Cavitation-facilitated AAV transduction of cardiac cells

Abstract

The present disclosure describes ultrasound-based methods for facilitating the selective transduction of cardiac cells with therapeutic AAV vectors in vivo.

Description

CAVITATION-FACILITATED AAV TRANSDUCTION OF CARDIAC CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 563, 171 filed on March 8, 2024, the content of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under HL171377 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] The adoption of gene therapy in clinical practice is predicated on the ability to safely and efficiently deliver genetic material to target tissues in a manner that favorably alters cell phenotype and organ function. Successful, high-efficiency, and safe gene transfer to cardiomyocytes and other cells of the heart could potentially revolutionize care for a wide variety of dilated, ischemic, arrhythmogenic, or infiltrative cardiomyopathies. The introduction of genetic material into cells has relied on either non-viral or viral vectors. Non-viral methods, including the use of “naked” plasmid DNA, is highly inefficient because of the low number of cells transfected even after direct injection, and short duration of expression. Viral vectors such as lentivirus and adenoviruses provide more efficient gene delivery (transduction) and longer duration of gene expression. However, lentivirus requires direct intramyocardial injection and adenoviral vectors are associated with serious safety issues from severe immune reactions. The advent of adeno- associated viruses (AAVs) as a safer vector has led to a resurgence in interest in gene therapy for the heart. AAVs are members of the parvovirus family surrounded by a protein shell (capsid) that have been engineered to enter cells and to be trafficked to the nucleus where they are maintained as episomes and generally not incorporated into host chromosomes.

[0004] Despite the success of AAVs in preclinical models and their promise in human diseases, a major limiting factor is the large vector doses required to achieve high levels of cardiomyocyte transduction and intended phenotypic response after systemic injection. These high doses are associated with serious dose-related adverse events (AEs) related to off-target effects and immune-mediated multi-organ damage in clinical trials. Methods to improve AAV transduction efficiency would allow dose-reduction, thereby increasing safety and reducing cost.SUMMARY

[0005] In an aspect, provided herein is a method for transducing cardiac cells with a therapeutic AAV vector in a subject in need thereof, the method comprising: administering the AAV vector to the subject; administering a microbubble (MB) composition to the subject; and administering ultrasound to the subject. The cardiac cells may comprise at least one of cardiomyocytes, myofibroblasts, endothelial cells, and smooth muscle cells. In embodiments, the cardiac cells comprise cardiomyocytes.

[0006] The ultrasound may be transthoracic ultrasound.

[0007] The MB composition may be administered via intravenous infusion, intracoronary infusion, or intracardiac infusion. The MB composition may comprise a lipid MB. The MB composition may comprise a perfluorocarbon MB. The perfluorocarbon may be selected from octafluoropropane and decafluorobutane.

[0008] The MB composition and the ultrasound may be administered in conjunction with the AAV vector. The MB composition may be administered at a dose of between about 5xl06and about 5xl09MB / kg. The MB composition may be administered at a dose of about 2xl08MB / kg. The MB composition may be administered for between about 30 seconds and about 30 minutes.

[0009] The ultrasound may be administered at a frequency of between about 1.0 MHz and about 2.5 MHz. The ultrasound may be administered at a mechanical index of between about 0.3 and about 1.8. The mechanical index may be about 1.2.

[0010] The ultrasound may be administered with at least one pulse of at least 100 microseconds (ps). The at least one pulse may be between about 100 ps and about 100 milliseconds (ms). The ultrasound is administered with about five pulses of about 20 ps per pulse. The ultrasound is administered with pulsing intervals of about 5 seconds. The ultrasound may be administered for 4 or more multipulses with a pulse repetition frequency of between about 1 and about 10 kHz, wherein each pulse of the multipulses is administered for between about 1 and about 15 microseconds.

[0011] The AAV vector may be administered via intravenous infusion, intracoronary infusion, or intracardiac infusion. The AAV vector may be administered at a dose of between about IxlO10and about 1x1013vector genomes (vg) / kg. The AAV vector may be an AA8 vector or an AAV9 vector.

[0012] The AAV vector may comprise a therapeutic polynucleotide operably linked to a cardiac-specific promoter. The cardiac-specific promoter may be a chicken cardiac troponin T (cTnT) promoter.

[0013] The ultrasound may be done using an ultrasound transducer; wherein the ultrasound transducer transmits the ultrasound at a medium line density of between about 20 and about 40 lines. The ultrasound transducer may transmit the ultrasound at a medium line density of about 32 lines. The ultrasound transducer may have a focal depth of between about 2 and 6 centimeters from the transducer.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or patent application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0015] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0016] FIGS. 1A-1E. (A) Transfection of luciferase (luc) reporter plasmid in the mouse. Leg exposed to US according to whether cDNA dose per MB, and whether MBs were charge coupled to cationic microbubbles (MB+) or non-conjugated with neutral agent (MBN). Optical imaging showing luc activity in the leg and the liver (in the shaded area), both of which were exposed to US, after I.V. injection of cationic MB combined with 2.5, 50, or 200 mcg / 108 MB (B-D), or with MBN combined with 200 mcg / 108 MB (E).

[0017] FIGS. 2A-2B. (A) Short-axis fluorescent microscopy of the LV of mice after AAV9+CFAT or AAV-9 alone (1 * 1013vg / kg) using a tdTomato (tdT) fluorescent reporter (pseudo-colored green) driven by the promiscuous hybrid CMV enhancer / chicken-beta-actin (CAG) promoter. Over 85% of cardiomyocytes were transduced by CFAT. Right panels show highmagnification of the area within the dashed lines, representing the lowest transduction area, after CFAT. Endothelial CD31 co-staining illustrates exclusive transduction of cardiomyocytes. (B) Confirmation of CFAT-mediated augmentation of transduction of a luciferase (luc) reporter gene with an AAV9 vector (l * 1013Vg / kg). The top images show in vivo bioluminescent acquisitions acquired under identical conditions (including dynamic range) 10 minutes after intraperitoneal injection of d-luciferin. Bioluminescent studies were performed 2 weeks after administration of vector. Luciferase activity in the region of the heart (arrows) is sufficient to be detected in the animal in whom CFAT was performed. The graphs illustrate ex vivo luciferase activity from the entire heart (normalized to heart) 4 weeks after administration of vector showing augmented transduction of the AAV9 with luc reporter at two different doses.

[0018] FIGS. 3A-3B. (A) Fluorescent microscopy of the LV of mice after AAV9 alone (3x l0nvg / kg) or AAV9+CFAT using a tdT reporter gene. (B) Fluorescent microscopy from the anteroseptal region of the LV from a Rhesus macaque after AAV9 alone (3 G O1 1vg / kg) or AAV9+CFAT using a tdT reporter gene (pseudo-colored red for primates) driven by the promiscuous hybrid CMV enhancer / chicken-beta-actin (CAG) promoter.

[0019] FIGS. 4A-4B. (A) Potential mechanisms for the ability of CFAT to augment transduction with AAV (top left). All mechanisms are likely to be more efficient if cavitation effects are performed at the capillary level where there are minimal other cellular (smooth muscle cell) and non-cellular (matrix) barriers. Permeability from microbubble cavitation at the capillary level has already been confirmed by in vivo fluorescent imaging of the limbs of mice whereby extravasation of a fluorescently-labeled macromolecule is much greater in the limb exposed to ultrasound at different acoustic pressures (mega-Pascals [MPa]) for 10 min after intravenous injection of microbubbles (bottom left). (B) Electron microscopy images show the ability to detect: trans-cytotic vesicles crossing coronary capillary endothelial cells (ECs) toward the extracellular space (ECS); tight junction integrity (TJ), and glycocalyx integrity by cationized ferritin (CF).

[0020] FIG. 5. Cardiac transduction efficiency measured by in vivo bioluminescent imaging 2 weeks after intravenous administration of AAV9 vector containing a d-luc reporter gene at a dose of 5xl012vg / kg. Control refers to AAV9 alone. All other conditions are variations of CFAT described in the text.

[0021] FIG. 6. Hydrophone data from different acoustic parameters on the Epiq imaging platform. The color coded images depict pressure spectra transducer (left to right), in theelevational and azimuthal dimensions at various distances from the probe (position 0 near probe surface). The right graphs show centerline pean negative acoustic pressure according to depth from The “Old Flash” represents the clinical build provided by the manufacturer for inertial cavitation whereas the “New Flash” was specifically designed for even field strength in the near-field for murine cardiac exposure.

[0022] FIG. 7. Ex vivo luminescence assays for heart and for adjacent liver 28 days after AAV9 administration (5xl012vg / kg) as a readout for transduction efficiency in the heart and a non-target organ.

[0023] FIG. 8. MicroSPECT imaging to detect NalS activity in a mouse 24 days after CFAT with an AAV9 vector in which the cTnT promoter drives the sodium iodide symporter (NalS) reporter gene. Note that the left ventricular myocardium demonstrates intense activity, with much lower activity in other organs, including the thyroid (where endogenous NalS activity is normally detected in control animals).

[0024] FIG. 9. High frequency transthoracic echocardiography data on LV systolic function in mice before (baseline') administration of AAV9 alone (red data) or AAV9 with CFAT-Sonos (blue data), and at various time intervals after administration.

[0025] FIGS. 10A-10C. (A) Schematic of ultrasound pulse amplitudes and simulated line density. (B) Stationary hydrophone data .(C) Single element transducer cavitation.DETAILED DESCRIPTION

[0026] As demonstrated herein, the inventors have revealed that cardiomyocyte transduction by AAV9, which has myocyte tropism, is markedly enhanced by cavitation of lipid microbubbles (MBs) using ultrasound in the diagnostic range for frequency and acoustic pressure. Cavitation- facilitated AAV transduction (CFAT) differs from previously published plasmid transfection in several important ways. AAV9 transduction does not require conjugation to the MB surface. Gene expression with AAV9 is naturally biased towards cardiomyocytes, and a vast majority of cardiomyocytes are transduced at a fraction of the usual dose. The inventors have identified the acoustic conditions, ultrasound pulse schemes, and MB doses that allow one to perform CFAT at the capillary level where transfer of AAV from the vascular space occurs, and to stimulate capillary endothelial transfer mechanisms for AAV that are not associated with serious adverse effects but are associated with 5-8-fold increases in efficiency of cardiomyocyte transduction.

[0027] Accordingly, in a first aspect, provided herein is a method for transducing cardiac cells with a therapeutic adeno-associated virus (AAV) vector in a subject in need thereof, the method comprising: administering the therapeutic AAV vector to the subject; administering a microbubble (MB) composition to the subject; and administering ultrasound to the subject. A “cardiac cell” refers to a cell localized in the heart of a subject. The cardiac cell may comprise at least one of cardiomyocytes, myofibroblasts, endothelial cells, and smooth muscle cells. In exemplary embodiments, the cells are cardiomyocytes.

[0028] Microbubbles (MBs) in the field of medical diagnostics are typically used as contrast agents for ultrasound imaging. Smaller than one hundredth of a millimeter in diameter, but larger than one micrometer, a MB is comprised of a gas encapsulated by a shell. Shell materials may comprise one or more of lipids, albumins, proteins, etc. In exemplary embodiments, the shell is comprised of lipids. The gas is typically air or a perfluorocarbon. In exemplary embodiments, the gas is a perfluorocarbon. In further exemplary embodiments, the gas is octafluoropropane and decafluorobutane. The gas may be any useful gas that can be used safely for the described methods. A microbubble composition may comprise a homogenous or heterogenous population of microbubbles. For example, each microbubble in the microbubble composition may be of the same or similar size, gas, shell, etc. In other examples, microbubbles in the microbubble composition may have different sizes, gases, shells, etc.

[0029] The microbubbles within the microbubble composition may be between about 0.5 to about 6 microns in size.

[0030] The ultrasound may be administered at a frequency of between about 1.0 MHz and about 3.5 MHz, between about 1.0 MHz and about 2.5 MHz, or any frequency or range in between. The ultrasound may be administered for between about five and about fifteen minutes, or any duration or range in between. The ultrasound may be administered at a mechanical index (MI) of between about 0.3 and about 1.8, or any MI or range in between. In exemplary embodiments, the mechanical index is 1.2.

[0031] “Ultrasound” is sound with frequencies greater than 20 kilohertz (kHz) generated by an ultrasonic transducer. The ultrasound may be a transthoracic ultrasound. The ultrasound may be administered with at least one pulse of at least 100 microseconds (ps). The ultrasound may be administered with pulses of between about 100 microseconds and about 100 milliseconds (ms) with pulsing intervals that allow complete replenishment of microbubbles between cavitationpulses, which is of about 5 seconds for the human heart. The ultrasound may be administered with about five pulses of about 20 ps per pulse. Alternatively, the ultrasound may be administered for 4 or more consecutive pulses (multipulses) with pulsing intervals of about 5 seconds, wherein each pulse of the multipulse is administered for between about 1 and about 15 ms. The ultrasound may be administered with a pulse repetition frequency of between about 1 and about 10 mHz.

[0032] In preferred embodiments, the transducer transmits the ultrasound at a medium line density. The ultrasound may be transmitted at a line density of between about 20 and about 40 lines. In exemplary embodiments, the ultrasound is transmitted at a line density of 32 lines. Line density is of utmost importance to ensure MB cavitation at the intended ultrasound pressure amplitude (FIG. 10). High line densities result in line overlap where neighboring ultrasound lines that are overlapping can produce ultrasound powers above the cavitation threshold (CT) but lower than the intended peak. Too low of a line density results in non-cavitated MBs between lines (FIG. 10A which schematically shows ultrasound pulse amplitudes as lines approach a microbubble (top) and simulated line density from the Philips transducer). Actual stationary hydrophone data placed in the center of the transducer at the acoustic focus indicate a gradual increase in ultrasound pressure as lines approach the hydrophone at a line density of 64, and a relatively low degree of inertial cavitation (broad band signal from in vitro cavitation the lipid microbubble agent (FIG. 10B). Single element transducer cavitation (FIG. 10C) which does not have neighboring lines has a much higher broadband signal. Hence, line density is medium in exemplary embodiments.

[0033] The ultrasound transducer will typically have a focal depth, the depth from the transducer where the ultrasound beam is most concentrated and focused, at between about 2 and about 6 centimeters from the transducer (depending upon the size of the subject, the depth of the target tissue, and the properties of the transducer). In exemplary embodiments shown in mice using a 1 to 3 MHz transducer, the focal depth is about 2 cm from the face of the transducer.

[0034] Adeno associated viruses (AAVs) are non-pathogenic viruses that belong to the genus Dependoparvovirus. AAV are small, nonenveloped viruses that have a linear single-stranded DNA genome that is approximately 4.7 kilobases (kb) in size. AAV viruses are replication defective, meaning that the production of AAV virus requires coinfection with helper virus(es). As used herein, the term "vector" refers to a virus particle that is used to deliver genetic material into cells. The term includes the vector as a nucleic acid genome structure packaged in a capsid for administration, as well as the vector genome after introduction into the nucleus of a host cell intowhich it has been introduced. AAVs offer several advantages for use as gene therapy vectors: AAV-based gene therapy vectors cause a very mild immune response, can infect both dividing and quiescent cells, and persist in an extrachromosomal state without integrating into the genome of the host cell. At least 11 AAV serotypes have been identified, the serotypes differing in their tropism, or the types of cells they infect. AAV serotype 9 (AAV9) has tropism for cardiomyocytes and has a relatively long plasma half-life compared to other AAV serotypes. Therefore, in exemplary embodiments, the AAV vector is an AAV9 vector. However, other AAV capsid serotypes (e.g. AAV8) could also be used, including hybrid capsids and capsids that have been artificially engineered to target specific cell types.

[0035] As used herein, the term “therapeutic AAV vector” refers to an AAV vector that includes a nucleic acid sequence encoding a therapeutic construct. The term “construct” refers to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic). For example, the therapeutic construct may comprise a portion of the coding region of a therapeutic polynucleotide operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) from the genome of a different species, or (3) is synthetic. As used herein, the term “promoter” refers to a DNA sequence that regulates the transcription of a polynucleotide. Typically, a promoter is a regulatory region that is capable of binding RNA polymerase and initiating transcription of a downstream sequence. Promoters may be derived in their entirety from a native gene, may be composed of elements derived from multiple regulatory sequences found in nature, or may comprise synthetic DNA segments. A promoter is “operably linked” to a polynucleotide if the promoter is connected to the polynucleotide such that it may affect transcription of the polynucleotide. Constructs can be generated using conventional recombinant DNA methods.

[0036] In embodiments, the therapeutic construct comprises a “selective promoter”, or a promoter that is only active or is primarily active in a specific cell type (“cell type-specific promoter”), a specific organ (“organ-specific promoter”), a specific tissue (“tissue-specific promoter”), a specific disease (“disease-specific promoter”), a specific tumor type (“tumorspecific promoter”), etc. The selective promoter may be at least 10-, 100-, 1000-fold more active in the selected cell type, tissue, organ, etc. relative to other cell types, tissues, organs, etc. The promoter used in the therapeutic AAVs described herein may be a cardiac tissue-specific promoter.In gene therapy applications involving AAV, it often becomes important to minimize the size of the promotor due to the packaging constraints of the viral capsid. The truncated chicken cardiac troponin T (cTnT) promoter is often preferred for such applications since it provides relatively strong, cardiomyocyte-specific transcription for its size (418 bp, see e.g. Prasad KM, Xu Y, Yang Z, Acton ST, French BA. Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution. Gene Ther. 2011 Jan;18(l):43-52. doi: 10.1038 / gt.2010.105. Epub 2010 Aug 12. PMID: 20703310, the content of which is incorporated herein by reference)). Other promoters with variable degrees of size, strength and cardiac-specificity include the human cTnT or the a-myosin heavy chain (a-MHC) promoters (see Schroder LC, Frank D, Muller OJ. Transcriptional Targeting Approaches in Cardiac Gene Transfer Using AAV Vectors. Pathogens. 2023 Oct 30; 12(11): 1301. doi: 10.3390 / pathogensl2111301. PMID: 38003766, the content of which is incorporated herein by reference). Cardiomyocyte-subtype-specific promoters have also been described, including the atrial natriuretic factor (ANF) and sarcolipin (SLN) promoters expressed in atrial contractile cardiomyocytes and the myosin light chain-2v (MLC-2v) that is restricted to ventricular cardiomyocytes (see Schroder et al.). Alternatively, certain applications may call for the high- level expression provided by non-tissue-specific, promiscuous promoters such as the cytomegalovirus (CMV) promoter, the hybrid CMV enhancer / chicken-beta-actin (CAG) promoter, the human eukaryotic translation factor la (EFl A) promoter, among others. Examples provided here include the use of both promiscuous promoters (CAG) and cardiac-specific promoters (cTnT).

[0037] A “therapeutic” refers to a polynucleotide or polypeptide that provides a therapeutic benefit to the subject. For example, the therapeutic may be DN , RNA, shRN A, oligonucleotides, antisense RNA, aptamers, protein, etc. that provides a beneficial or desirable biological or clinical result. The therapeutic may treat a disease or disorder, such as a cardiac disorder. The therapeutic AAV vector may be administered at a dose of at least about lxl()'!lJvector genomes (vg) / kg. The AAV vector may be administered at a dose of between about IxlO10and about 3xl014vg / kg, between about IxlO10and about IxlO13vg / kg, or any dose or range in between.

[0038] In embodiments, the vector further includes a reporter gene. Reporter genes that may be used in the described method include, but are not limited to, fluorescent or bioluminescent proteins, and proteins targeted by radionuclide agents and / or MRI contrast agents. In exemplaryembodiments, the reporter gene imaging agent is luciferase or a tdTomato (tdT) fluorescent reporter. In other exemplary embodiments, the reporter gene is sodium iodide symporter (“NIS” or “NalS”), and is imaged using techniques such as single-photon emission computed tomography (SPECT), positron-emission tomography (PET), or gamma scintigraphy.

[0039] The term “nucleic acid” as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which may contain natural, non-natural or altered nucleotides, and which may contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

[0040] The terms “polypeptide” or “peptide” or “protein” may be used interchangeably to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.

[0041] The term “subject” or “patient” are used herein interchangeably to refer to a mammal, preferably a human, to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. Preferably, the subject is a human. The subject may be a mammal in need of therapy for treating a cardiac disease or disorder. The term “subject” does not denote a particular age or sex.

[0042] As used herein, the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). "Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, ordefective function; or stimulating an inefficient process.

[0043] As used herein, the term "administering" is intended to refer to dispensing, delivering, or applying a substance or treatment to the intended target by any suitable route for delivery to the desired location. In terms of the therapeutic AAV vector, the microbubble, and the ultrasound, the term "administering" is intended to refer to dispensing, delivering, or applying the substance or treatment to a cardiac cell (i.e. cardiomyocyte and / or other cell type) in the heart of a subject. The AAV vector may be administered via intravenous, intracardiac, or intracoronary infusion. The MB may be administered via intravenous, intracardiac, or intracoronary infusion. Intracoronary infusion may be done antegrade or retrograde. In exemplary embodiments, administering of the AAV vector and the microbubble is done intravenously. In exemplary embodiments, administering the ultrasound is done by applying ultrasound externally to the chest area of the subject. In other embodiments, ultrasound is applied via alternative means, e.g. transesophageal or via catheter. The AAV vector may be administered before, during, or after the MB composition is administered for cavitation by ultrasound. In embodiments, ultrasound is administered during the MB injection and continued for a period of time no less than 10 minutes after MB injection concludes. In other embodiments, ultrasound is applied during a constant infusion of the MB composition. .

[0044] The MB composition may be administered at a dose of between about 5xl06and about 5xlO9MB / kg, or any dose or range in between. In exemplary embodiments, the MB composition is administered at a dose of about 2xl08MB / kg. The MB composition may be administered slowly for between about thirty seconds and about thirty minutes, between about one minute and about five minutes, or any duration or range in between, or infused at a constant rate for cavitation. The cavitation protocol, or administration of ultrasound, may be initiated after bolus infusion or at any time during prolonged infusion of the MB.

[0045] Miscellaneous

[0046] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0047] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus>10% of the particular term.

[0048] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0049] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0050] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0051] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0052] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0053] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.EXAMPLES

[0054] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.

[0055] Example 1

[0056] Background

[0057] Lipid or protein membrane-stabilized microbubbles (MBs) that contain high-molecular weight gases (perfluorocarbons, SF6) are currently used in humans for a variety of diagnostic US imaging applications. Contrast-enhanced ultrasound (US) (CEUS) imaging relies on the detection of acoustic signals produced by stable cavitation whereby volumetric oscillation of MBs, particularly non-linear oscillation, produces unique signatures that can be used to enhance MB signal relative to tissue.20Validated CEUS perfusion imaging algorithms employ kinetic analysis of tracer transit immediately after MBs in the beam volume are destroyed by inertial cavitation.21With this form of cavitation, a brief high-pressure US pulse (mechanical index >0.8 in the diagnostic frequency range) results in exaggerated non-linear oscillation, MB rupture, and release of free gas. Both stable and inertial cavitation are capable of producing pleiotropic bioeffects on surrounding tissues, including vascular cells and erythrocytes. These bioeffects have been leveraged to create novel therapeutic applications of CEUS including cavitation-induced clot lysis, shear-mediated flow augmentation, and site-targeted delivery of drugs and genes.22'2?

[0058] The premise for using US cavitation to enhance gene therapy is based on its ability to influence vascular or cell permeability, cell uptake, and intracellular trafficking. These effects are thought to be important in promoting transfection or transduction in a site-targeted manner. US when coupled with the co-administration of MB contrast agents has been shown to improve the otherwise extremely low efficiency or absence of transfection to target tissues when using plasmid DNA constructs or short-hairpin RNA alone.12 23'27The inventors previously pioneered the use of ultrasound (US) cavitation of lipid microbubble (MB) vectors that can markedly enhance plasmid DNA delivery in a site-targeted manner.10'12It has been shown that cavitation-mediatedtransfection in muscle is much more effective when plasmid cDNA is charge-coupled directly to the surface of MBs which can be accomplished with lipid-stabilized microbubbles with a positive zeta potential (cationic MBs) (FIG. I).10,12,13Inertial cavitation of these carriers by US produces physical forces on adjacent endothelial cells resulting in microporation, enhanced trans-cellular permeability, and active cellular uptake.10,1448Charge coupling results in protection of cDNA from endonucleases, concentration of cDNA at the site of cavitation, and perhaps the ability to enter cells from ballistic effects.10, 17,28,29While cavitation can increase plasmid reporter gene expression by several orders of magnitude, the percentage of cells that are transduced with plasmid DNA is still quite low.13,19Accordingly, plasmid DNA delivery through cavitation of cationic MBs is best suited for treating conditions where paracrine actions of gene product are desired, and not for the broad array of cardiomyocyte disorders where a majority of cells must be transduced to improve LV function. Equally important, for most published studies, by far most of cells transduced by cavitation of MBs with plasmid DNA are vascular cells, not cardiomyocytes.13,19Hence, cavitation -facilitated delivery of naked DNA such as plasmid, even when complexed to the MB surface, is poorly suited for treating disorders such as dilated, hypertrophic, infdtrative, and arrhythmogenic cardiomyopathies. Here, we describe how US cavitation can augment cardiomyocyte gene delivery when combined with AAV9 vectors that have an innate tropism for cardiomyocytes. In the attached Appendix, further background on the field of microbubbles for site-targeted delivery is provided.

[0059] Gene Therapy with AAVs

[0060] To address the low efficiency of transfection and the limited duration of gene expression offered by plasmid DNA and the high risk of immunoreactivity when using adenoviral vectors, the field of gene therapy has turned to the use of adeno-associated viruses (AAVs). Development of this vector type has led to a resurgence in the interest in gene therapy for rare or uncommon diseases. AAVs are members of the parvovirus family and are composed of a small, singlestranded DNA (approximately 4.7 kb) surrounded by a protein shell (capsid).6,7Ordinarily, AAVs rely on co-infection with other viruses, such as adenoviruses, to produce infection and initiate replication. Like their wild-type progenitors, recombinant AAVs enter cells and are trafficked to the nucleus where their genomes maintained as episomes (not incorporated into host DNA). Because adult cardiomyocytes rarely divide, “dilution” of the episomal AAV genomes in these cells is extremely slow.30AAV serotype 9 (AAV9) has innate tropism for cardiomyocytes and hasa relatively long plasma half-life compared to other AAV serotypes.30,31

[0061] Despite the success of AAVs in preclinical models and their promise in human diseases, there are still several major hurdles for their use in cardiovascular disease.8The most important limitation is that extremely high doses of AAV are needed to achieve a high percentage of cardiomyocyte transduction, which often is essential for producing the desired phenotypic response. These large doses are associated with heightened risks for serious AEs (SAEs) that occur secondary to inflammation, cytokine release, complement activation, immune complexes, and thrombosis.32,33Several Phase 1 trials examining AAV-based therapies overseen by the NHLBI Data Safety Monitoring Board for Gene and Cell Therapies have been prematurely terminated because of dose-related SAEs. A second limitation is that many patients may already carry antibodies against the AAV serotype being employed for gene therapy, which results in rapid clearance and low transduction efficiency even with high doses of vector. Methods for augmenting the efficiency of AAV-mediated gene delivery specifically to the target tissue of interest are needed to overcome both limitations discussed above. Improving transduction efficiency exclusively in the intended tissue target is vital for reducing AAV dose and associated dose-related SAEs. Preclinical models and human trials suggest that even a 5-fold reduction in dose would have a major impact on immune reactions.9,34A method that increases AAV extravasation as its mechanism for increasing transduction could also address the rapid immune clearance in patients with anti-AAV antibodies if that technique had a rapid effect immediately after AAV administration.

[0062] Mechanisms for Enhanced Gene Delivery

[0063] Stable and especially inertial cavitation of MBs in an US field produces oscillatory shear, high-pressure microstreaming, shock waves, and physical stretching or even suction of endothelial cells.35'37These biophysical events produce physical changes in adjacent cells that include membrane microporation and enhanced permeability through endothelial intercellular junctions.16,35,38,39Functional changes in permeability for vascular mural cells can also occur, including receptor-mediated transport through caveolin and clathrin-dependent endocytosis and transcytosis.16All of these events are thought to result in active and / or passive cellular uptake of macromolecules.10,14'18,39As mentioned above, because these structural and functional effects are localized to the region adjacent to the region of MB cavitation, transfection with plasmid DNA in the heart occurs primarily in vascular cells (endothelium, smooth muscle cells, perivascularfibroblasts).13,19,40In the heart, cavitation of microbubbles in conjunction with adenovirus vectors has demonstrated a different pattern whereby cardiomyocyte transduction tends to dominate.41

[0064] There is little previous knowledge regarding mechanisms responsible for preferential transduction of adenoviral vectors to cardiomyocytes rather than to cells that are directly adjacent to cavitation events. However, it is likely that the mechanisms are similar between adenoviral and AAV vectors. It is well known that different AAV vector serotypes use different receptors or pathways to gain access to cells beyond the vascular compartment. The ability to transit from the microvascular space is particularly important in optimizing AAV delivery to the brain which is protected by the poorly permeable blood-brain barrier,42or the heart where microvascular transit time is short (approximately 5 seconds). It has been suggested that AAV9 uses either active endothelial transcytosis or paracellular mechanisms to gain access to the myocardial extracellular space43Yet, this knowledge does not necessarily imply cavitation-facilitated AAV transduction (CFAT) would increase transduction by the same endogenous mechanism(s) since biophysical events that underly CFAT could act via parallel complimentary pathways, discussed below. Our preliminary data indicates that cavitation at the capillary level can stimulate transcytosis mechanisms.

[0065] Initial Experience with CFAT

[0066] The use of inertial cavitation to enhance AAV vector gene delivery has previously been reported in the ear. Studies using a dwell of highly concentrated commercially-produced microbubbles (Definity, Lantheus Medical Imaging) in the ear directly adjacent to the round window of the cochlea combined with direct non-attenuated US (1.5-2.0 MHz, 0.4 MPa peak negative acoustic pressure [PNAP]) demonstrated enhanced transduction with an AAV encoding a fluorescent reporter gene that was placed in gelfoam adjacent to the cochlea after cavitation. However, significant epithelial cell injury adjacent to the cavitation zone was observed in these studies. There are clearly many conditional differences of this study design compared to our approach where acoustic field, acoustic conditions, and method of MB administration promotes key cavitation events at the capillary level. Successful CFAT in the heart at a transduction rate that has any therapeutic potential has not previously been reported. However, one group did report achieving a low transduction rate (<20% cardiomyocytes transduced overall) in rat hearts by coupling an AAV9 reporter vector to the microbubble surface (Muller OJ, Schinkel S, Kleinschmidt JA, Katus HA, Bekeredjian R. Augmentation of AAV-mediated cardiac genetransfer after systemic administration in adult rats. Gene Ther. 2008 Dec; 15(23): 1558-65. doi: 10.1038 / gt.2008.111. Epub 2008 Jul 10. PMID: 18615116). The methods described here represent a significant advance over this report and are unique because: 1) we show that the injection of MBs can be decoupled from the injection of the AAV vector, thereby simplifying formulation and storage of both MBs and AAV, with the added benefit of enabling MB infusion to be employed for perfusion imaging in addition to CFAT therapy, 2) we demonstrate that the use of multi-pulse or long-pulse algorithms (or a combination of the two) results in a therapeutically relevant transduction rate (>85% of cardiomyocytes, see Examples 2&3) as compared to the <20% transduction rate evident in Fig 4 of Muller et al., and 3) we achieve a more homogenous distribution of cavitation in the heart by carefully selecting the most appropriate ultrasound line density, which ultimately translates to more homogeneous and efficient transduction by AAV9.

[0067] Our first exploration of CFAT in the heart of several mice was performed by injecting I x io13vector genomes (vg) per kg of an AAV9 encoding a tdTomato fluorescent reporter gene driven by the promiscuous C AG promoter by I.V. route followed immediately by injection of lipid- shelled decafluorobutane MBs (2x 108) manufactured in our lab and 15 min of transthoracic US (1.3 MHz, mechanical index 1.2, multipulse power Doppler transmission, pulsing interval [PI] of 5 seconds). In these mice, cardiomyocyte transduction efficiency with AAV9 was increased by 5- 10 fold in terms of percentage of cells transduced (FIG. 2A). CFAT was also performed in a single aged Rhesus macaque at the Oregon National Primate Research Center (ONPRC). Minimal cardiomyocyte transduction was seen with low-dose AAV9 alone (3x l0nvg / kg), whereas CFAT with 1 vial of Definity MBs and ultrasound (US) conditions similar to the murine experiments resulted in cardiomyocyte transduction of the CAG-driven tdTomato reporter in the short-axis region exposed to US (FIG. 3). However, the histologic approach taken in these studies was not conducive to quantitative analysis, thereby precluding a reliable comparison of efficiency in terms of percentage of cells. More recently we have used both in vivo bioluminescent imaging and ex vivo quantification of luciferase reporter activity to show a 5-10 fold increase in AAV transduction to the heart using identical acoustic conditions to those described above and AAV9 doses of 1x1012to 1 xlO13Vg / kg. The results show increase of luciferase activity with CFAT (FIG. 2B).

[0068] Example 2

[0069] FIG. 5 illustrates the transduction efficiency assessed by in vivo bioluminescent imaging in mice 2 weeks after administration of low-dose AAV9 (5xl012vg / kg) containing abioluminescent luciferase reporter gene driven by the cardiac-specific cardiac troponin T (cTnT) promoter. Image acquisition was standardized according to time after intraperitoneal (I P.) injection of d-luciferin. These data show a marked improvement in transduction efficacy compared to control conditions of AAV9 alone when AAV9 was coupled with CFAT. CFAT was performed after AAV9 administration by single plane trans-axial LV exposure at a pulsing interval of 5 seconds after injection of IxlO8decafluorobutane lipid-stabilized microbubbles produced in our laboratory.

[0070] The “Sonos” conditions are a power-harmonic Doppler scheme with medium packet size, medium line density, center frequency of 1.3 MHz and a mechanical index of 1.3. The marked variation in transduction efficiency was found to be attributable to variations in batch activity of AAV9. Much of this variation was from time dependent decay in the activity AAV9 which had to eventually be replenished with new vector with high activity. AAV9 stock used for administrations that resulted in the highest transduction efficiency with Sonos were temporally match with the highest control transduction (and vice-versa for low activity). When using a rolling average of control data to match stock conditions, the Sonos CFAT consistently produced a 5-8 fold increase in transduction.

[0071] We have matched these results with “Epiq new flash” conditions which were designed by our research team. Ultrasound was again transmitted at 1.3 MHz and a mechanical index of 1.1 using a programmable Epiq CVx system and a phased array transducer. For this method 4 packets of ultrasound were transmitted at a line density of 32 using long 16 cycle pulses. The variable focal depth allowed us to optimize the peak intensity at the level of the heart (2 cm from transducer) and to create a large and even exposure in the near-field for murine studies, evidenced by the hydrophone data in FIG. 6.

[0072] Data from in vivo bioluminescent imaging were validated by removing the hearts on day 28 post transduction and performing ex vivo bioluminescent assays on digested whole hearts. Data for these experiments are provided in FIG. 7 which shows an even greater relative degree of AAV9 transduction for CFAT, with both the Sonos and New Epiq flash conditions, relative to AAV9 alone, than was found with in vivo bioluminescence imaging. To show tissue specificity, a limited number of mice received CFAT with a novel AAV9 carrying the human-compatible sodium iodide symporter (NalS) reporter gene driven by the cTnT promoter. This strategy was used in order to perform whole body microSPECT imaging to demonstrate cardiac specificity forCFAT delivery of AAV9. Using 99mTc-pertechnetate (1 mCi) to detect NalS activity, microSPECT demonstrated superb cardiac specificity (FIG. 8). These data indicate that all of the luminescence signal on bioluminescence imaging is coming solely from the heart which is the target tissue.

[0073] Safety data has also been performed in mice receiving AAV9 alone or AAV9 coupled with CFAT (FIG. 9). These data indicate no adverse effects of CFAT on cardiac performance.

[0074] Acoustic Conditions for Successful CFAT

[0075] Exemplary key acoustic conditions for optimal AAV9 transduction in the heart by ensuring capillary level CFAT using acoustic conditions that are both safe and effective are as follows:1. Cavitation protocol initiated within 5 min of completing AAV9 infusion.2. Three to five minute slow administration of MBs at a dose of IxlO8to IxlO9MB / kg.3. Ten to fifteen min of CFAT using lipid shelled perfluorocarbon (octafluoropropane, decafluorobutane) MB which produce the intended bioeffects4. Use of either long duration US pulses (e.g. 100 ms or multipulse algorithm with 4 or more repetitive pulses per line)5. Mechanical index of 1.1 -1.56. US frequency of 1.3 to 2.0 MHz7. Pulsing interval between single or multi-frame destructive sequences of 5 seconds, or a time interval guided by complete microvascular refill with MB.8. Rastered exposure of the entire heart using low-MI myocardial enhancement to guide timing and location of rastered sequences

[0076] References1. Wolfram JA, Donahue JK. Gene therapy to treat cardiovascular disease. J Am Heart Assoc. 2013;2:e000119. doi: 10.1161 / JAHA.113.0001192. Nabel EG, Plautz G, Nabel GJ. Site-specific gene expression in vivo by direct gene transfer into the arterial wall. Science. 1990;249: 1285-1288. doi: 10.1126 / science.21190553. Rincon MY, VandenDriessche T, Chuah MK. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation. Cardiovasc Res. 2015;108:4-20. doi: 10.1093 / cvr / cvv2054. Wright MJ, Wightman LM, Lilley C, de Alwis M, Hart SL, Miller A, Coffin RS, Thrasher A, Latchman DS, Marber MS. In vivo myocardial gene transfer: optimization, evaluation and direct comparison of gene transfer vectors. Basic Res Cardiol. 2001;96:227-236. doi: 10.1007 / s0039501700535. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020;21 :255-272. doi: 10.1038 / s41576-019-0205-46. Naso MF, Tomkowicz B, Perry WL, 3rd, Strohl WR. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs. 2017;31 :317-334. doi: 10.1007 / s40259-017-0234-57. Au HKE, Isalan M, Mielcarek M. Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings. Front Med (Lausanne). 2021;8:809118. doi: 10.3389 / fmed.2021.8091188. Chowdhury EA, Meno-Tetang G, Chang HY, Wu S, Huang HW, Jamier T, Chandran J, Shah DK. Current progress and limitations of AAV mediated delivery of protein therapeutic genes and the importance of developing quantitative pharmacokinetic / pharmacodynamic (PK / PD) models. Adv Drug Deliv Rev. 2021;170:214-237. doi: 10.1016 / j.addr.202L 01.0179. Srivastava A. AAV Vectors: Are They Safe? Hum Gene Ther. 2020;31 :697-699. doi: 10.1089 / hum.2020.18710. Christiansen JP, French BA, Klibanov AL, Kaul S, Lindner JR. Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles. Ultrasound Med Biol. 2003;29: 1759- 1767. doi: S0301562903009761 [pii]11. Kobulnik J, Kuliszewski MA, Stewart DJ, Lindner JR, Leong-Poi H. Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection. J Am Coll Cardiol. 2009;54: 1735-1742. doi: S0735- 1097(09)02596-0 [pii]10.1016 / j.jacc.2009.07.02312. Xie A, Wu MD, Cigarroa G, Belcik JT, Ammi A, Moccetti F, Lindner JR. Influence of DNA-Microbubble Coupling on Contrast Ultrasound-Mediated Gene Transfection in Muscle and Liver. J Am Soc Echocardiogr. 2016;29:812-818. doi: 10.1016 / j.echo.2016.04.01113. Xie A, Belcik T, Qi Y, Morgan TK, Champaneri SA, Taylor S, Davidson BP, Zhao Y, Klibanov AL, Kuliszewski MA, et al. Ultrasound-mediated vascular gene transfection by cavitation of endothelial- targeted cationic microbubbles. JACC Cardiovasc Imaging.2012;5: 1253-1262. doi: 10.1016 / j.jcmg.2012.05.01714. van Warn el A, Kooiman K, Emmer M, ten Cate FJ, Versluis M, de Jong N. Ultrasound microbubble induced endothelial cell permeability. J Control Release. 2006;116:el00-102.15. Duvshani-Eshet M, Baruch L, Kesselman E, Shimoni E, Machluf M. Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy. Gene Ther. 2006;13: 163-172.16. Meij ering BD, Juffermans LJ, van Warn el A, Henning RH, Zuhorn IS, Emmer M, Versteilen AM, Paulus WJ, van Gilst WH, Kooiman K, et al. Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ Res. 2009;104:679-687.17. Mehier-Humbert S, Yan F, Frinking P, Schneider M, Guy RH, Bettinger T. Ultrasound- mediated gene delivery: influence of contrast agent on transfection. Bioconjug Chem. 2007;18:652-662.18. Sheikov N, McDannold N, Vykhodtseva N, Jolesz F, Hynynen K. Cellular mechanisms of the blood- brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol. 2004;30:979-989.19. Leong-Poi H, Kuliszewski MA, Lekas M, Sibbald M, Teichert-Kuliszewska K, Klibanov AL, Stewart DJ, Lindner JR. Therapeutic arteriogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle. Circ Res. 2007;101:295-303.20. Kaufmann BA, Wei K, Lindner JR. Contrast echocardiography. Curr Probl Cardiol. 2007;32:51-96.21. Wei K, Jayaweera AR, Firoozan S, Linka A, Skyba DM, Kaul S. Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion. Circulation. 1998;97:473-483.22. Unger E, Porter T, Lindner J, Grayburn P. Cardiovascular drug delivery with ultrasound and microbubbles. Adv Drug Deli v Rev. 2014;72: 110-126. doi: 10.1016 / j.addr.2014.01.01223. Mathias W, Jr., Tsutsui JM, Tavares BG, Fava AM, Aguiar MOD, Borges BC, Oliveira MT, Jr., Soeiro A, Nicolau JC, Ribeiro HB, et al. Sonothrombolysis in ST-Segment Elevation Myocardial Infarction Treated With Primary Percutaneous Coronary Intervention. J Am Coll Cardiol. 2019;73:2832-2842. doi: 10.1016 / j.jacc.2019.03.00624. Belcik JT, Davidson BP, Xie A, Wu MD, Yadava M, Qi Y, Liang S, Chon CR, Ammi AY,Field J, et al. Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling. Circulation. 2017;135: 1240-1252. doi: 10.1161 / CIRCULATION AHA.116.02482625. Bekeredjian R, Grayburn PA, Shohet RV. Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine. J Am Coll Cardiol. 2005;45:329-335. doi: 10.1016 / j.jacc.2004.08.06726. Korpanty G, Chen S, Shohet RV, Ding J, Yang B, Frenkel PA, Grayburn PA. Targeting of VEGF- mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles. Gene Ther. 2005;12:1305-1312.27. Haag P, Frauscher F, Gradl J, Seitz A, Schafer G, Lindner JR, Klibanov AL, Bartsch G, Klocker H, Eder IE. Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours. J Steroid Biochem Mol Biol. 2006;102: 103-113. doi: 10.1016 / j.jsbmb.2006.09.02728. Suzuki J, Ogawa M, Takayama K, Taniyama Y, Morishita R, Hirata Y, Nagai R, Isobe M. Ultrasound- microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice. J Am Coll Cardiol.55:904-913.29. Wang DS, Panje C, Pysz MA, Paulmurugan R, Rosenberg J, Gambhir SS, Schneider M, Willmann JK. Cationic versus neutral microbubbles for ultrasound-mediated gene delivery in cancer. Radiology. 2012;264:721-732. doi: 10.1148 / radiol.1211236830. Zacchigna S, Zentilin L, Giacca M. Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system. Circ Res. 2014;114: 1827-1846. doi: 10.1161 / CIRCRES AHA.114.30233131. Ambrosi CM, Sadananda G, Han JL, Entcheva E. Adeno- Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models. Front Physiol. 2019;10: 168. doi: 10.3389 / fphys.2019.0016832. Maurya S, Sarangi P, Jayandharan GR. Safety of Adeno-associated virus-based vector- mediated gene therapy-impact of vector dose. Cancer Gene Ther. 2022;29: 1305-1306. doi: 10.1038 / s41417-021- 00413-633. High-dose AAV gene therapy deaths. Nat Biotechnol. 2020;38:910. doi: 10.1038 / s41587- 020-0642-934. Ertl HCJ. Immunogenicity and toxicity of AAV gene therapy. Front Immunol. 2022;13:975803. doi: 10.3389 / fimmu.2022.97580335. Helfield B, Chen X, Watkins SC, Villanueva FS. Biophysical insight into mechanisms of sonoporation.Proc Natl Acad Sci U S A. 2016; 113 : 9983-9988. doi : 10.1073 / pnas.160691511336. Collis J, Manasseh R, Liovic P, Tho P, Ooi A, Petkovic-Duran K, Zhu Y. Cavitation microstreaming and stress fields created by microbubbles. Ultrasonics. 2010;50:273-279. doi: 10.1016 / j. ultras.2009.10.00237. Chen H, Kreider W, Brayman AA, Bailey MR, Matula TJ. Blood vessel deformations on microsecond time scales by ultrasonic cavitation. Phys Rev Lett. 2011;106:034301. doi: 10.1103 / PhysRevLett.106.03430138. Juffermans LJ, van Dijk A, Jongenelen CA, Drukarch B, Reijerkerk A, de Vries HE, Kamp O, Musters RJ. Ultrasound and microbubble-induced intra- and intercellular bioeffects in primary endothelial cells. Ultrasound Med Biol. 2009;35: 1917-1927. doi: 10.1016 / j .ultrasmedbio.2009.06.109139. Sirsi S, Feshitan J, Kwan J, Homma S, Borden M. Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice. Ultrasound Med Biol. 2010;36:935-948. doi: 10.1016 / j.ultrasmedbio.2010.03.01540. Kuliszewski MA, Kobulnik J, Lindner JR, Stewart DJ, Leong-Poi H. Vascular gene transfer of SDF-1 promotes endothelial progenitor cell engraftment and enhances angiogenesis in ischemic muscle. Mol Ther. 2011;19:895-902. doi: mt201118 [pii]10.1038 / mt.2011.1841. Shohet RV, Chen S, Zhou YT, Wang Z, Meidell RS, Unger RH, Graybum PA. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation. 2000;101 :2554-2556.42. Weber-Adrian D, Heinen S, Silburt J, Noroozian Z, Aubert I. The human brain endothelial barrier: transcytosis of AAV9, transduction by AAV2: An Editorial Highlight for 'Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells'. J Neurochem. 2017;140: 192-194. doi: 10.1111 / jnc.1389843. Duan D. Systemic delivery of adeno-associated viral vectors. Curr Opin Virol. 2016;21 : 16-25. doi: 10.1016 / j.coviro.2016.07.006

Claims

CLAIMS1. A method for transducing cardiac cells with a therapeutic AAV vector in a subject in need thereof, the method comprising: administering the AAV vector to the subject; administering a microbubble (MB) composition to the subject; and administering ultrasound to the subject.

2. The method of claim 1, wherein the cardiac cells comprise at least one of cardiomyocytes, myofibroblasts, endothelial cells, and smooth muscle cells.

3. The method of claim 2, wherein the cardiac cells comprise cardiomyocytes.

4. The method of any one of claims 1-3, wherein the ultrasound is transthoracic ultrasound.

5. The method of any one of claims 1-4, wherein the MB composition is administered via intravenous infusion, intracoronary infusion, or intracardiac infusion.

6. The method of any one of claims 1-5, wherein the MB composition comprises a lipid MB.

7. The method of any one of claims 1-6, wherein the MB composition comprises a perfluorocarbon MB.

8. The method of claim 7, wherein the perfluorocarbon is selected from octafluoropropane and decafluorobutane.

9. The method of any one of claims 1-8, wherein the MB composition and the ultrasound are administered in conjunction with the AAV vector.

10. The method of any one of claims 1-9, wherein the MB composition is administered at a dose of between about 5xl06and about 5xlO9MB / kg.

11. The method of claim 10, wherein the MB composition is administered at a dose of about 2xl08MB / kg.

12. The method of any one of claims 1-11, wherein the MB composition is administered for between about 30 seconds and about 30 minutes.

13. The method of any one of claims 1-12, wherein the ultrasound is administered at a frequency of between about 1.0 MHz and about 2.5 MHz.

14. The method of any one of claims 1-13, wherein the ultrasound is administered at a mechanical index of between about 0.3 and about 1.8.

15. The method of claim 14, wherein the mechanical index is about 1.2.

16. The method of any one of claims 1-15, wherein the ultrasound is administered with at least one pulse of at least 100 microseconds (ps).

17. The method of claim 16, wherein the at least one pulse is between about 100 ps and about 100 milliseconds (ms).

18. The method of claim 16 or 17, wherein the ultrasound is administered with about five pulses of about 20 ps per pulse.

19. The method of any one of claims 16-18, wherein the ultrasound is administered with pulsing intervals of about 5 seconds.

20. The method of any one of claims 1-19, wherein the ultrasound is administered for 4 or more multipulses with a pulse repetition frequency of between about 1 and about 10 kHz, wherein each pulse of the multipulses is administered for between about 1 and about 15 microseconds.

21. The method of any one of claims 1-20, wherein the AAV vector is administered via intravenous infusion, intracoronary infusion, or intracardiac infusion.

22. The method of any one of claims 1-21, wherein the AAV vector is administered at a dose of between about IxlO10and about IxlO13vector genomes (vg) / kg.

23. The method of any one of claims 1-22, wherein the AAV vector is an AA8 vector or an AAV9 vector.

24. The method of any one of claims 1-23, wherein the AAV vector comprises a therapeutic polynucleotide operably linked to a cardiac-specific promoter.

25. The method of claim 24, wherein the cardiac-specific promoter is a chicken cardiac troponin T (cTnT) promoter.

26. The method of any one of claims 1-25, wherein administering the ultrasound is done using an ultrasound transducer; and wherein the ultrasound transducer transmits the ultrasound at a medium line density of between about 20 and about 40 lines.

27. The method of claim 26, wherein the ultrasound transducer transmits the ultrasound at a medium line density of about 32 lines.

28. The method of claim 25 or 26, wherein the ultrasound transducer has a focal depth of between about 2 and 6 centimeters from the transducer.

Images