thereby inducing expression of the transgene in the first cell type and the second cell type, wherein the second expression cassette driving expression of the transgene from the LSECs prevents or reduces an immune response to an expression product of the transgene as measured at least a 5-fold reduction in antibody titers for an antibody binding the expression product in which the reduction in antibody titers is maintained for a period of at least 26 days, thereby maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder, as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

[0035] In some embodiments, expression of the transgene in both the first cell type and the second cell type maintains a therapeutic level of an expression product of the transgene in the subject and treats a genetic disorder. In some embodiments, inducing expression of the transgene in the first cell type and the second cell type in the subject reduces an immune response to the expression product of the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type. In some embodiments, the immune response is an adaptive immune response. In some embodiments, the immune response is an antibody immune response to an expression product of the transgene. In some embodiments, expression of the transgene in the second cell type tolerizes an immune system of a subject to an expression product of the transgene. In some embodiments, an amount of antibodies binding an expression product of the transgene are reduced. In some embodiments, the amount of antibodies binding an expression product of the transgene as measured by antibody titers are reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is measured in comparison to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. In some embodiments, the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is maintained for a period of at least 7, 14, 21, 26, or 30 days following administration of the nucleic acid delivery vector In some embodiments, an amount of antibodies binding an expression product of the transgene are within a range of plus or minus 10, 20, 30, 40 or 50 % of an amount of antibodies binding the transgene present in a naive subject not administered the nucleic acid delivery vector as measured by antibody titers In some embodiments, of antibodies binding an expression product of the transgene as measured by antibody titers is maintained within the range for a period of at least 7,14, 21, 28, 35, 42, 49, 56, 63, 70, 75, or 90 days following administration of the nucleic acid delivery vector. In some embodiments, the second cell type is a liver cell. In some embodiments, the first cell type is not a liver cell. In some embodiments, the second promoter sequence selectively drives expression in LSECs. In some embodiments, the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55. In some embodiments, the first promoter sequence comprises an ApoE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9. In some embodiments, the method further includes comprising administering to the subject a second dose of the sonoactive agent and the nucleic acid delivery vector, and applying ultrasound a second time. In some embodiments, the method further includes administering to the subject a third dose of the sonoactive agent and the nucleic acid delivery vector, and applying ultrasound energy a third time. In some embodiments, the second dose is administered at least 24 hours after the initial administration of the sonoactive agent and the nucleic acid delivery vector, and initial application of the ultrasound energy. In some embodiments, the third dose is administered at least 24 hours after the second administration of the sonoactive agent and the nucleic acid delivery vector, and second application of the ultrasound energy. In some cases, inducing expression of the transgene in LSECs provides a beneficial technical effect of preventing or reducing an immune response to the transgene expressed from a nucleic acid delivery vector, and maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder. In some embodiments, the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55. In some cases, the second promoter sequence of SEQ ID NO: 50 selectively driving expression in LSECs provides a beneficial technical effect of preventing or reducing an immune response to a transgene expressed from a nucleic acid delivery vector, and maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder. Without being bound to a particular theory, expressing the transgene in liver sinusoidal endothelial cells (LSECs) may exploits their intrinsically tolerogenic antigen-presentation pathway, which preferentially induces regulatory T cells and deletes reactive effector T cells, and prevents neutralizing antibody formation and cytotoxic T-cell responses against the therapeutic protein, enabling durable and higher systemic levels of expression without the need for broad immunosuppression.

[0036] Aspects disclosed herein provide a method of delivering a nucleic acid a first cell type and a second cell type in a subject using a nucleic acid delivery vector, the method comprising: administering to the subject a nucleic acid delivery vector comprising a first nucleic acid coding sequence encoding a first copy of a transgene and a second nucleic acid codingsequence encoding a second copy of a transgene; and administering to the subject a sonoactive agent and ultrasound energy, thereby inducing expression of the transgene in the first cell type and the second cell type. In some embodiments, the nucleic acid delivery vector comprises a first expression cassette comprising: the first nucleic acid coding sequence encoding the first copy of a transgene; and a first promoter sequence operably linked to the first nucleic acid coding sequence. In some embodiments, the nucleic acid delivery vector comprises a second expression cassette comprising: the second nucleic acid coding sequence encoding the first copy of a transgene; and a second promoter sequence operably linked to the first nucleic acid coding sequence. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector. In some embodiments, the nucleic acid delivery vector is at least 5, 7.5, 10, 12.5 or 15 kb in length kb in length. In some embodiments, the nucleic acid delivery vector lacks viral genomic coding sequences. In some embodiments, the nucleic acid delivery vector wherein the first expression cassette and the second expression cassette do not comprise any nucleic acid sequences which comprise 15 or more same nucleotides in sequence. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence are codon diversified. In some embodiments, the first nucleic acid coding sequence and the second nucleic acid coding sequence comprise different nucleic acid sequences but encode the same transgene. In some embodiments, the nucleic acid delivery vector is single stranded. In some embodiments, the nucleic acid delivery vector is double stranded. In some embodiments, the first expression cassette and the second expression cassette are both in a sense orientation the nucleic acid delivery vector. In some embodiments, the first expression cassette is in the sense orientation in the nucleic acid delivery vector, and wherein the second expression cassette is in an anti-sense orientation in the nucleic acid delivery vector. In some embodiments, the sonoactive agent comprises a microbubble or nanobubble filled with a perfluorinated gas. In some embodiments, the sonoactive agent comprises protein stabilized microstructures. In some embodiments, the sonoactive agent comprises a C3F8 gas encapsulated within a protein stabilized shell. In some embodiments, the sonoactive agent comprises lipid stabilized microstructures. In some embodiments, the sonoactive agent comprises an SF6 gas encapsulated within a lipid stabilized shell. In some embodiments, the sonoactive agent comprises a C4F10 gas encapsulated within a lipid stabilized shell. In some embodiments, ultrasound energy is applied at an MI of at least 0.8, 1.3, 1.8, 1.9, or 2.2. In some embodiments, administering to the subject ultrasound energy comprises applying an acoustic radiation force. In some embodiments, the first copy of the transgene is expressed in the first cell type, and wherein the second copy of the transgene isexpressed in the second cell type. In some embodiments, the first cell type is a hepatocyte. In some embodiments, the second cell type is a liver sinusoidal endothelial cell. Th In some embodiments, the first promoter sequence comprises an ApoE-AAT promoter sequence. In some embodiments, the second promoter sequence comprises an F8 promoter sequence. In some embodiments, the transgene is FVIII. In some embodiments, the delivery to the first cell type and the second cell type occurs simultaneously. In some embodiments, the expression in the first cell type and the second cell type occurs simultaneously. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45 over a minimum alignment length of at least 6500, 7000, or 7500 nucleotides. In some embodiments, the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the nucleic acid delivery vector comprises SEQ ID NO: 45. In some embodiments, the first coding sequence or the second coding sequence encodes an amino acid sequence of SEQ ID NO. 24. In some embodiments, the nucleic acid delivery vector is any nucleic acid delivery vector disclosed herein. In some embodiments, nucleic acid delivery vector further comprises a third expression cassette comprising a third nucleic acid coding sequence encoding a third copy of a transgene. In some embodiments, the third expression cassette comprises a third promoter sequence operably linked to the third nucleic acid coding sequence. In some embodiments, nucleic acid delivery vector further comprises a fourth expression cassette comprising: a fourth nucleic acid coding sequence encoding a fourth copy of the transgene. In some embodiments, the fourth expression cassette comprises a fourth promoter sequence operably linked to the fourth nucleic acid coding sequence.

[0037] Aspects disclosed herein provide method of treating a bleeding disorder in a subject comprising administering to the subject a nucleic acid delivery vector inducing expression of a transgene encoding a human clotting factor in both a first cell type and a second cell type in the subject, thereby maintaining a therapeutic level of the human clotting factor in a plasma of the subject. In some embodiments, the nucleic acid delivery vector comprises a first nucleic acid coding sequence encoding a first copy of a transgene and a second nucleic acid coding sequence encoding a second copy of the transgene. In some embodiments, the transgene comprises hFVIII, hFIX, or von-Willebrand factor, or Von Willebrand disease. In some embodiments, the bleeding disorder comprises hemophilia A, hemophilia B. In some embodiments, the transgene comprises hFVIII, wherein the bleeding disorder comprises hemophilia A, and wherein an average level of the human clotting factor in the plasma does not fall below thirty percent (30%) of a peak hFVIII plasma level induced by administration of the nucleic acid delivery vector. In some embodiments, the average level of the human clotting factor is assessed over one or more30-day periods, and the peak fFVIII plasma level is a maximum discreet value within said one or more 30-day periods, each following administration of the nucleic acid delivery vector. In some embodiments, the transgene comprises hFVIII, wherein the bleeding disorder comprises hemophilia A, and wherein an average level of the human clotting factor in the plasma does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state hFVIII plasma level induced by administration of the nucleic acid delivery vector. In some embodiments, the average level of the human clotting factor and the average steady state hFVIII plasma level are assessed over one or more 30-day periods following administration of the nucleic acid delivery vector. In some embodiments, the therapeutic level of the human clotting factor is maintained for at least 30, 60, 70, 80, 90,100, 110 or 120 days following administration of the nucleic acid delivery vector. In some embodiments, at least 20% of a total population of cells expressing the transgene are liver sinusoidal endothelial cells. In some embodiments, an average expression level of the transgene does not fall below thirty percent (30%) of an expression level of the transgene induced by administration of the nucleic acid delivery vector. In some embodiments, the average expression level of the transgene and the peak average expression level of the transgene over one or more 30-day periods, and a peak expression level is a maximum discreet value within said one or more 30-day periods, each following administration of the nucleic acid delivery vector. In some embodiments, an average expression level of the transgene does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state expression level of the transgene induced by administration of the nucleic acid delivery vector. In some embodiments, the average expression level of the transgene and the average steady state expression level of the transgene are assessed over one or more 30-day periods following administration of the nucleic acid delivery vector. In some embodiments, expression of the transgene is maintained for at least 30, 60, 70, 80, 90,100, 110 or 120 days following administration of the nucleic acid delivery vector. Without being bound to a particular theory, the nucleic acid delivery vectors disclosed herein which are configured to express the therapeutic transgene in multiple cell types can provide for more stable and durable gene expression due to immune tolerization of the therapeutic transgene being expressed in multiple cell types in the target organ. In some embodiments, expression level of the transgene is determined by mRNA production, a reporter gene, or transgene protein production. In some cases, inducing expression of the therapeutic transgene from the hepatocytes and the LSECs can lead to a reduced immune response to the therapeutic transgene as compared to expression for only or predominantly a single cell type, and provides a beneficial technical effect of improvedgene expression, including, for example, more stable gene expression and more durable gene expression.

[0038] Ultrasound refers to the application of electromagnetic energy in the range of greater than 20 kHz up to several gigahertz. Ultrasound is used in many different fields, most commonly in the field of diagnostics and medical imaging for producing images of tissue within the human body. Ultrasound energy can be generated at various frequencies within the 20 kHz up to several gigahertz range, most commonly within the range of about 1 to 10 megahertz when used for diagnostic imaging purposes. Ultrasound is commonly applied using ultrasonic transducers comprising one or more piezoelectric crystals which convert electrical energy into acoustic energy. In addition to imaging applications, ultrasound can also be used for a variety of other diagnostic and therapeutic applications, including determination of tissue elasticity and fibrosis, focused destruction of tissue using ultrasound ablation, and for the delivery of exogenous payloads (e.g., nucleic acids and therapeutic agents) to a cell. Sonoporation refers to the delivery of therapeutic agents, for example nucleic acids, using ultrasound and / or sonoactive microstructures to a cell. Provided in certain embodiments herein are methods of delivering a nucleic acid construct into a target cell or tissue (e.g., of a subject) by applying ultrasound energy to a cell, tissue, or organ (e.g., with sonoporation).

[0039] B mode ultrasound imaging refers to brightness mode imaging, in which ultrasonic waves are reflected from the tissue of a subject back to the ultrasound probe, and displayed on a 2 dimensional objects that are closer to the ultrasound transducer appear brighter, and objects which are farther away from the ultrasound transducer appear darker. B mode ultrasound imaging generally will focus ultrasound energy emitted from a plurality of ultrasound arrays comprising piezoelectric crystals into a focused ultrasound beam which penetrates into the tissue about a vertical axis which is perpendicular to the surface of the ultrasound probe. The focused ultrasound beam reflects off the tissue and back towards the ultrasound transducer, forming a scan line in an ultrasound image. By moving the ultrasound transducer about a surface of tissue, an image of the underlying tissue can be generated using a B mode ultrasound image. B mode ultrasound imaging is the most common form of ultrasound used in the United States for medical imaging, and is what is commonly referred to as diagnostic or imaging ultrasound.

[0040] Plane wave imaging refers to an ultrasound imaging technique in which a plurality of ultrasound arrays comprising piezoelectric crystals in an ultrasound transducer are simultaneously fired without directing ultrasound energy into a focused ultrasound beam, and which instead direct a large unfocused sheet or wave of ultrasound energy into a medium or tissue underlying an ultrasound probe. The primary difference between plane wave ultrasoundimaging and B mode ultrasound imaging is the number of transducer arrays which are fired. Plain wave imaging typically will fire all arrays within an ultrasound transducer Producing a much larger and less focused wave of ultrasonic energy, while B mode imaging will typically only fire a subset of arrays which focused the ultrasound into a beam producing what is commonly referred to as a scan line. In plane wave imaging, the acoustic radiation pressure is almost uniform over the entire field of view, and lower peak and negative pressures are typically experienced as compared to traditional beam mode focused ultrasound beam imaging.

[0041] Acoustic radiation force refers to a static or transient force applied by an acoustic wave on the propagation medium or to an object in the path of the acoustic wave. Acoustic radiation forces can be applied using an ultrasound transducer when applying ultrasound energy to a surface of a tissue or a propagation medium with sufficient ultrasound intensity. When applying a sufficient acoustic radiation force to a propagation medium or a tissue, the propagation medium or tissue under lying the ultrasound probe applying the acoustic radiation force may be displaced.

[0042] Shear waves, or secondary waves refer to transversely oriented waves which occur in elastic medium that is subjected to a periodic shear. Shear refers to a change in shape without a change of volume of a layer of a propagation medium or tissue produced by a pair of equal forces acting in opposite directions about two faces of the layer or the propagation medium. Shear waves are a type of elastic wave which move through the body of an object or a propagation medium. In an elastic medium, the layer or the tissue will resume its original shape following application of the shear force, adjacent layers will undergo subsequent shear, and the movement of particles within the medium or tissue will be propagated as a shear wave throughout the propagation medium or tissue. In an elastic medium, shear waves can be produced as a secondary wave following a compressional wave which is transmitted in the propagation medium or tissue. Ultrasound applying an acoustic radiation force can apply a compressional wave to a tissue, which can result in application of shear waves to a tissue when applied with sufficient intensity, at regular intervals, for sufficient periods of time to induce a regular shear in layers of a tissue. A compressional wave displaces tissue in a direction parallel to the propagation of the compressional waves. An ultrasound transducer can induce a compressional wave in a tissue which propagates from the ultrasound transducer about a vector normal to a surface of the ultrasound transducer. In some embodiments, applying the focused acoustic radiation force to the tissue comprises generating a compressional wave in the tissue. As an elastic tissue recovers from displacement due to a compressional wave, shear waves or secondary waves can be generated. In a shear wave, the direction of particle motion is parallel tothe direction of propagation of the compressional wave, and the direction of propagation of the shear wave is normal to the direction of propagation of the compressional wave. The direction of particle motion in a shear wave is also normal to the direction of shear wave propagation in an elastic medium. Further, a compressional wave may be followed by a rarefaction wave which is a negative acoustic force in the tissue.

[0043] Shear wave elastography refers to a diagnostic technique using ultrasound to determine the elastic modulus of tissue, which is indicative of its fibrotic quality. Diseased tissue with certain fibrotic conditions will result in a significantly reduced elastic modulus of the tissue, as compared to a healthy tissue which is reasonably elastic as compared to diseased tissue in a fibrotic state. Shear wave elastography is a diagnostic imaging technique which uses a combination of acoustic radiation force, plane wave imaging, and B mode imaging to provide a clinician with information as to the fibrotic quality of a tissue. Shear wave elastography applies an acoustic radiation force to displace the tissue underlying an ultrasound probe with a compressional wave, thereby generating shear waves in the tissue, applies a plane wave ultrasound to the tissue to monitor the propagation of the shear waves throughout the tissue thereby calculating the elastic modulus, and overlays this data atop a standard B mode ultrasound image in order to provide a visual representation of tissue stiffness. Using ultrasound machines configured to perform shear wave elastography, an acoustic radiation force to displace the tissue underlying an ultrasound probe with a compressional wave, thereby generating shear waves in the tissue, can be applied. In some cases, a standard B mode ultrasound image and / or plane wave in order to calculate or provide a visual representation of tissue stiffness may not be performed.

[0044] Provided in certain embodiments herein are methods of delivering a nucleic acid construct into a target cell or tissue (e.g., of a subject) by applying ultrasound energy to a cell, tissue, or organ. Provided in certain embodiments herein are methods of delivering a nucleic acid construct into a target cell or tissue (e.g., of a subject) by delivering an exogenous payload to the subject; and applying a focused acoustic radiation force (ARF) to the subject, thereby generating shear waves in the tissue of the subject, wherein the focused acoustic radiation force enhances delivery of the exogenous payload to the cell in the tissue of an organ of the subject. Disclosed herein are methods of sonoporation in which an exogenous payload is delivered to a cell in a tissue of a subject using a focused acoustic radiation force applied using ultrasound. Aspects of the sonoporation methods disclosed herein may also include inducing displacing the tissue of the subject with the acoustic radiation force to induce propagation of shear waves throughout the tissue of the subject thereby enhancing delivery of a nucleic acid payload to acell. Disclosed herein are methods of sonoporation in which an exogenous payload is delivered to a cell in a tissue of a subject using a focused acoustic radiation force applied using ultrasound. Aspects of the sonoporation methods disclosed herein may also include inducing displacing the tissue of the subject with the acoustic radiation force to induce propagation of shear waves throughout the tissue of the subject thereby enhancing delivery of a nucleic acid payload to a cell. The method may further include applying the acoustic radiation force to induce propagation of shear waves throughout the tissue in combination with other secondary ultrasound energies such as plane wave ultrasound or focused beam ultrasound in which the secondary ultrasound energy moves sonoactive microstructures toward an endothelial border of a tissue comprising the cell, while applying the focused acoustic radiation force during shear wave propagation induces inertial cavitation of sonoactive microstructures at the endothelial border of the tissue comprising the cell, thereby enhancing delivery of the therapeutic payload to a cell, and, in cases of a nucleic acid payload, resulting gene expression.

[0045] In some embodiments, the focused acoustic radiation force is applied using an ultrasound probe applying ultrasound energy to the tissue. In some embodiments, the ARF displaces the tissue of the subject. In some embodiments, the shear waves displace the tissue of the subject. In some embodiments, a tissue displacement is at least 0.001 mm. In some embodiments, a tissue displacement ranges from at least 0.001 mm to about 5 mm. In some embodiments, a tissue displacement ranges from 0.01 mm to about 1 mm.

[0046] In some embodiments, the focused acoustic radiation force is applied using an ultrasound probe applying ultrasound energy to the tissue. In some embodiments, the ultrasound energy is applied at a mechanical index greater than 0.4. In some embodiments, the ultrasound energy is applied at a mechanical index of about 1.4. In some embodiments, the ultrasound energy is applied at a mechanical index of at least 1.3. In some embodiments, the ultrasound energy is applied at a mechanical index of greater than 0.4 up to about 3.0. In some embodiments, the ultrasound energy is applied at a frequency of about 0.1 MHz to about 10 MHz. In some embodiments, the ultrasound energy is applied at a frequency of about 2.5 MHz. In some embodiments, the applying ultrasound energy to the tissue comprises applying the ultrasound energy at an ultrasound intensity of at least 100 mW / cm2. In some embodiments, the applying ultrasound energy to the tissue comprises applying the ultrasound energy at an ultrasound intensity of about 100 mW / cm2to about 10,000 mW / cm2. In some embodiments, the applying ultrasound energy to the tissue comprises applying the ultrasound energy at an ultrasound intensity of about 100 mW / cm2to about 5,000 mW / cm2. In some embodiments, the applying ultrasound energy to the tissue comprises applying the ultrasound energy at anultrasound intensity of about 100 mW / cm2to about 500 mW / cm2. In some embodiments, the applying ultrasound energy to the tissue comprises applying the ultrasound energy at an ultrasound intensity of about 110 mW / cm2to about 200 mW / cm2. In some embodiments, the applying ultrasound energy to the tissue comprises applying the ultrasound energy at an ultrasound intensity of about 188 mW / cm2. In some embodiments, the ultrasound intensity is a spatial-peak temporal average intensity. In some embodiments, the spatial-peak temporal average intensity is calculated in a focal region of the tissue. In some embodiments, the focused acoustic radiation force is applied in two or more pulses, with an interval between each of the two or more pulses. In some embodiments, a plane wave ultrasound is applied to the tissue during the interval. In some embodiments, the one or more pulses are up to 500 microseconds. In some embodiments, the one or more pulses are at least 100 microseconds. In some embodiments, the one or more pulses are about 100 microseconds to about 500 microseconds. In some embodiments, the interval is up to 500 milliseconds. In some embodiments, the interval is up to 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, or 5000 milliseconds. In some embodiments, the interval is from about 100 milliseconds to about 5000 milliseconds. In some embodiments, the applying the focused acoustic radiation force is performed in one or more sequences, wherein a sequence comprises two or more pulses and interval(s) therebetween. In some embodiments, a time between application of the one or more sequences is at least 5, 10, 20, 30, 60, 120, 180, 240, 300, 360, 420, 480, 540, or 600 seconds. In some embodiments, a time between application of the one or more sequences ranges from about 5 to about 300 seconds. In some embodiments, a time between application of the one or more sequences ranges from about 10 to about 60 seconds. In some embodiments, applying ultrasound energy to the tissue comprises applying the ultrasound energy at a focal depth of up to 10, 8, 6, or 4 cm from the ultrasound transducer. In some embodiments, applying ultrasound energy to the tissue comprises applying the ultrasound energy at a focal depth of about 1 to about 10 cm from the ultrasound transducer. In some embodiments, applying ultrasound energy to the tissue comprises applying the ultrasound energy at a focal depth of about 4 to about 10 cm from the ultrasound transducer. In some embodiments, applying ultrasound energy to the tissue comprises applying the ultrasound energy at a focal depth of about 4 cm from the ultrasound transducer. In some embodiments, applying ultrasound energy to the tissue comprises applying the ultrasound energy at a focal depth of about 6 cm from the ultrasound transducer.

[0047] In some embodiments, applying the focused wave ultrasound results in moving the sonoactive microstructures towards an endothelial border of the tissue comprising the cell. In some embodiments, the shear waves induce inertial cavitation the sonoactive microstructures atan endothelial border of the tissue comprising the cell, thereby enhancing delivery of the nucleic acid to the cell. In some embodiments, the focused acoustic radiation force increases internalization of the exogenous payload in the cell. In some embodiments, the shear waves increase internalization of the exogenous payload in the cell. In some embodiments, inducing inertial cavitation the sonoactive microstructures increases internalization of the exogenous payload in the cell.

[0048] In some embodiments, a process provided herein provides sonoporation at two or more different ultrasonic acoustic energies (e.g., a first and second ultrasound energy having a first and second MI, respectively). In certain embodiments, a process provided herein provides a process wherein an ultrasound energy is continuously applied (e.g., ultrasound energy transitions from the first ultrasound energy to the second ultrasound energy, without a period of no ultrasound energy being applied). In certain embodiments, a transitory (e.g., third, fourth, etc.) ultrasound energy is applied between application of the first and second ultrasonic acoustic energies. Provided in certain embodiments herein are methods of delivering a nucleic acid construct into a target cell or tissue (e.g., of a subject) by applying a first ultrasound energy to a cell, tissue, or organ, and applying a second ultrasound energy to the cell, tissue, or organ. In some embodiments herein are methods for transfecting a nucleic acid construct into a target cell or tissue by applying a first ultrasound energy having a first mechanical index (MI) and applying a second ultrasound energy having a second mechanical index (MI). The present disclosure provides methods for enhancing transfection of a nucleic acid construct into the target cell or tissue by applying alternating ultrasound energy, the alternating acoustic energy alternating between a first mechanical index (MI) and a second MI. Application of ultrasound energy can be repeated several times during sonoporation, such as to increase the efficiency of nucleic acid construct transfection and / or delivery.

[0049] In some embodiments, the sonoporation treatment comprises applying an ultrasound energy to the target cell (e.g., of a tissue or organ of the subject) (e.g., the ultrasound energy having a mechanical index (MI)). In some embodiments, applying an ultrasound energy to the target cell comprises applying a first ultrasound energy to the target cell and applying a second ultrasound energy to the target cell. In some embodiments, the (e.g., first or second) ultrasound energy has a first mechanical index (MI). In certain embodiments, (e.g., the other of the first or second) ultrasonic energy has a second mechanical index (MI). In some embodiments, the (e.g., first or second) MI is less than 0.4. In certain embodiments, the (e.g., the other of the first or second) MI is greater than 0.4 (e.g., and less than 2.0). In some embodiments, a first ultrasound energy and a second ultrasound energy are applied sequentially in a repeated manner. In some embodiments, afirst MI is a Low MI (e.g., less than 0.4). In certain embodiments, a second MI is a High MI (e.g., 0.4 or greater). In some embodiments, a first MI is a Low MI (e.g., less than 0.4) and a second MI is a High MI (e.g., 0.4 or greater). In some embodiments, a second MI naives a Low MI (e.g., less than 0.4). In certain embodiments, a first MI is a High MI (e.g., 0.4 or greater). In given embodiments, a second MI is a Low MI (e.g., less than 0.4) and a first MI is a High MI (e.g., 0.4 or greater). In some embodiments, a Low MI is less than 0.3. In given embodiments, a Low MI is less than 0.2. In more given embodiments, a Low MI is less than 0.1. In still more given embodiments, a Low MI is about 0.09. In still more given embodiments, a Low MI is about 0.04. In still more given embodiments, a Low MI is about 0.03. In some embodiments, a High MI is greater than 0.5. In given embodiments, a High MI is 0.5 to 2.0 or is between 0.5 and 2.0. In more given embodiments, a High MI is 0.5 to 1 or is between 0.5 and 2.0. In some embodiments, a High MI is 1.5. In some embodiments, a High MI is 1.8. In some embodiments, a High MI is 2.0. In some embodiments, a High MI is greater than 0.4. In some embodiments, a High MI is greater than 0.5. In more given embodiments, a High MI is 0.5 to 1 or is between 0.5 and 2.0. In some embodiments, a High MI is 1.5. In some embodiments, a High MI is 1.8. In some embodiments, a High MI is 2.0. In some embodiments, a High MI is 2.2. In some embodiments, a High MI is 2.5. In some embodiments, a High MI is 2.8. In some embodiments, a High MI is 3.0. In certain embodiments, any process provided herein (e.g., a sonoporation treatment) comprises administering of a continuous ultrasound energy (which may have varying energy levels) that alternates (e.g., in identical, similar, or variable periods) between Low MI and High MI. In some embodiments, a low MI (e.g., less than 0.1) (e.g., first) ultrasound energy (also referred to herein as a Low MI) is administered to the subject, and a set number pulses (e.g., of less than 30 seconds) of High MI (e.g., second) ultrasound energy (also referred to herein as a High MI) is administered to the subject. In some embodiments, a process provided herein comprises administration of a plurality of pulses of high MI (e.g., second) ultrasound energy, e.g., during an otherwise continuous administration of a low MI (e.g., first) ultrasound energy. In given embodiments, the number of High MI pulses is about 4 or more, 8 or more, 12, or more, 16 or more, 18 or more, 25 or more, such as up to about 30, or an unlimited number of pulses. In given embodiments, the number of High MI pulses is 6-30. In still more given embodiments, the number of High MI pulses is between 8, 9, 12, 15, 18, 27, 36, 45, or any number therebetween. In some embodiments, at least 8, 9, 12, 15, or 18 high MI pulses are administered to the subject in between applications of low MI ultrasound energy. In still more given embodiments, the number of High MI pulses applied is at least 8, 9, 12, 15, 18, 27, 36, or 45. In still more given embodiments, the number of High MIpulses applied in a given sequence of high MI ultrasound energy is at least 8, 9, 12, 15, 18, 27, 36, or 45.

[0050] In certain embodiments, the first (either High MI or Low MI) ultrasound energy is applied before or after administration of any other agent, such as the nucleic acid and / or sonoactive structure. In some embodiments, the first ultrasound energy is applied after administration of the sonoactive structure to the subject. In certain embodiments, the first ultrasound energy is applied after administration of the nucleic acid to the subject. In some embodiments, the first ultrasound energy is applied after administration of both the nucleic acid and the sonoactive structure(s).

[0051] In some embodiments, high MI ultrasound energy is administered in a pulse. In given embodiments, a pulse length is any suitable length, such as less than 30 seconds. In more given embodiments, a pulse length is less than 15 seconds. In still more given embodiments, a pulse length is less than 10 seconds. In yet more given embodiments, a pulse length is less than 5 seconds. In more given embodiments, a pulse length is less than 2 seconds. In still more given embodiments, a pulse length is less than 1 second and / or may be greater than or equal to 1 microsecond. In some embodiments, a pulse length ranges from 100 to 300 microseconds. In some embodiments, a pulse length is up to about 200 microseconds. In some embodiments, a pulse length is up to about 500 microseconds. In some embodiments, a pulse length ranges from 1 to 500 microseconds.

[0052] In various embodiments, a High MI ultrasound energy is provided first temporally (e.g., first in order). In other embodiments, a Low MI ultrasound energy is provided second temporally (e.g., second in order).

[0053] In some embodiments, any process provided herein further comprises administering (e.g., systemically administering, such as via infusion) a nucleic acid (e.g., any nucleic acid provided herein) to a subject (e.g., to whom the ultrasonic acoustic energies are applied).

[0054] In some embodiments, any process provided herein further comprises administering (e.g., systemically administering, such as via infusion) a sonoactive structure (e.g., any sonoactive structure or microbubble described herein) to a subject (e.g., to whom the ultrasonic acoustic energies are applied).

[0055] In certain embodiments, provided herein is a method of delivering a nucleic acid payload in a target cell (e.g., of a tissue or organ) of a subject, the method comprising: (a) administering to the subject a nucleic acid construct comprising the nucleic acid payload; (b) administering to the subject a plurality of sonoactive microstructures; and (c) administering an ultrasound energy, thereby delivering a sonoporation treatment.

[0056] In some embodiments, a sonoporation treatment (e.g., application of a first ultrasound energy, a second ultrasound energy, a single cycle of a first ultrasound energy and a second ultrasound energy, or series of cycles comprising a plurality of applications of a first ultrasound energy and a plurality of applications of a second acoustic energy) can last for a few seconds (e.g., 1-100 seconds) or more, such as up to a few minutes (e.g., 1-3 minutes). In given embodiments, a sonoporation treatment lasts for 1-30 seconds. In some given embodiments, a sonoporation treatment lasts for 5-100 seconds. In certain embodiments, a sonoporation treatment lasts for at least 1 minute (e.g., 1-30 minutes).

[0057] In some embodiments, the first ultrasound energy is administered within 60 minutes of administration of the nucleic acid and / or sonoactive structure(s). In given embodiments, the first ultrasound energy is administered within 30 minutes of administration of the nucleic acid and / or sonoactive structure(s). In more given embodiments, the first ultrasound energy is administered within 5 minutes of administration of the nucleic acid and / or sonoactive structure(s). In still more given embodiments, the first ultrasound energy is administered within 2 minutes of administration of the nucleic acid and / or sonoactive structure(s). In still more given embodiments, the first ultrasound energy may be applied simultaneously with administration of the nucleic acid and / or sonoactive structure(s).

[0058] In given embodiments, the first (e.g., High MI) ultrasound energy is applied immediately upon administration (e.g., infusion) or a period of time after administration (e.g., infusion) of the sonoactive structure(s) and / or nucleic acid.

[0059] In some embodiments, either the first or second ultrasound energy is an ultrasound energy (e.g., Low MI) that when applied to a cell, tissue, or organ of a subject results in stable cavitation (or stable vibrational cavitation) of the sonoactive structure and / or a change in the average diameter of the sonoactive structure(s), for example, due to inherent resonance properties of the microbubbles.

[0060] In certain embodiments, the first or second ultrasound energy is an ultrasound energy (e.g., High MI) that when applied to a cell, tissue, or organ of a subject results in inertial cavitation or the collapse of the sonoactive structures and / or disruption of cell membrane and / or vascular endothelial integrity.

[0061] In certain embodiments, either the first or second ultrasound energy is an ultrasound energy (e.g., Low MI) that when applied to a cell, tissue, or organ of a subject results in stable cavitation (or stable vibrational cavitation) and / or a change in the average diameter of the sonoactive structure(s), and the other of the first or second ultrasound energy is an ultrasound energy (e.g., High MI) that when applied to a cell, tissue, or organ of a subject results in inertialcavitation or the collapse of the sonoactive structures and / or disruption of cell membrane and / or vascular endothelial integrity.

[0062] In some instances, disruption of cell membrane allows target cells to become permeable to circulating agents such as nucleic acid constructs. In certain instances, such circulating agents can then enter the target cells, tissues or organs, such as in a more rapid manner (e.g., relative to either Low MI or High MI ultrasound energy application alone, or in the absence of ultrasound energy application).

[0063] In some embodiments, the methods herein comprise alternating the ultrasound energy applied between a first ultrasound energy having a first MI and a second ultrasound energy having a second MI. In some embodiments, applying alternating ultrasound energy administered to a subject between a first MI and a second MI is performed repeatedly over a number of times, such as to enhance gene transfection into the target cells, tissue or organ (e.g., relative to a similar process wherein a first and second ultrasound energy are not used and / or are not alternately applied and / or are not alternately applied repeatedly).

[0064] In certain embodiments, changing parameters of the ultrasound energy or MI can be performed to induce and / or enhance an expression of a transgene in a cell or an organ of a subject. In one aspect, provided herein are methods of transfection by alternating the ultrasound energy using a first MI and a second MI. In some embodiments, the first MI that results in stable vibrational cavitation are applied prior to the second MI, which results in inertial cavitation. In some embodiments, the first MI that results in stable vibrational cavitation is a low MI ultrasound energy and is applied prior to the second MI which is a high MI ultrasound energy which results in inertial cavitation. In some embodiments, the ultrasound energy using the first MI and the second MI are reapplied for a number of times to increase transfection efficiency at the target cell. In some embodiments, during the application of sonoporation, the ultrasound energy is applied at the first MI continuously except for when the ultrasound energy is applied at the second MI. For example, applying an ultrasound energy to the target cell at the first MI then applying an ultrasound energy to the target cell at the second MI are repeated between 4 to 18 times. In some embodiments, applying an ultrasound energy to the target cell at the first MI then applying an ultrasound energy to the target cell at the second MI are repeated an unlimited number of times. In one aspect, during this time, the ultrasound energy of the first MI is applied continuously except for when the ultrasound energy of the second MI is applied. In some embodiments, applying an ultrasound energy to the target cell at the includes applying an ultrasound energy to the target cell at a high MI at least 2, 3, 4, or 5 times in a given sonoporation treatment session.

[0065] In some embodiments, the first MI ranges from about 0.05 to less than 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 less than 0.4. In some embodiments, the first MI ranges from about 0.09 to about 0.3.

[0066] In some embodiments, the second MI ranges from about 0.4 to about 2.0. 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.

[0067] In some embodiments, the second MI ranges from about 0.4 to about 2.8. In some embodiments, the second MI ranges from about 0.5 to about 2.8. In some embodiments, the second MI ranges from greater than 1.4 to about 2.8. In some embodiments, the second MI ranges from greater than 1.4 to about 2.8. In some embodiments, the second MI ranges from about 1.5 to about 2.8.

[0068] In some embodiments, the second MI ranges from about 0.4 to about 3.0. In some embodiments, the second MI ranges from about 0.5 to about 3.0. In some embodiments, the second MI ranges from greater than 1.4 to about 3.0. In some embodiments, the second MI ranges from greater than 1.4 to about 3.0. In some embodiments, the second MI ranges from about 1.5 to about 3.0.

[0069] In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated at least 4, 6, 8, 12, 18, 20, 25, 30, 40, or 50 times. In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated between 4 and 18 times. In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated between 6 and 12 times. In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated between 8 and 10 times. In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated between 8 and 18 times.

[0070] In some embodiments, the applying the ultrasound energy comprises applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI, without ceasing applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI. In some embodiments, the applying the ultrasound energy comprises applying the ultrasound energy at the first MI except for when the ultrasound energy is applied at the second MI. In some embodiments, an ultrasound probe applying the ultrasound energy is in constantcontact with the surface of the subject’s skin at the location of application (e.g., abdomen, chest wall, skull, etc.). In some embodiments, an ultrasound transducer that applies the ultrasound energy to the target cell is continuously in contact with tissue of the subject and is continuously either (1) applying the ultrasound energy to the subject or (2) receiving reflected ultrasound energy from the subject. In certain embodiments, a transitory (e.g., third, fourth, etc.) ultrasound energy is applied between application of the first and second ultrasonic acoustic energies39ertainin embodiments, applying the ultrasound energy comprises applying the ultrasound energy without regard to an EKG gating signal regulating the application of the ultrasound energy. In certain embodiments, applying the ultrasound energy comprises applying the ultrasound energy without turning off power to the ultrasound transducer off. In some embodiments, applying the ultrasound energy comprises an ultrasound transducer sending ultrasound energy or receiving reflected ultrasound energy at least 95% of a period of time in which an ultrasound transducer continuously is contacting the subject.

[0071] In some instances, the ultrasound energy applied at the second MI (e.g., high MI) is applied using a pulse. In some instances, a pulse comprises applying the ultrasound energy in a short pulse (e.g., microsecond length pulse). In some cases, the high MI is applied with the pulse, results in induces inertial cavitation and destruction of the sonoactive microstructure, resulting in the disruption of cell membrane and vascular endothelial integrity, transducing the nucleic acid payload to the cell. In some instances, the pulse is applied with a duration of about 1 ps to about 200 ps. In some instances, the pulse is applied with a duration of about 1 ps to about 200 ps or greater.

[0072] In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse. In some instances, the duration of the second MI applied ranges from 0.1 ps to about 200 ps. In some instances, the duration of the second MI applied ranges from 1 ps to about 200 ps or greater. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration of about 1 ps to about 200 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration of up to 200 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration of about 1 ps to about 500 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration of up to 500 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with aduration of about 2.3 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration of at least 2.3 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration ranging from 1-500 ps. In some embodiments, applying the ultrasound energy at the second MI comprises applying the ultrasound energy at the second MI using a pulse with a duration ranging from 0.1-500 ps.

[0073] In some embodiments, the ultrasound energy is applied using an ultrasound probe applying ultrasound energy to the tissue. In some embodiments, the acoustic radiation force is applied using an ultrasound probe applying ultrasound energy to the tissue. In some embodiments, the ultrasound probe comprises a plurality of piezoelectric elements configured to emit ultrasound energy. In some embodiments, portions of the plurality of piezoelectric elements are arranged in one or more arrays. In some embodiments, the ultrasound probe is a phased array transducer comprising a plurality of piezoelectric elements configured to emit ultrasound energy. In some embodiments, the ultrasound probe is a phased array ultrasound probe, a linear ultrasound probe, a curvilinear ultrasound probe, a convex array ultrasound probe, an endocavitary ultrasound probe, a 3D ultrasound probe, a 4D ultrasound probe, a Doppler ultrasound probe, or a color doppler ultrasound probe.

[0074] In some embodiments, the method comprises administering ultrasound energy transcutaneously to the subject in proximity to one or more target cells. In some embodiments, the one or more target cells are hepatic cells. In some embodiments, the one or more target cells are renal 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 myocytes. 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 cancerous cells.

[0075] In some embodiments, the one or more target cells are comprised in 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 nervous tissue. In some embodiments, the tissue is diseased tissue, e.g., cancerous tissue, fibrotic tissue, or tissue otherwise in need of gene therapy.

[0076] In some embodiments, the target tissue is comprised in an organ. In some embodiments, the organ is the liver. In some embodiments, the organ is a kidney. In someembodiments, 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 comprised in a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a liquid tumor.

[0077] In some embodiments, cells, tissue, or organ are those of the liver. In some embodiments, cells, tissue, or organ are those of the kidney.

[0078] In certain embodiments, a subject herein is a mammal. In some embodiments, the mammal is, by way of non-limiting example, a human, rat, mouse, monkey, and other non-human primates.

[0079] In some cases, alternating the ultrasound energy between the first MI and the second MI for a number of times also allows reperfusion of the sonoactive microstructures and the nucleic acid constructs to the target cell, tissue, or organ, following disruption of the sonoactive microstructures within or proximal to the target cell, tissue, or organ.

[0080] In some embodiments, the repeating application of ultrasound energy between the first MI and the second MI comprises applying the ultrasound energy at the first MI for an amount of time sufficient to permit reperfusion of the sonoactive microstructures in a tissue comprising the target cell before reapplying the ultrasound energy at the second MI.

[0081] In some embodiments, the method comprises applying the ultrasound energy at the first MI for 1-30 seconds before repeating the applying the ultrasound energy of (d). In some embodiments, the method comprises applying the ultrasound energy at the first MI for 5-15 seconds before repeating the applying the ultrasound energy of reapplying the ultrasound energy at the second MI. In some embodiments, the method comprises applying the ultrasound energy at the first MI for 10 seconds before repeating the applying the ultrasound energy of reapplying the ultrasound energy at the second MI.

[0082] In some instances, the duration of the first MI applied ranges from about 2 s to about 30 s. In some embodiments, applying the ultrasound energy at the first MI comprises initially applying the ultrasound energy at the first MI from about 2 s to about 30 s.

[0083] In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated for a total amount of time ranging from about 1 s to about 60 m. In some embodiments, applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI are repeated for a total amount of time ranging from about 60 s to about 120 s.

[0084] In some embodiments, applying the ultrasound energy at the first MI induces stable vibration cavitation of the sonoactive microstructures. In some embodiments, applying theultrasound energy at the first MI does not induce substantial disruption of the sonoactive microstructures. In some embodiments, applying the ultrasound energy at the first MI does not induce substantial disruption of the sonoactive microstructures in a vasculature space and an extravascular space, or induces stable vibration cavitation of the sonoactive microstructures in a vasculature space and an extravascular space.

[0085] In some embodiments, applying the ultrasound energy at the first MI induces formation of an intercellular gap or an interendothelial gap or endocytosis. In some embodiments, the intercellular gap or the interendothelial gap ranges from about 10 nm to about 10 um. In some embodiments, the stable vibration cavitation of the sonoactive microstructures moves the nucleic acid construct from an intravenous space into an interstitial space or into the cytoplasm.

[0086] In some embodiments, applying the ultrasound energy at the second MI induces inertial cavitation of the sonoactive microstructures to disrupt the sonoactive microstructures. In some embodiments, applying the ultrasound energy at the second MI induces inertial cavitation of the sonoactive microstructures to disrupt the sonoactive microstructures in a vasculature space and an extravascular space. In some embodiments, the extravascular spaces comprise an interstitial space, a subcutaneous space, intramuscular or a lymphatic space. In some embodiments, the extravascular spaces comprise an extravascular tissue. In some embodiments, the extravascular tissue comprises an interstitial space, a cytoplasmic space, a subcutaneous, a lymph tissues, muscular or combinations thereof.

[0087] In some embodiments, applying the ultrasound energy at the second MI induces formation of a pore in a membrane of the cell. In some embodiments, the formation of a pore in a membrane of the cell ranges from about 10 nm to about 10 um.

[0088] In some embodiments, administration of the sonoactive microstructures and nucleic acid constructs occurs simultaneously in that the sonoactive microstructures are mixed with a solution comprising the nucleic acid constructs prior to delivery to the subject. Such mixtures can comprise of 50% v / v of the sonoactive microstructures (e.g., Optison) and 50% v / v of a solution comprising a nucleic acid construct. Such mixtures can comprise varying percentages 5-90% v / v of the sonoactive microstructures.

[0089] In some embodiments, the nucleic acid payload comprises an expression cassette. In some embodiments, the expression cassette comprises a transgene. In some embodiments, the nucleic acid payload comprises a transgene (endogenous or non-endogenous). In some embodiments, the transgene comprises a therapeutic transgene. In some embodiments, inducing expression of the nucleic acid payload comprises inducing expression of the therapeutic transgene. In some embodiments, the transgene comprises a detectible marker. In some embodiments, thetransgene comprises luciferase. In some embodiments, inducing expression of the nucleic acid payload comprises inducing expression of luciferase.

[0090] In some embodiments, a nucleic acid payload comprises a regulatory element such as a promoter, (e.g., APOE-ATT). In some embodiments, a total amount (e.g., dose) of DNA administered to a subject for purposes of sonoporation can range from 100 microgram to 200 mg.

[0091] In some embodiments, the therapeutic payload is a nonendogenous gene. In some embodiments, the nucleic acid payload is configured to perform gene augmentation, gene replacement, gene editing, gene knockdown, or gene knockout.

[0092] In some embodiments, the nucleic acid construct comprises one or more regulatory elements, such as a promoter, enhancer, ribosome binding site, or transcription termination signal. Examples of promoters contemplated herein include, but are not limited to, e.g., ApoE promoter, ApoE-AATl promoter, a F8 promoter, an NPHS1 promoter, an NPHS2 promoter, and combinations thereof. In some embodiments, the nucleic acid construct comprises a promoter sequence comprising ApoE. In some embodiments, the nucleic acid construct comprises a promoter sequence comprising ApoE-AAT. In some embodiments, the nucleic acid construct comprises a promoter sequence comprising NPHS1. In some embodiments, the nucleic acid construct comprises a promoter sequence comprising NPHS2.

[0093] In some embodiments, inducing expression of the nucleic acid payload comprises inducing production of RNA encoded by the payload. In some embodiments, inducing expression of the nucleic acid payload comprises inducing production of protein encoded by the payload.

[0094] In some embodiments, the nucleic acid construct is configured to perform gene augmentation, gene replacement, base editing, base knockdown, gene editing gene knockdown, or gene knockout. In some embodiments, delivering the nucleic acid payload to the target cell of the subject increases or decreases expression of a gene in the target cell.

[0095] 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 a Cas protein or homologs or variants thereof, or a nucleic acid molecule encoding the Cas protein or homologs or variants thereof. In some embodiments, the payload comprises a TALEN or a nucleic acid molecule encoding the TALEN. In some embodiments, the payload comprises a zinc-finger nuclease (ZFN) or a nucleic acid encoding the ZFN. In some embodiments, the nuclease is an engineered nuclease. In some embodiments, the engineerednuclease is catalytically inactive. In some embodiments, the engineered nuclease is a fusion protein comprising the engineered nuclease a regulatory protein or an enzyme, or a functional domain thereof (e.g., a nuclease fused to a transcriptional regulatory domain or a nuclease fused to a deaminase) In some embodiments, the payload may further comprise a template DNA molecule suitable for knock-in to the subject’s genome via non-homologous end joining (NHEJ) or homology directed repair (HDR).

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

[0097] The sonoactive microstructures can be administered prior to, after, or simultaneous (e.g., co-administered) with the administration of the nucleic acid construct (or nucleic acid payload). In some embodiments, the nucleic acid construct and the sonoactive microstructures are coadministered. In some embodiments, the administering of the nucleic acid construct and the sonoactive microstructures occurs serially, concurrently, sequentially, or continuously. In some embodiments, the administering of the nucleic acid construct and the sonoactive microstructures occurs serially. In some embodiments, the administering of the nucleic acid construct and the sonoactive microstructures occurs concurrently. In some embodiments, the administering of the nucleic acid construct and the sonoactive microstructures occur sequentially. In some embodiments, the administering of the nucleic acid construct and the sonoactive microstructures occurs continuously.

[0098] In some embodiments, the sonoactive microstructures are administered at a dosage of about 1-50 mL, for example 1 mL of Optison. The sonoactive microstructures may be administered at a concentration of about 5M to about 8M microstructures per mL. In some embodiments, the sonoactive microstructures are administered at a concentration of about 5x 10A8 to about 1.2x 10A9 microstructures / mL, for example lx 10A9 of Definity RT. In someembodiments, the sonoactive microstructures are administered at a concentration of about 0.1 to about 0.8 mg / kg. In some embodiments, the sonoactive microstructures are administered at a concentration of about 0.1 to about 1.0 mL / kg. In some embodiments, the sonoactive microstructures are administered at a concentration of about 10A9 microstructures / mL. In some embodiments, the sonoactive microstructures are administered at a concentration of about 5x 10A8 to about 8x 10A8 microstructures / mL.

[0099] As used herein, concentrations of microstructures / mL refer to the concentration of the sonoactive microstructures in a pharmaceutical composition immediately prior to administration to the subject. In some embodiments, the sonoactive microstructures are administered at a concentration of about 5 / I 08to about L2 / I O10microstructures / mL. In some embodiments, the sonoactive microstructures are administered at a dosage of about 1-50 mL, for example 1 mL of a protein-stabilized sonoactive microstructure (e.g., Optison). In some embodiments, the protein-stabilized sonoactive microstructure (e.g., Optison) has a diameter of 3-4.5 micrometers. The sonoactive microstructures may be administered at a concentration of about 5M (million) to about 8M microstructures per mL. In some embodiments, 1 * 109of phospholipid stabilized sonoactive microstructures (e.g., Sonazoid) are administered. In some embodiments, the phospholipid stabilized sonoactive microstructures (e.g., Sonazoid) comprise a diameter of 1-5 micrometers. In some embodiments, the sonoactive microstructures are administered at a concentration of about 0.1 to about 0.8 mg / kg. In some embodiments, the sonoactive microstructures are administered at a concentration of about 0.1 to about 1.0 mL / kg. In some embodiments, the sonoactive microstructures are administered at a concentration of about 10A9 microstructures / mL. In some embodiments, the sonoactive microstructures are administered at a concentration of at least 5x 10A8 microstructures per mL. In some embodiments, the sonoactive microstructures are administered at a concentration of up to 1.2 x 10Al 0 microstructures / mL. In some embodiments, the sonoactive microstructures are administered at a concentration of 5x 10A8 to 8x 10A8 microstructures / mL.

[0100] In some embodiments, the nucleic acid construct and the sonoactive microstructures are mixed prior to being coadministered. In some instances, the sonoactive microstructures are mixed with the nucleic acid constructs before administering to the subject. In some instances, the sonoactive microstructures are mixed with the nucleic acid constructs along with additional buffers or agents such as saline or other biocompatible solutions with varying electrostatic charges and surface chemistries and ligands before administering to the subject. For example, Optison sonoactive microstructures can be mixed with a Nanoplasmid comprising a promoter operatively linked to a transgene (e.g., APOE-Fluc) and saline and are administered together.

[0101] In some embodiments, the administering of the nucleic acid construct and the sonoactive microstructures is by intravenous administration or subcutaneous or intramuscular or intra-arterial or inter-osseus or direct organ puncture.

[0102] In some embodiments, after administering of the nucleic acid construct and sonoactive microstructures, the ultrasound energy is applied at the target cell, tissue, or organ.

[0103] Once the nucleic acid constructs are inside the target cell, expression of the nucleic acid payload is induced. In some embodiments, the nucleic acid payload comprises luciferase. In some embodiments, inducing expression of the nucleic acid payload using the miniplasmid construct comprises inducing expression inducing an average radiance of at least 2xlOA4 p / sec / cm2 / sr. In some embodiments, inducing expression of the nucleic acid payload comprises inducing an average radiance of from about 2xlOA4 p / sec / cm2 / sr to about 5xl0A5 p / sec / cm2 / sr. In some embodiments, inducing expression of the nucleic acid payload comprises inducing a flux of at least 10A6 p / s. In some embodiments, inducing expression of the nucleic acid payload comprises inducing a flux of about 10A6 p / s to about 10A9 p / s.

[0104] In some embodiments, inducing expression of the nucleic acid payload comprises inducing a flux which is 2, 3, 4, or 5x greater than expression induced without repeating applying the ultrasound energy at the first MI, and the applying the ultrasound energy at the second MI.

[0105] In some embodiments, inducing expression of the nucleic acid payload comprises inducing expression within about 3 to about 12 hours of administering the payload. In some embodiments, inducing expression of the nucleic acid payload comprises inducing expression within about 3 hours of administration. In some embodiments, inducing expression of the nucleic acid payload comprises inducing expression within about 6 hours of administration. In some embodiments, inducing expression of the nucleic acid payload comprises inducing expression within about 12 hours of administration.

[0106] Undesirable effects on living cells or tissues can occur due to ultrasound applications. In some embodiments, the present disclosure provides methods for improvement of gene transfection and not result in substantial DNA or cell damage in the target cells, tissues, or organs, using sonoporation by alternating ultrasound energy between the first MI and the second MI. In some embodiments, the method does not result in substantial cellular damage to the target cell. In some embodiments, the method results in less than 1%, 5%, or 10% of target cells undergoing apoptosis.

[0107] Cellular damage can be detected using apoptotic biomarkers. For example, in liver, detection of released hepatocellular transaminases, e.g., serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST), can be an indicator of apoptotic hepatocytes. For example,when assessing potential damage to the kidney, serum creatinine, blood urea nitrogen level, urine albumin-to-creatinine ratio, electrolyte levels, and serum cystatin C levels can be measured and assessed relative to standard or patient normal physiological levels as measures of impact to kidney function. Additional apoptotic biomarkers comprise interleukin 6 (IL6) or B-cell lymphoma 2 (BCL2 or BCL2 apoptosis regulator). In some embodiments, the following biomarkers for cellular damage are not detected at apoptotic levels following delivering the nucleic acid payload to the target cell of the subject: ALT, AST, IL6, BCL2, or combinations thereof. In some embodiments, the following biomarkers for cellular damage are not clinically elevated following delivering the nucleic acid payload to the target cell of the subject: ALT, AST, IL6, BCL2, or combinations thereof. In some embodiments, the following biomarkers for cellular damage are not detected at apoptotic levels following delivering the nucleic acid payload to the target cell of the subject: ALT, AST, IL6, BCL2, or combinations thereof, and, optionally wherein the target cell is in a liver. In some embodiments, the following biomarkers for cellular damage are not clinically elevated following delivering the nucleic acid payload to the target cell of the subject: ALT, AST, IL6, BCL2, or combinations thereof, and, optionally wherein the target cell is in a liver. In some embodiments, the following biomarkers for cellular damage are not clinically elevated following delivering the nucleic acid payload to the target cell of the subject: creatinine levels in urine, albumin to creatine ratio in urine, creatinine levels in blood, a glomerular filtration rate, blood in urine, protein levels in urine, or an osmolality of urine, and, optionally wherein the target cell is in a kidney. In some embodiments, the following biomarkers for cellular damage are not clinically elevated following delivering the nucleic acid payload to the target cell of the subject: troponin levels in blood, or creatinine phosphokinase, and, optionally wherein the target cell is in a heart or skeletal muscle.

[0108] A sonoporation treatment using the methods described herein can be used to induce expression of a nucleic acid payload in a cell in a liver, a cell in a kidney, a cell in the heart, or a cell in the pancreas.

[0109] A sonoporation treatment using the methods described herein can be used to treat a subject in need of gene therapy or enzyme replacement treatment. In another aspect, the present disclosure provides methods of treating a subject having a liver condition. In some embodiments, the liver condition treated is: Wilso”s Disease, Cholestasis progressive familial intrahepatic, Von Willebrand disease, Hemophilia A, Hemophilia B, Factor 5 deficiency, Alpha-Mannosidosis, Gauche”s (glucocerebrosidase deficiency, glucocerebrosidosis), Niemann Pick Disease A / B, Carbamoylphosphate Synthetase I Deficiency, Glycogen Storage Disease Type III, Cystinosis, A1AT deficiency, Citrullinemia Type I & II. In some embodiments, the methods comprise (a)administering to the subject a nucleic acid construct comprising the nucleic acid payload (e.g., a therapeutic transgene); (b) administering to the subject a plurality of sonoactive microstructures; and (c) administering ultrasound energy, thereby administering a sonoporation treatment.

[0110] In some embodiments, the present disclosure provides methods of treating a subject having a liver condition with a therapeutic transgene. In some embodiments, the therapeutic transgene encodes one or more of: ATP7B (copper-transporting P-type ATPase); ABCB11 (bile salt export pump protein); ABCB4; ATP8B1 (ATPase phospholipid transporting 8B1); TJP2; VWF (Von Willebrand Factor); FVIII (factor VIII); FIX (factor IX); F5 (factor V); MAN2B1 (mannosidase alpha class 2B member 1); GBA (Glucocerebrosidase); SMPD1 (sphingomyelin phosphodiesterase 1); CPS1 (carbamoyl phosphate synthetase); GDE / AGL (glycogen debrancher enzyme); CTNS (cystinosin, lysosomal cystine transporter); SERPINA1 (alpha-1 antitrypsin); ASS1 (argininosuccinate synthase 1), and / or SLC25A13 (citrin).[OHl] In some embodiments, the present disclosure provides methods of treating a subject having a liver condition with 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 (PFIC1-4) and the therapeutic transgene encodes one or more ofABCBl 1, 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 FV. In some embodiments, the liver condition is Alpha-Mannosidosis, and the therapeutic transgene encodes MAN2B1. In some embodiments, the liver condition is Gauche’ 's (glucocerebrosidase deficiency, glucocerebrosidosis), and the therapeutic transgene encodes GBA. In some embodiments, the liver condition is Niemann Pick Disease A / B, and the therapeutic transgene encodes SMPD1. In some embodiments, the liver condition is Carbamoylphosphate Synthetase I Deficiency, and the therapeutic transgene encodes CPS1. In some embodiments, the liver condition is Glycogen Storage Disease Type III, and the therapeutic transgene encodes GDE / AGL. In some embodiments, the liver condition is Cystinosis, and the therapeutic transgene encodes CTNS. In some embodiments, the liver condition is Al AT deficiency, and the therapeutic transgene encodes SERPINA. In some embodiments, the liver condition is Citrullinemia Type I & II, and the therapeutic transgene encodes one or more of ASS1 and / or SLC25A13. In some embodiments, the method comprises delivering the nucleic acid payload and the plurality of sonoactive microstructures systemically (e.g., by intravenous administration).

[0112] 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 a plurality of sonoactive microstructures; applying an ultrasound energy to the target cell. 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 a plurality of sonoactive microstructures; applying an ultrasound energy to the target cell. In one aspect, using the methods described herein, the present disclosure provides methods of treating a subject having a kidney condition. In some embodiments, the kidney condition treated is: Alport Syndrome, or Autosomal Dominant Polycystic Kidney Disease. In some embodiments, the present disclosure provides methods of treating a subject having a kidney condition with a therapeutic transgene. In some embodiments, the therapeutic transgene encodes one or more of COL4A3, COL4A4, COL4A5, PKD1 and / or PKD2. In some embodiments, the present disclosure provides methods of treating a subject having a kidney condition with a therapeutic transgene. In some embodiments, the kidney condition is Alport Syndrome, and the therapeutic transgene encodes one or more of COL4A3, COL4A4, and / or COL4A5. In some embodiments, the kidney condition is Autosomal Dominant Polycystic Kidney Disease, and the therapeutic transgene encodes one or more of PKD1 and / or PKD2. In some embodiments, 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 plurality of sonoactive microstructures; applying an ultrasound energy to the target cell. 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 plurality of sonoactive microstructures are administered systemically (e.g., by intravenous administration). 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 plurality of sonoactive microstructures; applying an ultrasound energy to the target cell. In some embodiments, applying an ultrasound energy to the target cell comprises applying a focused acoustic radiation force (ARF) to the subject, thereby generating shear waves in the tissue of the subject, wherein the focused acoustic radiation force enhances delivery of the exogenous payload to the cell in the tissue of an organ ofthe subject. In some embodiments, applying an ultrasound energy to the target cell comprises applying ultrasound energy at a first MI and a second MI.

[0113] In another aspect, the present disclosure provides a kit to perform the methods described herein. In some embodiments, the kit comprises: (a) a first container comprising microbubbles for sonoporation; and (b) a second container comprising nucleic acids (e.g., miniplasmids) comprising a transgene and a mixture chamber (reservoir, syringe, Y-port, etc.). In some embodiments, the kit further comprises instructions for administration of ultrasound energy in connection with administration of the microbubbles and the nucleic acids.

[0114] In some embodiments, the miniplasmid further comprises an expression cassette. As used herein, an expression cassette comprises nucleic acid sequences encoding nucleic acid payload, e.g., an expression cassette comprising a transgene. The expression cassette further comprises a regulatory element such as a promoter, enhancer, ribosome binding site, or transcription termination signal.

[0115] In some embodiments, the first container and second container are configured to induce the expression of the transgene in the target cell of the subject within 20 hours after the transfection.

[0116] In some embodiments, the method further includes inducing expression of the nucleic acid payload and maintaining expression of a protein encoded by the nucleic acid payload for at least 1, 2, 3, 4, 5, 6, or 7 days following administration of the nucleic acid construct, the sonoactive microstructures, and application of the ultrasound energy to the target cell. In some embodiments, the method further includes inducing expression of the nucleic acid payload and maintaining expression of a protein encoded by the nucleic acid payload for at least 1, 2, 3, 4, 5, 6, or 7 days following administration of the nucleic acid construct, the sonoactive microstructures, and application of the ultrasound energy to the target cell.

[0117] In some embodiments, the method further includes increasing expression of the nucleic acid payload by increasing the dosage of the nucleic acid payload administered to the subject. In some embodiments, the method further includes increasing expression of the nucleic acid payload by increasing the dosage of the nucleic acid payload administered to the subject in a linear manner. In some embodiments, the method further includes increasing expression of the nucleic acid payload by administering at least 5, 50, 250, or 500 ug of the nucleic acid payload to the subject.

[0118] In some embodiments, ALT is not detected at levels exceeding 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 U / L following delivering the nucleic acid payload to the target cell of the subject. In some embodiments, AST is not detected at levels exceeding 225, 250, 275, or 300 U / L following delivering the nucleic acid payload to the target cell of the subject. In someembodiments, IL6 is not detected at 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 following delivering the nucleic acid payload to the target cell of the subject.

[0119] In some embodiments, the kit further comprises instructions for software and hardware directions for the safe and effective operation of an ultrasound machine sufficient to disrupt the sonoactive microstructures to generate the sonoporation processes which include but are not limited to the following: disrupting the microstructures, inducing inertial and stable cavitation, promoting endocytosis and inter-endothelial gap formation, microstreaming at cell surfaces, thereby increasing transfection of a nucleic acid payload to a cell. In some embodiments, the instructions described methods for improvement of gene transfection using sonoporation by applying alternating ultrasound energy applying an acoustic radiation force to a subject and inducing a shear wave in a tissue comprising the target cell. In some embodiments, the instructions described methods for improvement of gene transfection using sonoporation by applying alternating ultrasound energy between a first MI then a second MI. In some embodiments, the kit further comprises instructions for administration of the first container and the second container.

[0120] Aspects disclosed herein provide a use of a nucleic acid delivery vector for treating a genetic disorder in a subject in need thereof, comprising administering the nucleic acid delivery vector to the subject in accordance with the method of any one of the preceding embodiments , thereby inducing expression of the transgene and treating the genetic disorder.

[0121] Aspects disclosed herein provide a nucleic acid having a sequence of any one of SEQ ID NO: 1-55. Aspects disclosed herein provide a nucleic acid having a sequence of SEQ ID NO: 9 or 50. In some embodiments, the sequence is a promoter sequence. In some embodiments, the sequence is SEQ ID NO: 50, wherein the sequence selectively drives gene expression in LSECs.III. KITS

[0122] Aspects disclosed herein provide a kit comprising a nucleic acid delivery vector as disclosed herein, a sonoactive agent, and instructions for administering to a subject the sonoactive agent and ultrasound energy. Aspects disclosed herein provide a kit comprising a nucleic acid delivery vector as disclosed herein, and a sonoactive agent.IV. DEFINITIONS

[0123] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or forready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0124] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0125] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0126] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0127] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0128] The term “zzz vivo" is used to describe an event that takes place in a subject’s body.

[0129] The term “ex vivo" is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “zzz vitro" assay.

[0130] The term “zzz vitro" is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which livingor dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

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

[0132] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0133] As used herein the term “sequence identity” refers to the percentage identity calculated as the matching residues divided by the total number of residues in the total alignment when performing a consensus alignment of two sequences, with gaps in the alignment scored as a mismatching residue.

[0134] As used herein, when referring to a “first copy of a transgene” and a “second copy of a transgene,” the first and second copies of the transgene encode the same protein or a protein capable of performing the same biological function, regardless of whether the first copy of the transgene and the second copy of the transgene use the same codons for encoding the same or the protein capable of performing the same biological function.

[0135] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.V. EXAMPLES

[0136] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Cloning of Bicistronic Vector for Inducing Expression in Multiple Cell Types

[0137] In this Example, a bicistronic vector for inducing expression in multiple cell types is cloned for subsequent administration and testing. In brief, a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 46 was chemically synthesized, and the sequence verified by next-gen sequencing methods.

[0138] The following reagents were combined and incubated at 37 °C for 2 hours.

[0139] Following incubation, fragments were separated on agarose gel using a gel electrophoresis system, the fragment of interest was cut from agarose gel, and DNA was purified using NEB Monarch Gel extraction kit and eluted in 20 uL of water. A measured DNA sample concentration was using collected a NanoDrop benchtop spectrophotometer.

[0140] The fragment of interest was then amplified using a 4x 50 uL PCR reaction using the following reagents and amplification conditions.

[0141] Following amplification, 10 uL of the PCR reaction product was loaded on agarose gel, concentrated using a PCR purification kit, eluted in 30 uL of water, loaded on agarose gel and separated using a gel electrophoresis system, and fragments were excised from the gel usinga UV transmitter to illuminate bands. Then, DNA was purified using a NEB gel purification kit and eluted in 20 uL of sterile water.

[0142] Purified fragments were then assembling in a HiFi assembly reaction in which a miniplasmid backbone fragment was combined with the purified fragment and NEBuilder® HiFi DNA Assembly Master Mix, samples were incubated at 50 C for 30 min, transformed into host strains of E. coli bacteria engineered for stable high quality production of large plasmids, cells were streaked onto an agar plate with ampicillin, and left to incubate at 37 °C overnight. 6 mL Luria broth and antibiotic culture was inoculated with 5 uL of cells from the successful clones. DNA was extracted from cultures using the Qiagen mini purification preparation kit, DNA was eluted by 50 uL of EB buffer, and DNA concentration was assessed by Qubit. Selected mini preps were verified for fidelity by next-gen sequencing methods. It was observed that the fidelity of the original nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 46 was maintained in the cloning process, that that the codon diversification between the first and second expression cassettes (SEQ ID NO: 47 and 49) prevents recombination during the cloning process, with each expression cassette encoding a protein having an amino acid sequence of SEQ ID NO. 24. An exemplary vector map of the nucleic acid delivery vector is shown in FIG. 5.Example 2: Evaluation of Bicistronic Vector for Inducing Expression in Multiple Cell Types in a Murine Model of Ultrasound Mediated Nucleic Acid Transfection to the Liver

[0143] In this example, the bicistronic vector described in Example 1 was tested against a monocistronic vector (SEQ ID NO. 39) comprising one of the open reading frames of the bicistronic vector in a dose escalation study. The bicistronic vector described in Example 1 comprises two open reading frames encoding the same FVIII transgene having an amino acid sequence of SEQ ID NO. 24. The two open reading frames in the bicistronic vector described in Example 1 were configured to produce expression in multiple liver cell types, hepatocytes and liver sinusoidal epithelial cells (LSEC).

[0144] In this example, four groups of RAG 2 mice were evaluated in a murine model of ultrasound mediated nucleic acid transfection to the liver. The first group was administered 5 ug of a nucleic acid composition comprising the monocistronic vector (SEQ ID NO. 39). The second group was administered 5 ug of the nucleic acid composition comprising the bicistronic vector described in Example 1 having the nucleic acid sequence of SEQ ID NO: 46. A third group was administered 50 ug of a nucleic acid composition comprising the monocistronic vector (SEQ ID NO. 39). A fourth group was administered 50 ug of a nucleic acid compositioncomprising the nucleic acid composition comprising the bicistronic vector described in Example 1 having the nucleic acid sequence of SEQ ID NO: 46.

[0145] All mice were treated with identical dosages of sonoactive agents, and identical applications of ultrasound. A dose of sonoactive microstructure and DNA solution was readied by first preparing the sonoactive microstructures (Optison) as instructed on the label: remove from 4 °C storage and roll between the palms for 20 seconds; removing protective plastic and aluminum covering from Optison vial; placing 25G needle through the rubber gasket to provide a pressure vent; and using 1.5 inch 18G needle to draw up 150 uL of Optison into a syringe (dead space of the needle (about 50 microliters (uL)) included in the calculations). With the same needle and syringe, 50 uL of solution comprising either 250 ug or 100 ug (depending on experimental group) of pDNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenous at a 1 :4 volumetric ratio of DNA to microbubble solution. The DNA + Optison solution was drawn out of the needle dead space. Then the 18G needle was exchanged for a 25G blunt needle for injection into a subject JVC.

[0146] The microbubble-DNA solution was administered to the subject in a single bolus injection over about 30 seconds through the jugular vein catheter, and ultrasound energy was applied to the subject over the liver using an alternating mechanical index protocol. Ultrasound energy was transcutaneously applied to the liver region of the subject with a Cl -6 ultrasound probe, and scans liver of each mouse were performed with a GE LOGIQ elO ultrasound system equipped with a Cl -6 probe, and use of the “ELASTO” software, Shear Wave, using shear wave elastography parameters: GE LOGIQelO probe Cl-6, Abdomen, CHI, Frame rate 55, MI 1.4, Frequency 2.5MHz, Gain 44, Depth 4cm, AO%100, Gain 55, T 8, SVD 6.0, AO%100%, +50- 400Hz, GEN. 100% push output and 100% Track output. Mice were administered 150 pL of protein stabilized Optison sonoactive microstructures and 250pg of nucleic acid in 50 uL of solution, for a total injection volume of about 200 uL, and an acoustic radiation force protocol was applied for 10 seconds, followed by 20 seconds with no application of ultrasound during which the ultrasound probe was removed from the subject, followed again by 10 seconds of an acoustic radiation force protocol, for 40 total seconds of ultrasound acoustic radiation force application. The shear wave ultrasound acoustic radiation force protocol was applied at an ultrasound intensity of 117.5 to 187.9 mW / cm2(ISPTA) (spatial-peak temporal average intensity), at a mechanical index of 1.4, and a frequency of 2.5 MHz. Ultrasound energy was applied using an alternating imaging technique applying focused ultrasound and generating a B-modeultrasound image at a mechanical index of 1.4, and a frequency of 2.5 MHz with the ultrasound probe removed from contacting the subject as indicated.

[0147] Subjects were administered three total sonoporation treatments and dosages of nucleic acids compositions, with each sonoporation treatment administered 48 hours apart. Following the sonoporation treatments, FVIII expression and secretion was measured in subject plasma samples collected following the sonoporation treatment and frozen at -80 °C.

[0148] Transgenic FVIII level in mouse plasma was measured by MSD assay. Briefly capture antibody (GMA-8024) was loaded to the 96-well plate overnight at 4 °C. Next the plate was washed three times with wash buffer and incubated with blocking buffer for 30 min at room temperature. Eight point serial dilution standard were prepared using Xinta® ranging from 0.92 lU / ml to 0.01 lU / ml. 2-fold diluted samples and standards were added to the wells in 96-well plate, incubated 2 hours at room temperature, and washed 3 times. The detection was performed by incubating samples with GMA-8023 antibody for 2 hours following triple wash. Signal was developed by Sulfo-TAG and detected by MSD machine.

[0149] Results are shown in FIG. 1 and FIG. 2. In FIG. 1, among the 5 ug groups (Groups 1 and Group 2) it was shown that over the study period during which plasma samples were collected at 12 days following the treatments, that the bicistronic “Monster” vector was expressed at levels approaching 0.1 lU / mL at each time point, and was more than double (2x) the FVIII levels detected in the mice which were administered the monocistronic vector comprising only one of the open reading frames in the bicistronic vector and expressing FVIII levels of about 0.04 lU / mL. In FIG. 2, it is illustrated that among the 50 ug groups (Group 3 and Group 4), that the bicistronic “Monster” vector was expressed at levels of about 0.4 lU / mL at 14 days following treatment, and that the and was approximately double (2x) the FVIII levels detected in the mice which were administered the monocistronic vector comprising only one of the open reading frames in the bicistronic vector expressing FVIII levels averaging about 0.2 lU / mL at 14 days following treatment.

[0150] Among the 50 ug groups (Group 3 and Group 4), the mice were followed for collection up to 57 days to assess durability of gene expression. Results are illustrated in FIG. 3 in which it was observed that at 57 days post treatment, Group 4 administered the bicistronic “Monster” vector maintained expression of FVIII levels at about 0.3 lU / mL, and that the Group 3 administered the monocistronic vector expressed FVIII levels averaging about 0.2 lU / mL at 57 days following treatment.Example 3: Evaluation of Bicistronic Vector for Inducing Expression in Multiple Cell Types in a Murine Model of Ultrasound Mediated Nucleic Acid Transfection to the Liver

[0151] In this example, the bicistronic vector described in Example 1 was evaluated in an expression stability study. The bicistronic vector described in Example 1 comprises two open reading frames encoding the same FVIII transgene having an amino acid sequence of SEQ ID NO. 24. The two open reading frames in the bicistronic vector described in Example 1 were configured to produce expression in multiple liver cell types, hepatocytes and liver sinusoidal epithelial cells (LSEC).

[0152] In this example, a single group of RAG 2 mice was evaluated in a murine model of ultrasound mediated nucleic acid transfection to the liver. The group was administered 50 ug of the bicistronic vector described in Example 1 having the nucleic acid sequence of SEQ ID NO: 46.

[0153] All mice were treated with identical dosages of sonoactive agents, and identical applications of ultrasound. A dose of sonoactive microstructure and DNA solution was readied by first preparing the sonoactive microstructures (Optison) as instructed on the label: remove from 4 °C storage and roll between the palms for 20 seconds; removing protective plastic and aluminum covering from Optison vial; placing 25G needle through the rubber gasket to provide a pressure vent; and using 1.5 inch 18G needle to draw up 150 uL of Optison into a syringe (dead space of the needle (about 50 microliters (uL)) included in the calculations). With the same needle and syringe the DNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenous at a 1 :4 volumetric ratio of DNA to microbubble solution. The DNA + Optison solution was drawn out of the needle dead space. Then the 18G needle was exchanged for a 25G blunt needle for injection into a subject JVC.

[0154] The microbubble-DNA solution was administered to the subject in a single bolus injection over about 30 seconds through the jugular vein catheter, and ultrasound energy was applied to the subject over the liver using an alternating mechanical index protocol. Ultrasound energy was transcutaneously applied to the liver region of the subject with a Cl -6 ultrasound probe, and scans liver of each mouse were performed with a GE LOGIQ elO ultrasound system equipped with a Cl -6 probe, and use of the “ELASTO” software, Shear Wave, using shear wave elastography parameters: GE LOGIQelO probe Cl-6, Abdomen, CHI, Frame rate 55, MI 1.4, Frequency 2.5MHz, Gain 44, Depth 4cm, AO%100, Gain 55, T 8, SVD 6.0, AO%100%, +50- 400Hz, GEN. 100% push output and 100% Track output. Mice were administered 150 pL of protein stabilized Optison sonoactive microstructures and 50pg of nucleic acid in 50 uL ofsolution, for a total injection volume of about 200 uL, and an acoustic radiation force protocol was applied for 10 seconds, followed by 20 seconds with no application of ultrasound during which the ultrasound probe was removed from the subject, followed again by 10 seconds of an acoustic radiation force protocol, for 40 total seconds of ultrasound acoustic radiation force application. The shear wave ultrasound acoustic radiation force protocol was applied at an ultrasound intensity of 117.5 to 187.9 mW / cm2(ISPTA) (spatial-peak temporal average intensity), at a mechanical index of 1.4, and a frequency of 2.5 MHz. Ultrasound energy was applied using an alternating imaging technique applying focused ultrasound and generating a B-mode ultrasound image at a mechanical index of 1.4, and a frequency of 2.5 MHz with the ultrasound probe removed from contacting the subject as indicated.

[0155] Subjects were administered three total sonoporation treatments and dosages of nucleic acids compositions, with each sonoporation treatment administered 24 hours apart. Following the sonoporation treatments, FVIII expression and secretion was measured in subject plasma samples collected following the sonoporation treatment and frozen at -80 C. Samples were collected approximately every 7 days for about 60 days.

[0156] Transgenic FVIII level in mouse plasma was measured by MSD assay. Briefly capture antibody (GMA-8024) was loaded to the 96-well plate overnight at 4 °C. Next the plate was washed three times with wash buffer and incubated with blocking buffer for 30 min at room temperature. Eight point serial dilution standard were prepared using Xinta® ranging from 0.92 lU / ml to 0.01 lU / ml. 2-fold diluted samples and standards were added to the wells in 96-well plate, incubated 2 hours at room temperature, and washed 3 times. The detection was performed by incubating samples with GMA-8023 antibody for 2 hours following triple wash. Signal was developed by Sulfo-TAG and detected by MSD machine.

[0157] Results are shown in FIGS. 4A-4B. FIG. 4B shows the same data as FIG. 4 A but in bar graph format. Results are shown in FIGS. 4A-4B show that FVIII expression levels continue to rise up to about 20 days following treatment, at which point a steady state expression level of about 0.3 lU / mL was maintained for the duration of the study, maintaining stable expression thought the entire 60 day period. As is shown in FIGS. 4A-4B, an average level of the human clotting factor in the plasma does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state hFVIII plasma level induced by administration of the nucleic acid delivery vector.Example 4: Evaluation of Bicistronic Vector Expression in Multiple Cell Types in a Murine Model of Ultrasound Mediated Nucleic Acid Transfection to the Liver

[0158] In this example, liver samples which were transfected with the bicistronic vector evaluated in Example 3 were analyzed using RNAscope to determine percentage of hepatocytes and liver sinusoidal epithelial cells (LSECs) expressing the first expression cassette and the second expression cassette respectively. Liver samples collected 19 days following the final dose administered in Example 3 were used for this analysis.

[0159] RNAscope analysis was used to identify the expression of a FVIII transgene contained within two different expression cassettes in the bicistronic vector, the ApoE-FVIII expression cassette and the pF8(factor 8 promoter)-FVIII expression cassette. The FVIII transgene expression resulted from administration of the bicistronic vector disclosed herein to a subject, and the RNAscope analysis distinguished their expression in hepatocytes and liver sinusoidal endothelial cells (LSECs). RNAscope, is an advanced in situ hybridization (ISH) technique which allows for the detection of specific RNA molecules at single-molecule resolution, making it ideal for identifying the expression of these expression cassettes in particular liver cell types. It employs specially designed probes that bind to target RNA sequences, allowing precise localization of gene expression at the cellular level. The method provides a highly sensitive and specific approach to studying RNA, offering insights into gene expression patterns in various tissues and cell types. RNAscope is particularly useful in distinguishing between closely related transcripts, detecting low-abundance RNA, and analyzing spatial gene expression in both normal and diseased tissues. Its multiplex capability enables simultaneous detection of multiple RNA targets within the same sample.

[0160] Liver samples which were transfected with the bicistronic vector evaluated in Example 3 were utilized. Fresh frozen samples were fixed with 4% paraformaldehyde (PF A) and treated with RNAscope Protease to ensure adequate probe penetration.

[0161] RNAscope probes were prepared targeting the ApoE-FVIII sequence, which was expected to be expressed in hepatocytes, and the pF8-FVIII sequence, expected to be expressed in LSECs, and hybridized to the tissue sections. To ensure correct identification of the cells expressing these expression cassettes, Asgrl was used as the hepatocyte-specific marker, and Mcam was employed to label LSECs. Both markers were co-hybridized with the respective plasmid probes, allowing for simultaneous detection of nucleic acid expression and cell identity.

[0162] Hybridization was carried out in a humidified oven at 40°C for 2 hours, ensuring efficient binding of the probes to their RNA targets. After hybridization, signal amplification was performed following the steps outlined in the RNAscope Multiplex Fluorescent DetectionKit, which includes sequential application of amplifiers and label probes. Fluorescent dyes were then applied to visualize the probes. The ApoE-FVIII probe was tagged with a green fluorophore (e.g., Alexa Fluor 488), the pF8-FVIII probe with a red fluorophore (e.g., Alexa Fluor 594), Asgrl for hepatocytes was labeled with a suitable fluorescent dye (e.g., Alexa Fluor 647), and Mcam for LSECs was tagged with another distinct fluorophore (e.g., Alexa Fluor 555).

[0163] Once signal amplification was completed, the slides were counterstained with DAPI to label nuclei. The tissue sections were then mounted and visualized under a fluorescence or confocal microscope. The distinct fluorescent signals allow for clear identification of plasmid expression in hepatocytes and LSECs. Hepatocytes, identified by co-localization with the Asgrl probe, displayed green fluorescence for ApoE-FVIII expression, while LSECs, marked by colocalization with the Mcam probe, displayed red fluorescence for pF8-FVIII expression.

[0164] For data analysis, HALO image analysis software (Indica Labs) was used quantify the number of hepatocytes expressing the ApoE-FVIII expression cassette and LSECs expressing the pF8-FVIII expression cassette. The quantification was based on the intensity and area of fluorescence signals. To ensure reliable results, negative controls (without plasmids or probes) to detect non-specific binding were included, as well as positive control slides with known expression cassette-expressing cells, were utilized. Analysis was performed using Indica Labs - ISH v4.2.3. The Image Zoom was set to 1 to access the full range of magnification used during scanning. The stain selection was configured by specifying one probe in the Number of Probes setting and adjusting the nuclear stain by selecting an unstained nucleus in the tissue to define the stain characteristics.

[0165] For the RNAscope probes, each probe was named and adjusted to capture the average color of a puncta. The Exclusion Stain setting was used to ensure that unwanted material was excluded from the analysis. Once satisfied with these settings, the user navigated to Cell Detection to ensure that the Segmentation Type was set to Al Custom, using the nuclear segmentation classifier created for the analysis. Following these configurations, the analysis was performed. Results provided detailed parameters and data for identifying of the RNAscope probe hybridization identifying expression in hepatocytes and LSECs of the APOE-hFVIII and the pF8-hFVIII expression cassettes, respectively.

[0166] Results are shown in FIG. 6, in which it was found that, of the total cells expressing FVIII, 72.17% were hepatocytes, and 27.83% were LSECs, and 0.18% were cells of an unknown type. The results shown herein demonstrate that bicistronic vectors disclosed herein are effective in inducing expression of hF VIII in hepatocytes and LSECs cell populations, andprovide a beneficial technical effect of successfully and simultaneously driving expression in multiple cell types.Example 5: Evaluation of Long Term Expression of Bicistronic Vector in a Murine Model of Ultrasound Mediated Nucleic Acid Transfection to the Liver

[0167] In this example, the bicistronic vector described in Example 1 was evaluated in an expression stability study. The bicistronic vector described in Example 1 comprises two open reading frames encoding the same FVIII transgene having an amino acid sequence of SEQ ID NO. 24. The two open reading frames in the bicistronic vector described in Example 1 were configured to produce expression in multiple liver cell types, hepatocytes and liver sinusoidal epithelial cells (LSEC).

[0168] In this example, a single group of RAG 2 mice was evaluated in a murine model of ultrasound mediated nucleic acid transfection to the liver. The group was administered 100 ug of the bicistronic vector described in Example 1 having the nucleic acid sequence of SEQ ID NO: 46.

[0169] All mice were treated with identical dosages of sonoactive agents, and identical applications of ultrasound. A dose of sonoactive microstructure and DNA solution was readied by first preparing the sonoactive microstructures (Optison) as instructed on the label: remove from 4 °C storage and roll between the palms for 20 seconds; removing protective plastic and aluminum covering from Optison vial; placing 25G needle through the rubber gasket to provide a pressure vent; and using 1.5 inch 18G needle to draw up 150 uL of Optison into a syringe (dead space of the needle (about 50 microliters (uL)) included in the calculations). With the same needle and syringe the DNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenous at a 1 :4 volumetric ratio of DNA to microbubble solution. The DNA + Optison solution was drawn out of the needle dead space. Then the 18G needle was exchanged for a 25G blunt needle for injection into a subject JVC.

[0170] The microbubble-DNA solution was administered to the subject in a single bolus injection over about 30 seconds through the jugular vein catheter, and ultrasound energy was applied to the subject over the liver using an alternating mechanical index protocol. Ultrasound energy was transcutaneously applied to the liver region of the subject with a Cl -6 ultrasound probe, and liver scans of each mouse were performed with a GE LOGIQ elO ultrasound system equipped with a Cl -6 probe, and use of the “ELASTO” software, Shear Wave, using shear wave elastography parameters: GE LOGIQelO probe Cl-6, Abdomen, CHI, Frame rate 55, MI 1.4, Frequency 2.5MHz, Gain 44, Depth 4cm, AO%100, Gain 55, T 8, SVD 6.0, AO%100%, +50-400Hz, GEN. 100% push output and 0% Track output. Mice were administered 150 pL of protein stabilized Optison sonoactive microstructures and 50pg of nucleic acid in 50 uL of solution, for a total injection volume of about 200 uL, and an acoustic radiation force protocol was applied for 10 seconds, followed by 20 seconds with no application of ultrasound during which the ultrasound probe was removed from the subject, followed again by 10 seconds of an acoustic radiation force protocol, for 40 total seconds of ultrasound acoustic radiation force application. The shear wave ultrasound acoustic radiation force protocol was applied at an ultrasound intensity of 117.5 to 187.9 mW / cm2(ISPTA) (spatial-peak temporal average intensity), at a mechanical index of 1.4, and a frequency of 2.5 MHz. Ultrasound energy was applied using an alternating imaging technique applying focused ultrasound and generating a B-mode ultrasound image at a mechanical index of 0.4, and a frequency of 2.5 MHz with the ultrasound probe removed from contacting the subject as indicated.

[0171] Subjects were administered three total sonoporation treatments and dosages of nucleic acids compositions, with each sonoporation treatment administered 24 hours apart. Following the sonoporation treatments, FVIII expression and secretion was measured in subject plasma samples collected following the sonoporation treatment and frozen at -80 C. Samples were collected approximately every 7 days for about 85 days, and then samples were collected at 112 and 203 days following treatment to evaluate the long term durability of gene expression induced using the vector.

[0172] Transgenic FVIII level in mouse plasma was measured by immunoassay (MESO SCALE DIAGNOSTICS, LLC). Briefly capture antibody (GMA-8024) was loaded to the 96- well plate overnight at 4 °C. Next the plate was washed three times with wash buffer and incubated with blocking buffer for 30 min at room temperature. Eight point serial dilution standard were prepared using Xinta® ranging from 0.92 lU / ml to 0.01 lU / ml. 2-fold diluted samples and standards were added to the wells in 96-well plate, incubated 2 hours at room temperature, and washed 3 times. The detection was performed by incubating samples with GMA-8023 antibody for 2 hours following triple wash. Signal was developed by Sulfo-TAG and detected by MSD machine.

[0173] Results are shown in FIGS. 7A-7B. FIG. 7B shows the same data as FIG. 7A but in bar graph format. In FIGS. 7A-7B in which it was shown that FVIII expression levels continue to rise up to about 13 days following treatment, at which point a peak expression level of about 0.28 lU / mL was achieved, with expression continuing for the duration of the study, including levels of 0.18 lU / mL at day 56, 0.15 lU / mL at day 112, and 0.1 lU / mL at day 203. As is shown in FIGS. 7A-7B, an average level (among all subjects) of the human clotting factor in the plasmathroughout the study does not fall below thirty percent (30%) of an average (among all subjects) peak hFVIII plasma level induced by administration of the nucleic acid delivery vector.Example 6: Evaluation of Immunological Effects of a Bicistronic Vector in a Murine Model of Ultrasound Mediated Nucleic Acid Transfection to the Liver

[0174] In this example, a bicistronic vector cloned as described in Example 1 was evaluated in an expression stability and immunogenicity study. The bicistronic vector has a sequence of SEQ ID NO: 53, comprises two open reading frames encoding the same FVIII transgene having an amino acid sequence of SEQ ID NO. 24. The two open reading frames in the bicistronic vector described are as described in Example 1 and were configured to produce expression in multiple liver cell types: hepatocytes and liver sinusoidal epithelial cells (LSEC). The bicistronic vector (aka Monster vector) was evaluated against a monocistronic vector having a sequence of SEQ ID NO: 54 comprising only the first open reading frame encoding the FVIII transgene having an amino acid sequence of SEQ ID NO. 24

[0175] In this example, three groups of 129-S F8 factor VIII knock-out mice were evaluated in a murine model of ultrasound mediated nucleic acid transfection to the liver. Group 1 was administered three ultrasound mediated nucleic acid delivery treatments administering 50 ug of the bicistronic vector having a sequence of SEQ ID NO: 53 in each treatment, with each treatment delivered 48 hours apart. Group 2 was administered three ultrasound mediated nucleic acid delivery treatments administering 50 ug of the monocistronic vector having a sequence of SEQ ID NO: 54 comprising only the first open reading frame in each treatment, with each treatment delivered 48 hours apart. Group 3 was administered no nucleic acid and served as a negative control for the immunogenicity study.

[0176] All mice were treated with identical dosages of sonoactive agents, and identical applications of ultrasound. A dose of sonoactive microstructure and DNA solution was readied by first preparing the sonoactive microstructures (Optison) as instructed on the label: remove from 4 °C storage and roll between the palms for 20 seconds; removing protective plastic and aluminum covering from Optison vial; placing 25G needle through the rubber gasket to provide a pressure vent; and using 1.5 inch 18G needle to draw up 150 uL of Optison into a syringe (dead space of the needle (about 50 microliters (uL)) included in the calculations). With the same needle and syringe the DNA payload was drawn into the syringe to combine the DNA and Optison. The Optison microbubbles and DNA payload were mixed in the syringe by rolling the syringe between the fingers until the solution was homogenous at a 1 :4 volumetric ratio of DNA to microbubble solution. The DNA + Optison solution was drawn out of the needle dead space. Then the 18G needle was exchanged for a 25G blunt needle for injection into a subject JVC.

[0177] Ultrasound energy was applied using a C-16 probe on a GE LOGIQ E10 system in research mode for the acoustic radiation force ultrasound application. The ultrasound parameters are described below.

[0178] The B-mode ultrasound was applied at focal depth setting was set to 5 cm, and the zoom to 0. Ultrasound was delivered at a mechanical index (MI) value of 0.09 and at a frequency of 2.5 MHz to visual the liver tissue and confirm the presence of the sonoactive agent in the tissue and vasculature.

[0179] The acoustic radiation force ultrasound was applied to the liver region of each subject with the same GE LOGIQ elO ultrasound system, equipped with a Cl -6 probe operating using the “ELASTO” software, Shear Wave mode. The ARF was applied immediately following the visualization of the organ with the B-mode ultrasound. The acoustic radiation force was applied with ultrasound acoustic energy applied at an ultrasound intensity of at least 180mW / cm2(ISPTA) (spatial-peak temporal average intensity), a frequency of 2.5 MHz, at a mechanical index of 2.1, and tracking output reduced to about 0%, at a pulse length of about 600 us, a pulse repetition frequency of about 0.9 Hz, a pulse repetition period of about 1.1s and a duty cycle of about 0.05%. Upon disruption of the sonoactive agent with application the ARF ultrasound, serial bolus injections of approximately 1.0-4.0 mL of sonoactive microstructure and DNA solution were re-administered to the subject about every 30 seconds, and the acoustic radiation force ultrasound was applied until the 10 mg of DNA and sonoactive agent solution was fully administered, with the total infusion and sonication treatment time occurring over about 10 minutes. A rapid loss of ultrasound contrast (due to inertial cavitation of the sonoactive agent) occurred following the application of the acoustic radiation force.

[0180] Plasma samples were collected from each group approximately every 7 days for the duration of the study, with Group 2 (monocistronic, SEQ ID NO: 54) being discontinued after bout 19 days due to lack of noted expression. Group 1 (bicistronic, SEQ ID NO: 53) and Group 3 (immunological control) were continued through day 75.

[0181] Transgenic FVIII level in mouse plasma was measured by immunoassay (MESO SCALE DIAGNOSTICS, LLC). Briefly capture antibody (GMA-8024) was loaded to the 96- well plate overnight at 4 °C. Next the plate was washed three times with wash buffer and incubated with blocking buffer for 30 min at room temperature. Eight point serial dilution standard were prepared using Xinta® ranging from 0.92 lU / ml to 0.01 lU / ml. 2-fold diluted samples and standards were added to the wells in 96-well plate, incubated 2 hours at room temperature, and washed 3 times. The detection was performed by incubating samples withGMA-8023 antibody for 2 hours following triple wash. Signal was developed by Sulfo-TAG and detected by MSD machine.

[0182] Results are shown in FIGS. 8A-8B in which it was shown that FVIII expression levels in Group 1 administered the bicistronic (SEQ ID NO: 53) continue to rise up to about 19 days following treatment, at which point a peak expression level of about 0.1 lU / mL was achieved, with therapeutic expression continuing through Day 33 at 0.59 lU / mL and ongoing expression continuing for the duration of the study at day 75. Group 2 administered the monocistronic vector (SEQ ID NO: 54) only comprising the first expression cassette, exhibited increased FVIII expression levels peaking at 0.43 lU / mL at day 19, before exhibiting a rapid decline in FVIII levels to 0.016 at day 19, with no detectable FVIII at day 26, with the rapid decline due to an immune response to the foreign FVIII protein, as further described below.

[0183] To evaluate the loss of the FVIII expression, an immunogenicity study was conducted to determine an adaptive immune response was mounted against FVIII protein expression product. MSD assay for detection of anti-FVIII antibodies was performed on blood samples collected from each of Groups 1-3 at day 26, and on Groups 1 and 3 on Day 75. In brief, an assay was developed to quantify anti-FVIII antibody titers in mouse plasma or serum using a sandwich-format Meso Scale Discovery (MSD) platform and was performed by the Center for Biomedical Testing (Illinois). Plates were first coated with an antihemophilic factor capture antibody and incubated overnight. Serial dilutions of a monoclonal anti-FVIII antibody were prepared to generate a standard curve, alongside quality control samples and diluted mouse test samples. Detection was performed using a labeled secondary antibody specific to mouse immunoglobulin. After a final series of washes and addition of a read buffer, the plates were analyzed using an MSD instrument. Results were interpreted by comparison to baseline or untreated control levels to assess the presence and magnitude of anti-FVIII antibodies.

[0184] Results are shown in FIGS. 9-10. In FIG. 9, it is shown that: Group 3 naive negative control exhibited anti-FVIII levels ranging from about 160-320 establishing a background signal for the assay; Group 2 administered the monocistronic vector (SEQ ID NO: 54) only comprising the first expression cassette, exhibited anti-FVIII antibody titer levels of about 1700-2000 ng / mL depending on the dilution factor; and Group 1 administered the bicistronic (SEQ ID NO: 53) exhibited anti-FVIII antibody titer levels of about 150-220 ng / mL consistent with the Group 3 naive negative control. In FIG. 10, it is shown that Group 3 naive negative control exhibited anti-FVIII levels ranging from about 200-340 establishing a background signal for the assay; and Group 1 administered the bicistronic (SEQ ID NO: 53) exhibited anti-FVIII antibody titer levels of about 200-411 ng / mL representing a slight elevation above the negative control. Theresults of this immunogenicity study support the conclusion that the bicistronic nucleic acid delivery vector disclosed herein is effective at preventing or reducing an immune response to a transgene in a subject with approximately a 10-fold reduction in antibody titer levels for antibodies targeting the transgene expression product observed, when inducing expression of the transgene from a second cell type (here, the LSECs), as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type, or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette. It is further observed that an amount of antibodies binding an expression product of the transgene are within a range of plus or minus about 30% of an amount of antibodies binding the transgene present in anaivee subject not administered the nucleic acid delivery vector as measured by antibody titers.

[0185] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to 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 may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.VI. SEQUENCE LISTING

Claims

CLAIMSWhat is Claimed is:

1. A nucleic acid delivery vector comprising: a. a first expression cassette comprising: i. a first nucleic acid coding sequence encoding a first copy of a transgene; ii. a first promoter sequence operably linked to the first nucleic acid coding sequence; b. a second expression cassette comprising: i. a second nucleic acid coding sequence encoding a second copy of the transgene; ii. a second promoter sequence operably linked to the second nucleic acid coding sequence; c. wherein the nucleic acid delivery vector is at least 10.5 kb in length, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector.

2. A nucleic acid delivery vector comprising: a. a first expression cassette comprising: i. a first nucleic acid coding sequence encoding a first copy of a transgene; ii. a first promoter sequence operably linked to the first nucleic acid coding sequence; b. a second expression cassette comprising: i. a second nucleic acid coding sequence encoding a second copy of the transgene; ii. a second promoter sequence operably linked to the second nucleic acid coding sequence; c. wherein the nucleic acid delivery vector is at least 10.5 kb in length, wherein the nucleic acid delivery vector lacks viral genomic coding sequences.

3. A nucleic acid delivery vector configured to induce expression of a transgene in multiple cell types, the nucleic acid delivery vector comprising: a. a first expression cassette comprising: i. a first nucleic acid coding sequence encoding a first copy of the transgene; ii. a first promoter sequence operably linked to the first nucleic acid coding sequence;b. a second expression cassette comprising: i. a second nucleic acid coding sequence encoding a second copy of the transgene; ii. a second promoter sequence operably linked to the second nucleic acid coding sequence, c. wherein the first expression cassette is configured to induce expression of the transgene in a first cell type, and wherein the second expression cassette is configured to induce expression of the transgene in a second cell type, wherein the second cell type is an LSEC.

4. A nucleic acid delivery vector for expressing a transgene in multiple cell types, comprising: a. a first expression cassette comprising a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in a first cell type; b. a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein the first and second cell types are different.

5. A nucleic acid delivery vector for preventing or reducing an immune response to a transgene, comprising: a. a first expression cassette encoding the transgene, wherein the first expression cassette selectively drives expression in a first cell type; b. a second expression cassette encoding the transgene, wherein the second expression cassette selectively drives expression in a second cell type, wherein expression of the transgene from the second cell type prevents or reduces the immune response to the transgene, as compared to (i) a nucleic acid delivery vector inducing expression of the transgene in a single cell type, or (ii) a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

6. A nucleic acid delivery vector for treating a genetic disorder, comprising: a. a first expression cassette encoding the transgene, wherein the first expression cassette selectively drives expression in a first cell type; b. a second expression cassette encoding the transgene, wherein the second expression cassette selectively drives expression in a second cell type, wherein the nucleic acid delivery vector maintains expression of the transgene at a therapeutic level suitable for treating the genetic disorder, as compared to a nucleic acid delivery vectorinducing expression of the transgene in a single cell type, or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

7. A nucleic acid delivery vector, comprising: a. a first expression cassette comprising a first nucleic acid sequence encoding a transgene operably linked to a first promoter, wherein the first expression cassette selectively drives expression in a first cell type; b. a second expression cassette comprising a second nucleic acid sequence encoding the transgene, wherein the second nucleic acid sequence is operably linked to a second promoter that selectively drives expression of the transgene in a second cell type, wherein: i. the nucleic acid delivery vector is at least 15 kb in length; ii. the first cell type and the second cell type are different; iii. the second cell type is a liver sinusoidal epithelial cell (LSEC); iv. the second promoter selectively drives expression of the transgene in the LSEC; and v. the second expression cassette driving expression of the transgene in the LSEC prevents or reduces an immune response to an expression product of the transgene, wherein the reduction in the immune response comprises at least a 5-fold reduction in antibody titers for an antibody binding the expression product in which the reduction in antibody titers is maintained for a period of at least 26 days, thereby maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder, as compared to (i) a nucleic acid delivery vector inducing expression of the transgene in a single cell type or (ii) a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

8. The nucleic acid delivery vector of any one of claims 5-6, wherein the first expression cassette comprises a first nucleic acid sequence encoding the transgene operably linked to a first promoter that selectively drives expression in the first cell type, and wherein the second expression cassette comprises a second nucleic acid sequence encoding the transgene operably linked to a second promoter that selectively drives expression of the transgene in the second cell type.

9. The nucleic acid delivery vector of any one of claims 1-4 or 6, wherein the nucleic acid delivery vector maintains expression of the transgene at a therapeutic level as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type.

10. The nucleic acid delivery vector of any one of claims 1-5, wherein expression of the transgene from the first cell type prevents or reduces the immune response to the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type.

11. The nucleic acid delivery vector of claim 4, wherein the first nucleic acid sequence encoding the transgene comprises a first nucleic acid coding sequence, wherein the second nucleic acid encoding the transgene comprises a second nucleic acid coding sequence.

12. The nucleic acid delivery vector of any one of claims 2 or 3 or 6-11, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector.

13. The nucleic acid delivery vector of any one of claims 1-12, wherein the nucleic acid delivery vector lacks viral genomic coding sequences.

14. The nucleic acid delivery vector of any one of claims 1-3, wherein the first expression cassette is configured to preferentially induce expression of the transgene in a first cell type, and wherein the second expression cassette is configured to preferentially induce expression of the transgene in a second cell type.

15. The nucleic acid delivery vector of any one of the preceding claims, wherein the first promoter sequence and the second promoter sequence comprise different nucleic acid sequences.

16. The nucleic acid delivery vector of any one of the preceding claims, wherein the first expression cassette and the second expression cassette do not comprise any nucleic acid sequences which comprise 15 or more same nucleotides in sequence.

17. The nucleic acid delivery vector of any one of the preceding claims, wherein the immune response is an adaptive immune response.

18. The nucleic acid delivery vector of any one of the preceding claims, wherein the immune response is an antibody immune response to an expression product of the transgene.

19. The nucleic acid delivery vector of any one of claims 17-18, wherein the nucleic acid delivery vector tolerizes an immune system of a subject to an expression product of the transgene.

20. The nucleic acid delivery vector of any one of claims 17-19, wherein an amount of antibodies binding an expression product of the transgene are reduced.

21. The nucleic acid delivery vector of claim 20, wherein the amount of antibodies binding an expression product of the transgene as measured by antibody titers are reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold.

22. The nucleic acid delivery vector of claim 21, wherein the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is measured in comparison to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

23. The nucleic acid delivery vector of claims 21-22, wherein the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is maintained for a period of at least 7, 14, 21, 26, or 30 days following administration of the nucleic acid delivery vector.

24. The nucleic acid delivery vector of any one of claims 17-18, wherein an amount of antibodies binding an expression product of the transgene are within a range of plus or minus 10, 20, 30, 40 or 50 % of an amount of antibodies binding the transgene present in a naive subject not administered the nucleic acid delivery vector as measured by antibody titers.

25. The nucleic acid delivery vector of claim 24, wherein of antibodies binding an expression product of the transgene as measured by antibody titers is maintained within the range for a period of at least 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 75, or 90 days following administration of the nucleic acid delivery vector.

26. The nucleic acid delivery vector of any one claims 1-2, 12-16, wherein the vector is configured to induce expression of the transgene in different cell types.

27. The nucleic acid delivery vector of any one of the preceding claims, wherein the first expression cassette and second expression cassette are configured to induce expression of the transgene in different cell types.

28. The nucleic acid delivery vector of claim 27, wherein the first expression cassette is configured to express of the first nucleic acid coding sequence in a first cell type, and wherein the second expression cassette is configured to express the second nucleic acid coding sequence in a second cell type.

29. The nucleic acid delivery vector of claim 28, wherein the second cell type is a liver cell.

30. The nucleic acid delivery vector of claim 29, wherein the first cell type is not a liver cell.

31. The nucleic acid delivery vector of claim 28, wherein the first cell type is a hepatocyte.

32. The nucleic acid delivery vector of any one of claims 28 or 31, wherein the second cell type is a liver sinusoidal endothelial cell (LSEC).

33. The nucleic acid delivery vector of any one of claims 26-32, wherein the second promoter sequence selectively drives expression in LSECs.

34. The nucleic acid delivery vector of any one of claims 26-32, wherein the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55.

35. The nucleic acid delivery vector of any one of claims 26-33, wherein the first promoter sequence comprises an ApoE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9.

36. The nucleic acid delivery vector of any one of claims 26-35, wherein the second promoter sequence comprises a F8 promoter sequence.

37. The nucleic acid delivery vector of any one of claims 31-36, wherein the transgene is a FVIII transgene.

38. The nucleic acid delivery vector of claim 37, wherein at least 20% of a total transfected cell population expressing the FVIII transgene are liver sinusoidal endothelial cells.

39. The nucleic acid delivery vector of any one of claims 26-35, wherein at least 20% of a total transfected cell population expressing the transgene are liver sinusoidal endothelial cells.

40. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector is at least 12.5 or 15 kb in length.

41. The nucleic acid delivery vector of any one of the preceding claims, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence are codon diversified relative to one another.

42. The nucleic acid delivery vector of claim 41, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence comprise different nucleic acid sequences but encode the same transgene.

43. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector is single stranded.

44. The nucleic acid delivery vector of any one of claims 1-42, wherein the nucleic acid delivery vector is double stranded.

45. The nucleic acid delivery vector of claim 44, wherein the first expression cassette and the second expression cassette are both in a sense orientation in the nucleic acid delivery vector.

46. The nucleic acid delivery vector of claim 44, wherein the first expression cassette is in the sense orientation in the nucleic acid delivery vector, and wherein the second expression cassette is in an anti-sense orientation in the nucleic acid delivery vector.

47. The nucleic acid delivery vector of claim 44, wherein the first expression cassette is upstream or positioned 5’ of the second expression cassette.

48. The nucleic acid delivery vector of claim 44, wherein the first expression cassette is downstream or positioned 3’ of the second expression cassette49. The nucleic acid delivery vector of any one of the preceding claims, wherein the first expression cassette further comprises an intron sequence.

50. The nucleic acid delivery vector of any one of the preceding claims, wherein the second expression cassette further comprises an intron sequence.

51. The nucleic acid delivery vector of claim 49 or 50, wherein the intron sequence comprises a hemoglobin subunit gamma intron (hBGi) sequence.

52. The nucleic acid delivery vector of claim 50, wherein the intron sequence in the first expression cassette and the intron sequence in the second expression cassette comprise different nucleic acid sequences.

53. The nucleic acid delivery vector of any one of the preceding claims, wherein the first expression cassette further comprises a posttranscriptional regulatory element.

54. The nucleic acid delivery vector of any one of the preceding claims, wherein the second expression cassette further comprises a posttranscriptional regulatory element.

55. The method of claim 54, wherein the posttranscriptional regulatory element in the second expression cassette and the posttranscriptional regulatory element in the first expression cassette comprise different nucleic acid sequences.

56. The nucleic acid delivery vector of claim 53 or 54, wherein the posttranscriptional regulatory element comprises a woodchuck hepatitis posttranscriptional regulatory element.

57. The nucleic acid delivery vector of claim 53 or 54, wherein the posttranscriptional regulatory element comprises a polyadenylation signal.

58. The nucleic acid delivery vector of claim 53 or 54, wherein the posttranscriptional regulatory element comprises a polyadenylation signal coupled downstream to a woodchuck hepatitis posttranscriptional regulatory element.

59. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector further comprises a nuclear targeting sequence.

60. The nucleic acid delivery vector of any one of the preceding claims, wherein nuclear targeting sequence is positioned downstream of the first expression cassette and upstream of the second expression cassette.

61. The nucleic acid delivery vector of any one of the preceding claims, wherein nuclear targeting sequence is positioned downstream of the second expression cassette.

62. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector increases expression of the transgene when administered to a subject as compared to a nucleic acid delivery vector comprising either the first expression cassette or the second expression cassette alone.

63. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector is a non-viral vector.

64. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector is a DNA vector.

65. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector is not comprised within a viral capsid.

66. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector is configured to be administered to a subject as unencapsulated DNA.

67. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector comprises any one of SEQ ID NO: 1-2, 4-6, 9, 16, 32, 33, or 39.

68. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector comprises any one of SEQ ID NO: 47 or 49.

69. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector comprises SEQ ID NO: 47 and 49.

70. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45 over a minimum alignment length of at least 6500, 7000, or 7500 nucleotides.

71. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45.

72. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector comprises SEQ ID NO: 45.

73. The nucleic acid delivery vector of any one of the preceding claims, wherein the first coding sequence or the second coding sequence encodes an amino acid sequence of SEQ ID NO.24.

74. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector further comprises a third expression cassette comprising a third nucleic acid coding sequence encoding a third copy of a transgene.

75. The nucleic acid delivery vector of any one of the preceding claims, wherein the third expression cassette comprises a third promoter sequence operably linked to the third nucleic acid coding sequence.

76. The nucleic acid delivery vector of any one of the preceding claims, wherein the nucleic acid delivery vector further comprises a fourth expression cassette comprising: a fourth nucleic acid coding sequence encoding a fourth copy of the transgene.

77. The nucleic acid delivery vector of any one of the preceding claims, wherein the fourth expression cassette comprises a fourth promoter sequence operably linked to the fourth nucleic acid coding sequence.

78. A method of delivering a nucleic acid to a subject comprising administering to the subject the nucleic acid delivery vector of any one of the preceding claims.

79. A method of treating a bleeding disorder in a subject in need thereof comprising administering to the subject the nucleic acid delivery vector of claim 37.

80. The method of claim 79, wherein the bleeding disorder is Hemophilia A.

81. A kit comprising the nucleic acid delivery vector of any one of the preceding claims, and a sonoactive agent.

82. The kit of claim 81, further comprising instructions for administering to a subject the sonoactive agent and ultrasound energy.

83. Use of the nucleic acid delivery vector any one of the preceding claims for treating a genetic disorder in a subject in need thereof, comprising administering the nucleic acid delivery vector to the subject, thereby inducing expression of the transgene and treating the genetic disorder.

84. A method of delivering a nucleic acid to both a first cell type and a second cell type in a subject using a nucleic acid delivery vector, the method comprising: a. administering to the subject a nucleic acid delivery vector comprising a first nucleic acid coding sequence encoding a first copy of a transgene and a second nucleic acid coding sequence encoding a second copy of the transgene; and b. administering to the subject a sonoactive agent and ultrasound energy, thereby inducing expression of the transgene in both the first cell type and the second cell type.

85. A method of treating a bleeding disorder in a subject comprising administering to the subject a nucleic acid delivery vector inducing expression of a transgene encoding a human clotting factor in both a first cell type and a second cell type in the subject, thereby maintaining a therapeutic level of the human clotting factor in a plasma of the subject.

86. A method of preventing or reducing an immune response to an expression product of a transgene in a subject, comprising: administering to the subject a nucleic acid delivery vector inducing expression of the transgene in a first cell type and a second cell type in the subject, thereby reducing the immune response to the expression product of the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type.

87. A method of treating a genetic disorder in a subject in need thereof, comprising: administering to the subject a nucleic acid delivery vector, wherein the nucleic acid delivery vector induces expression of a transgene in a first cell type and a second cell type in the subject, thereby maintaining a therapeutic level of an expression product of the transgene in the subject and treating the genetic disorder.

88. A method of inducing expression of a transgene in a subject, comprising: a. administering to the subject a sonoactive agent; b. administering to the subject a nucleic acid delivery vector, comprising; i. a first expression cassette comprising a first nucleic acid sequence encoding the transgene operably linked to a first promoter, wherein the first expression cassette selectively drives expression in a first cell type; ii. a second expression cassette comprising a second nucleic acid sequence encoding the transgene operably linked to a second promoter, wherein the second expression cassette selectively drives expression of the transgene in a second cell type, wherein: the first cell type and second cell type are different, the second cell type is a liver sinusoidal epithelial cell (LSEC), the second promoter selectively drives expression of the transgene in the LSECs, and the nucleic acid delivery vector is at least 15 kb in length; and c. applying ultrasound energy to tissue(s) of the subject comprising the first cell type and the second cell type, thereby inducing expression of the transgene in the first cell type and the second cell type, wherein the second expression cassette driving expression of the transgene from the LSECs prevents or reduces an immune response to an expression product of the transgene as measured by at least a 5-fold reduction in antibody titers for an antibody binding the expression product in which the reduction in antibody titers is maintained for a period of at least 26 days, thereby maintaining expression of the transgene at a therapeutic level suitable for treating a genetic disorder, as compared to (i) a nucleic acid delivery vector inducing expression of the transgene in a single cell type or (ii) a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

89. The method of any one of claims 84-87, wherein expression of the transgene in both the first cell type and the second cell type maintains a therapeutic level of an expression product of the transgene in the subject and treats a genetic disorder.

90. The method of any one of claims 84-87, wherein inducing expression of the transgene in the first cell type and the second cell type in the subject reduces an immune response to theexpression product of the transgene as compared to a nucleic acid delivery vector inducing expression of the transgene in a single cell type.

91. The method of any one of claims 86, 88, or 90, wherein the immune response is an adaptive immune response.

92. The method of claim 91, wherein the immune response is an antibody immune response to an expression product of the transgene.

93. The method of claim 91 or 92, wherein expression of the transgene in the second cell type tolerizes an immune system of a subject to an expression product of the transgene.

94. The method of any one of claims 92-93, wherein an amount of antibodies binding an expression product of the transgene are reduced.

95. The method of any one of claims 92-94, wherein the amount of antibodies binding an expression product of the transgene as measured by antibody titers are reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold.

96. The method of claim 95, wherein the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is measured in comparison to a nucleic acid delivery vector inducing expression of the transgene in a single cell type or a nucleic acid delivery vector comprising only the first expression cassette or the second expression cassette.

97. The method of any one of claims 95-96, wherein the reduction of antibodies binding an expression product of the transgene as measured by antibody titers is maintained for a period of at least 7, 14, 21, 26, or 30 days following administration of the nucleic acid delivery vector98. The method of any one of claims 91-93, wherein an amount of antibodies binding an expression product of the transgene are within a range of plus or minus 10, 20, 30, 40 or 50 % of an amount of antibodies binding the transgene present in a naive subject not administered the nucleic acid delivery vector as measured by antibody titers99. The method of claim 98, wherein of antibodies binding an expression product of the transgene as measured by antibody titers is maintained within the range for a period of at least 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 75, or 90 days following administration of the nucleic acid delivery vector.

100. The method of any one of claims 84-99, wherein the second cell type is a liver cell.

101. The method of any one of claims 84-100, wherein the first cell type is not a liver cell.

102. The method of any one of claims 84-101, wherein the second promoter sequence selectively drives expression in LSECs.

103. The method of any one of claims 84-102, wherein the second promoter sequence comprises a promoter sequence any one of SEQ ID NO: 50, or 55.

104. The method of any one of claims 84-103, wherein the first promoter sequence comprises an ApoE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9.

105. The method of any one of claims 84-88, wherein the transgene encodes a human clotting factor, and the administration of the nucleic acid delivery vector induces and maintains a therapeutic level of the human clotting factor in a plasma of the subject106. The method of any one of claims 85-105, wherein the therapeutic level of the human clotting factor is maintained for at least 30, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days following administration of the nucleic acid delivery vector.

107. The method of any one of claims 85-106, wherein the nucleic acid delivery vector comprises a first nucleic acid coding sequence encoding a first copy of a transgene and a second nucleic acid coding sequence encoding a second copy of the transgene.

108. The method of any one of claims 85, further comprising administering to the subject ultrasound energy.

109. The method of claim 108, further comprising administering to the subject a sonoactive agent.

110. The method of any one of claims 85-109, wherein the transgene comprises hFVIII, hFIX, or von-Willebrand factor.

111. The method of any one of claims 85-110, wherein the bleeding disorder comprises hemophilia A, hemophilia B or Von Willebrand disease.

112. The method of any one of claims 85-111, wherein the transgene comprises hFVIII, wherein the bleeding disorder comprises hemophilia A, and wherein an average level of the human clotting factor in the plasma does not fall below thirty percent (30%) of a peak hFVIII plasma level induced by administration of the nucleic acid delivery vector.

113. The method of claim 112, wherein the average level of the human clotting factor is assessed over one or more 30-day periods, and the peak hFVIII plasma level is a maximum discreet value within said one or more 30-day periods, each following administration of the nucleic acid delivery vector.

114. The method of any one of claims 85-113, wherein the transgene comprises hFVIII, wherein the bleeding disorder comprises hemophilia A, and wherein an average level of the human clotting factor in the plasma does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state hFVIII plasma level induced by administration of the nucleic acid delivery vector.

115. The method of claim 114, wherein the average level of the human clotting factor and the average steady state hFVIII plasma level are assessed over one or more 30-day periods following administration of the nucleic acid delivery vector.

116. The method of claim 84 or 85, wherein the nucleic acid delivery vector comprises a first expression cassette comprising: the first nucleic acid coding sequence encoding the first copy of a transgene; and a first promoter sequence operably linked to the first nucleic acid coding sequence.

117. The method of claim 116, wherein the nucleic acid delivery vector comprises a second expression cassette comprising: the second nucleic acid coding sequence encoding the second copy of a transgene; and a second promoter sequence operably linked to the second nucleic acid coding sequence.

118. The method of claim 117, wherein the first copy of the transgene is expressed relative to the second copy of the transgene, at a higher level in the first cell type and the second copy of the transgene is, relative to the first copy of the transgene, expressed at a higher level in the second cell type.

119. The method of claim 117, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence collectively comprise at least 50% of total nucleotides in the nucleic acid delivery vector.

120. The method of any one of claims 84 or 85, wherein the nucleic acid delivery vector is at least 5, 7.5, 10, 12.5 or 15 kb in length.

121. The method of any one of claims 84-120, wherein the nucleic acid delivery vector lacks viral genomic coding sequences.

122. The method of any one of claims 84-121, wherein the nucleic acid delivery vector wherein the first expression cassette and the second expression cassette do not comprise any nucleic acid sequences which comprise 15 or more same nucleotides in sequence.

123. The method of any one of claims 84-122, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence are codon diversified relative to one another.

124. The method of any one of claims 84-123, wherein the first nucleic acid coding sequence and the second nucleic acid coding sequence comprise different nucleic acid sequences but encode the same transgene.

125. The method of any one of claims 84-124, wherein the nucleic acid delivery vector is single stranded.

126. The method of any one of claims 84-124, wherein the nucleic acid delivery vector is double stranded.

127. The method of any one of claims 84-125, wherein the first expression cassette and the second expression cassette are both in a sense orientation in the nucleic acid delivery vector.

128. The method of any one of claims 84-127, wherein the first expression cassette is in the sense orientation in the nucleic acid delivery vector, and wherein the second expression cassette is in an anti-sense orientation in the nucleic acid delivery vector.

129. The method of any one of claims 84-128, wherein the sonoactive agent comprises a microbubble or nanobubble filled with a perfluorinated gas.

130. The method of any one of claims 84-129, wherein the sonoactive agent comprises protein stabilized microstructures.

131. The method of any one of claims 84-130, wherein the sonoactive agent comprises a C3F8 gas encapsulated within a protein stabilized shell.

132. The method of any one of claims 84-131, wherein the sonoactive agent comprises lipid stabilized microstructures.

133. The method of any one of claims 84-132, wherein the sonoactive agent comprises a SFe gas encapsulated within a lipid stabilized shell.

134. The method of any one of claims 84-133, wherein the sonoactive agent comprises a C4F10 gas encapsulated within a lipid stabilized shell.

135. The method of any one of claims 84-134, wherein ultrasound energy is applied at an MI of at least 0.8, 1.3, 1.8, 1.9, or 2.2.

136. The method of any one of claims 84-135, wherein administering to the subject ultrasound energy comprises applying an acoustic radiation force.

137. The method of any one of claims 84-136, further comprising administering to the subject a second dose of the sonoactive agent and the nucleic acid delivery vector, and applying ultrasound a second time.

138. The method of any one of claims 84-137, further comprising administering to the subject a third dose of the sonoactive agent and the nucleic acid delivery vector, and applying ultrasound energy a third time.

139. The method of any one of claims 136-138, wherein the second dose is administered at least 24 hours after the initial administration of the sonoactive agent and the nucleic acid delivery vector, and initial application of the ultrasound energy.

140. The method of claim 139, wherein the third dose is administered at least 24 hours after the second administration of the sonoactive agent and the nucleic acid delivery vector, and second application of the ultrasound energy.

141. The method of any one of claims 84-140, wherein the first copy of the transgene is expressed in the first cell type, and wherein the second copy of the transgene is expressed in the second cell type.

142. The method of claim 141, wherein the first cell type is a hepatocyte.

143. The method of any one of claims 141-142, wherein the second cell type is a liver sinusoidal endothelial cell.

144. The method of claim 143, wherein at least 20% of a total population of cells expressing the transgene are liver sinusoidal endothelial cells.

145. The method of any one of claims 141-143, wherein the first promoter sequence comprises an ApoE-AAT promoter sequence, or a promoter sequence of SEQ ID NO: 9.

146. The method of any one of claims 141-145, wherein the second promoter sequence comprises a F8 promoter sequence.

147. The method of claim 141, wherein the transgene is a FVIII transgene.

148. The method of claim 141, wherein the delivery to the first cell type and the second cell type occurs simultaneously.

149. The method of claim 141, wherein the expression in the first cell type and the second cell type occurs simultaneously.

150. The method of claim 141, wherein an average expression level of the transgene does not fall below thirty percent (30%) of a peak expression level of the transgene induced by administration of the nucleic acid delivery vector.

151. The method of claim 150, wherein the average expression level of the transgene and the peak average expression level of the transgene over one or more 30-day periods, and a peak expression level is a maximum discreet value within said one or more 30-day periods, each following administration of the nucleic acid delivery vector.

152. The method of claim 141, wherein an average expression level of the transgene does not vary by more than ± (plus or minus) thirty percent (30%) of an average steady state expression level of the transgene induced by administration of the nucleic acid delivery vector.

153. The method of claim 152, wherein the average expression level of the transgene and the average steady state expression level of the transgene are assessed over one or more 30-day periods following administration of the nucleic acid delivery vector.

154. The method of any one of claims 150-153, wherein expression level of the transgene is determined by mRNA production, a reporter gene, or transgene protein production.

155. The method of any one of claims 84-153, wherein expression of the transgene is maintained for at least 30, 60, 70, 80, 90,100, 110 or 120 days following administration of the nucleic acid delivery vector.

156. The method of any one of claims 84-155, wherein the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45 over a minimum alignment length of at least 6500, 7000, or 7500 nucleotides.

157. The method any one of claims 84-156, wherein the nucleic acid delivery vector comprises at least 90% sequence identity to SEQ ID NO: 45.

158. The method any one of claims 84-157, wherein the nucleic acid delivery vector comprises SEQ ID NO: 45.

159. The method any one of claims 84-158, wherein the first coding sequence or the second coding sequence encodes an amino acid sequence of SEQ ID NO. 24.

160. The method of claim 84, wherein the nucleic acid delivery vector is the nucleic acid delivery vector of any one of claims 1-73.

161. Use of a nucleic acid delivery vector for treating a genetic disorder in a subject in need thereof, comprising administering the nucleic acid delivery vector to the subject in accordance with the method of any one of the preceding claims.

162. A nucleic acid having a sequence of any one of SEQ ID NO: 1-55.

163. A nucleic acid having a sequence of SEQ ID NO: 9 or 50.

164. The nucleic acid of claim 163, wherein the sequence is a promoter sequence.

165. The nucleic acid of claim 163, wherein the sequence is SEQ ID NO: 50, wherein the sequence selectively drives gene expression in LSECs.

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