Method to noninvasively deliver lipid nanoparticles to the brain using ultrasound-mediated blood brain barrier opening

Abstract

Provided herein are compositions, methods and systems, for non-invasive delivery of lipid nanoparticles (LNPs) encapsulating nucleic acids across the blood-brain barrier (BBB), facilitated by BBB opening (BBBO), using microbubbles and focused ultrasound (FUS). The compositions, methods and systems enable delivery of therapeutic nucleic acid to brain tissue of a subject, for treatment of various conditions, including brain cancers, such as glioblastoma (GBM).

Description

[0001] METHOD TO NONINVASIVELY DELIVER LIPID NANOPARTICLES TO THE BRAIN USING ULTRASOUND-MEDIATED BLOOD BRAIN BARRIER OPENING

[0002] TECHNICAL FIELD

[0003] The present disclosure generally relates to compositions, methods and systems for delivery of lipid nanoparticles to brain target region, using ultrasound mediated blood brain barrier (BBB) opening.

[0004] BACKGROUND OF THE INVENTION

[0005] The blood-brain barrier (BBB) is a highly selective physiological barrier that protects the central nervous system (CNS) from toxins and pathogens. While essential for brain homeostasis, the BBB poses a major obstacle to the delivery of therapeutic agents, particularly large molecules such as nucleic acids, antibodies, and nanoparticles. As a result, the effective treatment of disorders, such as, cancer, remains a formidable clinical challenge.

[0006] Glioblastoma is the most aggressive and common form of primary brain cancer. Standard-of-care therapies include surgical resection, radiotherapy, and chemotherapy with temozolomide. However, these approaches achieve only limited survival benefits, with high recurrence rates and median survival times of approximately 15 months. One major reason for poor therapeutic efficacy is the inability of large therapeutic molecules and carriers to penetrate the BBB and reach tumor tissue. Although the BBB is sometimes disrupted in glioblastoma, such permeability is heterogeneous, inconsistent, and insufficient to enable reliable delivery of large therapeutic modalities.

[0007] Lipid nanoparticles (LNPs) can be used for nucleic acid delivery. LNPs possess favorable features such as biodegradability, low immunogenicity, high encapsulation efficiency, and the ability to carry diverse nucleic acid cargos. Despite these advantages, the application of LNPs in brain therapies has been severely limited due to the impermeability of the BBB. Current strategies often rely on invasive intracranial injections, which are clinically impractical and carry significant risks.

[0008] Focused ultrasound (FUS) in combination with intravenously administered microbubbles has emerged as a promising non-invasive technique for transiently and reversibly opening the BBB. By inducing oscillations of circulating microbubbles under ultrasound exposure, localized and temporary permeability of the BBB can be achieved, thereby facilitating the entry of therapeutic agents. This approach has been explored in preclinical and clinical studies for small molecules and antibodies. However, effective delivery of large nanoparticles across the BBB using FUS has not been demonstrated.

[0009] Accordingly, there is a need in the art for a non-invasive, safe, and effective methods and systems to deliver nucleic acid-loaded lipid nanoparticles across the blood-brain barrier and into target brain regions.

[0010] SUMMARY OF THE INVENTION

[0011] According to embodiments, there are provided herein advantageous systems, methods and compositions, for delivering lipid nanoparticles (LNPs) carrying a nucleic acid payload to target brain regions, using ultrasound mediated blood brain barrier (BBB) opening, in non- invasive, safe and efficient manner. The systems, methods and compositions can be used to effectively deliver therapeutic nucleic acids to target brain regions, for the treatment of various brain related conditions, including, for example, glioblastoma and other brain cancers, Alzheimer’s disease (AD), Parkinson’s disease (PD), psychiatric disorders, genetic diseases of the CNS, and the like, or any combinations thereof.

[0012] According to some embodiments, there are provided herein methods, systems, and compositions for the non-invasive delivery of lipid nanoparticles (LNPs) encapsulating nucleic acids, such as, RNA molecules, across the blood-brain barrier (BBB). More particularly, the disclosure relates to the use of focused ultrasound (FUS) in combination with microbubbles to transiently and reversibly open the BBB, thereby enabling the systemic administration of the nucleic-acid containing LNPs to reach brain tissue target regions, including tumors, such as glioblastoma.

[0013] According to some embodiments, there is provided herein a therapeutic platform in which focused ultrasound in combination with microbubbles is applied under defined acoustic conditions to transiently and reversibly disrupt the BBB, permitting systemically administered lipid nanoparticles to reach brain parenchyma and tumor tissue.

[0014] According to some embodiments, advantageously as demonstrated herein below, ionizable lipid nanoparticles (LNPs) carrying nucleic acid molecules (such, siRNA and mRNA) can be delivered non-invasively across the blood-brain barrier (BBB) into both healthy brains and glioblastoma (GBM) tumors, using low-frequency focused ultrasound (FUS) with microbubbles. According to some embodiments, advantageously, the systems, compositions and methods disclosed herein enable achieving safe delivery of nanoparticles while balancing sufficient ultrasound intensity to enable BBB permeabilization with the need to avoid vascular damage, microhemorrhage, and neurotoxicity.

[0015] According to some embodiments, advantageously, as demonstrated herein below, the systems, methods and compositions disclosed herein can facilitate safe BBB opening at low acoustic pressures (e.g., 100-220 kPa), which avoids microvascular damage and hemorrhage observed at higher pressures (e.g., over 400 kPa).

[0016] In some embodiments, the systems, methods and compositions disclosed herein enables efficient delivery of relatively large nanoparticles (e.g., about 65-120 nm size), which are significantly larger than small molecules or antibodies shown to be delivered using FUS.

[0017] In some embodiments, advantageously, as exemplified hereinbelow, systemic delivery of siRNA nucleic-acid loaded LNPs results in a significant increase (e.g., at least 2-fold, at least 3-fold, at least 6-10 fold) in accumulation within brain tissue compared to controls, demonstrating enhanced permeability and delivery efficiency.

[0018] In some embodiments, advantageously, as exemplified hereinbelow, systemic delivery of mRNA-loaded LNPs results in a significant increase (e.g., at least 2-fold, at least 4-fold, at least 6-fold, at least 12-fold) in protein expression in brain tissue compared to untreated controls, demonstrating not only efficient delivery but also functional expression of nucleic acid cargo.

[0019] In some embodiments, advantageously, as exemplified hereinbelow, delivery into glioblastoma tumor models results in a significant increase (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 6-fold) in nanoparticle uptake into tumors and surrounding stromal and immune cells, thereby overcoming the heterogeneous and inconsistent permeability of the tumor-associated BBB.

[0020] In some embodiments, advantageously, as exemplified hereinbelow, the compositions, systems and methods disclosed herein, provide a non-invasive alternative to intracranial injections, reducing patient risk and increasing clinical translatability.

[0021] In some embodiments, advantageously, as exemplified hereinbelow, BBB permeability can remain open for an extended period of time (e.g. 10-60 minutes, 1-6 hours) post-ultrasound treatment, thereby enabling flexible timing of nanoparticle administration and improving treatment protocols. In some embodiments, advantageously, the systems, compositions and methods disclosed herein enable repeatable, safe cycles of BBB opening, supporting chronic treatment regimens for various conditions, such as, neurodegenerative and genetic CNS disorders.

[0022] According to some embodiments, there is provided a method for transiently opening blood-brain barrier (BBB) of a subject and facilitating introduction of a nucleic acid into brain tissue of the subject, the method includes: administering to the subject lipid nanoparticles (LNPs) encapsulating the nucleic acid; administering microbubbles; and applying focused ultrasound at a frequency below about 1 MHz and a peak negative pressure between 150 and 220 kPa; wherein the blood-brain barrier is transiently and reversibly opened, thereby enabling the lipid nanoparticles to enter the brain tissue and release the nucleic acid.

[0023] According to some embodiments, the focused ultrasound is applied at a frequency of about 850 kHz or less. According to some embodiments, the focused ultrasound is applied at a frequency of about 250 kHz or less.

[0024] According to some embodiments, the nucleic acid is a therapeutic nucleic acid, selected from mRNA, siRNA, miRNA, antisense oligonucleotides, plasmid DNA, cDNA, or CRISPR- associated nucleic acids.

[0025] According to some embodiments, the nucleic acid is configured to: reduce or inhibit expression of a gene of interest in a brain tissue, increase expression of a gene of interest in a brain tissue, or express a gene of interest in a brain tissue.

[0026] According to some embodiments, the lipid nanoparticles include an ionizable lipid, cholesterol, a helper phospholipid, a PEG-lipid, or any combinations thereof.

[0027] According to some embodiments, the lipid nanoparticles include: an ionizable lipid in an amount of 30-60 mol%, cholesterol in an amount of 20-45 mol%, a helper phospholipid in an amount of 5-15 mol%, and a PEG-lipid in an amount of 0.5-5 mol%. According to some embodiments, the ionizable lipid may be selected from DLin-MC3- DMA, SM-102, Lipid- 14, derivatives thereof, or any combinations thereof.

[0028] According to some embodiments, the helper phospholipid may include DSPC, DOPE, DOPC, DMPC, DSPE and / or DPPC.

[0029] According to some embodiments, the PEG-lipid may include DMG-PEG2000, DSPE- PEG2000, and / or PEG-ceramide.

[0030] According to some embodiments, the lipid nanoparticles have a surface charge (zeta potential) of between about -10 mV and about +10 mV at physiological pH.

[0031] According to some embodiments, the lipid nanoparticles may have an average particle size of about 65-120 nm.

[0032] According to some embodiments, the microbubbles have an average diameter in the range of about 250-2000nm.

[0033] According to some embodiments, the microbubbles include a phospholipid shell and a perfluorocarbon gas core.

[0034] According to some embodiments, the nucleic acid is capable of being introduced into neurons, astrocytes, endothelial cells and / or tumor cells in the brain.

[0035] According to some embodiments, the blood-brain barrier opening may persist for between at least about 1 and 6 hours.

[0036] According to some embodiments, the LNPs may be administered before, concomitantly with, or after administration of the microbubbles.

[0037] According to some embodiments, the LNPs may be administered before, during, or after ultrasound application.

[0038] According to some embodiments, the LNPs and the microbubbles may be formulated in a single or separate compositions.

[0039] According to some embodiments, the LNPs and / or the microbubbles may be administered systemically.

[0040] According to some embodiments, the method may be repeated in multiple treatment cycles. According to some embodiments, the method may be for treating brain cancer in a subject in need thereof.

[0041] According to some embodiments, the cancer may be Glioblastoma (GBM).

[0042] According to some embodiments, the nucleic acid may target a gene selected from EGFR (Epidermal Growth Factor Receptor), EGFRvIII (Epidermal Growth Factor Receptor variant III), TP53 (Tumor Protein 53), PTEN (Phosphatase and Tensin Homolog), APP (Amyloid Precursor Protein), SNCA (Synucl ein Alpha), HTT (Huntingtin), SOD1 (Superoxide Dismutase 1), SMN1 (Survival of Motor Neuron 1), MECP2 (Methyl CpG Binding Protein 2), CCND1 (Cyclin DI), CCND2 (Cyclin D2), CCND3 (Cyclin D3), FMRI (Fragile X Mental Retardation 1), or any combinations thereof. Each possibility is a separate embodiment.

[0043] According to some embodiments, there is provided a combination including a composition including lipid nanoparticles (LNPs) encapsulating a nucleic acid, and a composition including microbubbles, for use in a method of transiently and reversibly opening blood-brain barrier (BBBO) of a subject and enabling introduction of the nucleic acid into brain tissue of the subject, wherein the method includes administering the combination to the subject and applying focused ultrasound at a frequency below about 1 MHz and a peak negative pressure between 150 and 220 kPa.

[0044] According to some embodiments, there is provided a system for transiently opening blood-brain barrier (BBB) of a subject and facilitating introduction of a nucleic acid into brain tissue of the subject, the system includes a focused ultrasound transmitter configured to emit ultrasound (US) at a frequency of below about 1 MHz and a peak negative pressure between 150 and 220 kPa, towards the brain tissue of the subject, wherein the subject has been administered with microbubbles and lipid nanoparticles (LNPs) encapsulating nucleic acid molecules; wherein said US causes the microbubbles to induce BBBO and facilitate the introduction of the nucleic acid molecules into the brain tissue.

[0045] Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the figures, descriptions and claims included herein.

[0046] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions. BRIEF DESCRIPTION OF THE FIGURES

[0047] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0048] Fig. 1A shows a schematic illustration of overview of experimental setting for non- invasive brain delivery of lipid nano particles (LNPs), by ultrasound-mediated BBB opening (BBBO), according to some embodiments. Fig. IB- shows fluorescent microscopy and bioluminescence experiments demonstrating extravasation of the LNPs into health brain tissue and GBM brain tissue of a model animal. The results presents fluorescent microscopy images of healthy and GBM-brain tissue samples. Further shown are quantification graphs, quantifying the mean intensity of the under various US pressure conditions, and in the absence or presence of MBs;

[0049] Figs. 2A-G - EB extravasation following FUS-mediated BBBO as a function of centerfrequency. Fig. 2A shows an in-vivo FUS setup that includes a fixed-laser indicator to target the RH (right hemisphere). Figs. 2B-2G show the evaluation of BBBO at three centerfrequencies: Images of Evans blue (EB) extravasation in brains treated with a center frequency of 850 kHz (Fig. 2B), 250 kHz (Fig. 2C), 80 kHz (Fig. 2D); Fig. 2E, Fig. 2F and Fig. 2G show fluorescence images of the brain slices of Figs. 2B-2D, respectively. EB extravasation was detected in the red channel. The images were acquired with 20x objective lens. Scale bar: 2 mm;

[0050] Figs. 3A-H -EB extravasation as a function of the PNP at a center-frequency of 850 kHz. Fig. 3A- EB extravasation in extracted brains positioned prone and in coronal cuts. Red and Blue lines represent the X- and Z-axes directions, respectively; Fig. 3B- Comparison of opening width and height [mm] in X and Z axis as a function of PNP; Fig. 3C- Ellipsoid representation of the opening area, as a function of pressure; Fig. 3D- EB extravasation imaged using fluorescent microcopy (EB / DAPI Merge). Full brain images were captured in 20x magnification and used to quantify the size of opening. Scale bar: 2 mm. Fig. 3E-3F- Comparison of histological evaluation of microhemorrhages (in H&E staining) at 330 kPa (Fig. 3E) and 180 kPa (Fig. 3F); Fig. 3G- Quantification of blood presented in brain slices as a metric to evaluate microhemorrhage area out of the total brain area (%). One-way ANOVA with Tukey's multiple comparison (*** for p<0.001, **** for p<0.0001); Fig. 3H- No BBBO was detected when using an 850 kHz center-frequency and a PNP of 124 kPa. (Panel i) Top view of an extracted brain. Fluorescence microscopy images of brains treated with: (Panel ii) 124 kPa and center frequency of 850 kHz, and (Panel iii) Non-treated control (NTC) brain (injected with EB only). All microscopy images were stained with DAPI and acquired at x20 magnification. Scale bar: 2 mm.

[0051] Figs. 4A-C - Delivery of EB and 4-150 kDa fluorescent dextran. Fig. 4A- Fluorescence microcopy images (EB / D API Merge) of brain slices following BBBO showing delivery of: left column: controls of EB only, dextrans only (mix of all three dextrans: 4,70 and 150 kDa), middle and right columns: Treated brains with MB+FUS and: EB (~ 1 kDa), 4 kDa Dextran Antonia Red, 70 kDa Dextran Antonia Red and 150 kDa Dextran FITC. Images were captured in 20x magnification. Scale bar: 2 mm; Fig. 4B- Size of the opening in the X and Z axes as a function of delivered particle. Fig. 4C - Fluorescence marker’s intensity comparison between the different molecules and their controls (* for p<0.05, *** for p<0.001, **** for p< 0.0001.; Two-Way ANOVA with Tukey's multiple comparison);

[0052] Figs. 5A-H- SM-102 LNP brain delivery in healthy mice. Fig. 5A- Fluorescence microcopy images of brain slices after BBBO showing delivery of siRNA-Cy5-LNP (~70 nm) as a function of PNP. Images were captured at a 20x magnification. Scale bar: 2 mm. Fig. 5B Comparison of siRNA-Cy5-LNP mean intensity in the BBBO region as a function of the PNP. Fig. 5C - Size of the opening in the X and Z axes as a function of the PNP. Fig. 5D Comparative analysis of all BBBO delivery of 1,4,70,150 kDa particles and siRNA-Cy5-LNP (~70 nm); Fig. 5E- The graph in Fig. 5D presented as the fold increase in intensity compared to the controls; Fig. 5F Bioluminescence images of whole brain mRNA-LUC-LNP expression 24 hours post BBBO; Fig. 5G) Comparison of total flux for the treated group (MB + FUS + LNP) vs controls (FUS + LNP, MB + LNP, and LNP only) in brains; Fig. 5H - shows as Illustration of the fabricated LNPs (structure and cargo). Two-Way ANOVA with Tukey's multiple comparison was used in Fig. 5D. Figs. 5B, 5E and 5G- One-way ANOVA with Tukey's multiple comparison. (* for p<0.05, ** for p<0.01, *** for p<0.001 and **** for p<0.0001) were used;

[0053] Figs. 6A-F - Direct comparison of brain delivery using two mRNA-LUC-LNP formulations via bioluminescence imaging. Fig. 6A shows structures of SM-102 lipid; Fig. 6B shows structures of Lipid 14, Fig. 6C shows Bioluminescence images of whole brains for the treated groups (MB + FUS + LNP) versus LNP only controls; i: SM-102, ii: Lipid 14); Fig. 6D shows Liver signal from the same mice. Figs. 6E and 6F show Comparison of total flux for the treated group (MB + FUS + LNP) vs control group (LNP only) in brains (Fig. 6E), and Liver (Fig. 6F). Two-way ANOVA with Tukey's multiple comparison. (* for p<0.05, ** for p<0.01) was used;

[0054] Figs. 7A-F - EB extravasation in the 005 glioma tumor model. Representative fluorescence images of coronal brain sections showing GFP -labeled 005 glioma tumors (green) (Fig. 7A and Fig. 7D), EB accumulation (red) (Fig. 7B and Fig. 7E), and the merged channels (Fig. 7C and Fig. 7F). Top row (Figs. 7A-7C)- No-FUS control. Bottom row (Figs. 7D-7F)- FUS + MB group. Scale bar: 2 mm.; and

[0055] Figs. 8A-C - FUS-mediated BBBO for the delivery of siRNA-Cy5-LNP into GBM tumors. Fig. 8A- Fluorescence microcopy images of brain slices after BBBO showing delivery of siRNA-Cy5-LNP (~70 nm) in treated (FUS+LNP+MB) vs. control (FUS+LNP) GBM brains. Images were captured with 20x magnification. Scale bar: 2mm; Fig. 8B- Bar graph showing Quantification of siRNA-Cy5-LNP fluorescence in treated compared to controls brains; Fig. 8C- Confocal microscopy (x60 magnification) of the tumor region stained by DAPI. Green channel shows the GFP positive tumor cells and red channel is the siRNA-Cy5- LNP. Co-localization in the GFP / Cy5 merge indicates LNP uptake by 005 glioma cells, while the DAPI / Cy5 merge represents uptake by both 005 glioma cells and surrounding non-tumor cells. The FUS+MB panels show representative images from two different treated mice. Scale bar: 100 pm. Unpaired students t-test (** p<0.01) was used.

[0056] DETAILED DESCRIPTION

[0057] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0059] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the stated object, unless the context clearly dictates otherwise. By way of example, “a treatment” means one or more treatments.

[0060] As used herein, the term "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0061] The term “may” refer to an optional or possible, approach or possibility, but not a requirement. The term “can” refer to a permissible or plausible, approach or possibility, but not a requirement.

[0062] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0063] As used herein, the term “plurality” is directed to include more than one. In some embodiments, the term plurality includes at least two.

[0064] As used herein, the term “comprising” is synonymous with the terms "including," "containing," or "characterized by," and is inclusive or open-ended i.e. does not exclude additional, unrecited elements. According to some embodiments, the term comprising may be replaced with the term “consisting of’ which excludes any element, step, or ingredient not specified in the claim. According to some embodiments, the term comprising may be replaced with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0065] As used herein, the term “blood-brain barrier” or “BBB” refers to the highly selective, semipermeable physiological barrier formed primarily by endothelial cells lining the cerebral microvasculature. The BBB regulates the passage of substances between the systemic circulation and the central nervous system (CNS), permitting the diffusion of essential nutrients and gases while restricting the entry of pathogens, toxins, large molecules, and many therapeutic agents.

[0066] The term “blood-brain barrier opening” or “BBBO” refers to a temporary, reversible, or permanent disruption or modulation of the BBB that increases its permeability, thereby allowing substances, compounds, compositions, to enter brain regions (e.g. brain parenchyma) from the bloodstream.

[0067] According to some embodiments, the terms “treat,” “treating,” and “treatment” refer to the administration of composition(s) (e.g., LNP and MBS) and performance of a method (e.g. US), to a subject (e.g., a human or other mammal) for the purpose of obtaining a beneficial or desired clinical result. The beneficial or desired result may include, but is not limited to prevention (preventing the onset or development of a disease or disorder); Delay (slowing or delaying the progression of a disease or disorder); Reduction (reducing the severity, extent, or recurrence of a disease or disorder); Amelioration (alleviating one or more symptoms associated with a disease or disorder); and / or Cure (achieving partial or complete eradication of the disease or disorder). In some embodiments, treatment encompasses prophylactic treatment in a subject at risk of developing a brain-related condition, therapeutic administration in a subject already diagnosed, and adjuvant administration in combination with other therapies.

[0068] According to some embodiments, there is provided a method of delivering a nucleic acid (e.g., RNA molecules) payload to the brain of a subject by administering a lipid nanoparticle encapsulating the nucleic acid; administering microbubbles, and applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between about 150 and 220 kPa, thereby inducing BBB opening (BBBO), sufficient to permit delivery of the lipid nanoparticle into brain parenchyma or tumor tissue.

[0069] According to some exemplary embodiments, the focused ultrasound may be applied at a frequency of about 850 kHz and a duty cycle of about 0.1%., or about 250KkHz. In other embodiments, the lipid nanoparticle may have a particle size of about 65-120 nm and may include an ionizable lipid such as SM-102, Lipid 14, or derivatives thereof. In some embodiments, the nucleic acid payload is an RNA, wherein the RNA payload may be siRNA, mRNA, microRNA, a CRISPR-associated RNA, and the like, or any combinations thereof. Each possibility is a separate embodiment.

[0070] According to some embodiments, there are further provided systems which includes a focused ultrasound transducer configured to operate under suitable frequency, peak negative pressure and duty cycle parameters, to effect BBB opening by administered microbubbles, wherein the induced BBBO facilitates extravasation of lipid nanoparticles encapsulating nucleic acids to brain target region. According to some embodiments, there are provided pharmaceutical compositions including lipid nanoparticles encapsulating nucleic acid formulated together with (in the same or different composition) microbubbles suitable for ultrasound activation.

[0071] According to some embodiments, there is provided the use of lipid nanoparticles encapsulating nucleic acids for treating a brain cancer or neurological disorder by administration in combination with microbubbles and focused ultrasound as described herein. Exemplary brain cancers and neurological disorders may include glioblastoma, Alzheimer’s disease, Parkinson’s disease, psychiatric disorders, genetic brain diseases, and the like, or any combinations thereof.

[0072] According to some embodiments, as exemplified herein, there is provided the use of low-frequency FUS-mediated BBBO with MBs to enhance the delivery of large particles, including, mRNA-based LNP as a platform for advanced non-invasive local brain therapies. As exemplified herein, there is provided a robust platform for the noninvasive, safe, and efficient delivery of LNPs with diameters of about 70-100 nm to both healthy brain tissue and GBM tumors. The platform successfully enable the delivery of large LNPs to GBM tumors using FUS-mediated BBBO. As exemplified herein, LNPs, with their ability to encapsulate RNA-based therapeutics such as siRNA and mRNA, can be safely delivered to the brain using a noninvasive approach.

[0073] According to some embodiments, as exemplified herein, US application, operating at a center-frequency of 850 kHz or less (e.g., about 250kHz), in a PNP of 180 kPa-120kPa establishes a consistent threshold for safe and effective delivery of particles of various sizes, while preserving tissue integrity. According to some embodiments, MBs used are size-selected to remove bubbles larger than about 5 pm, thereby reducing size heterogeneity and enhancing safety.

[0074] According to some embodiments, the results presented herein demonstrate distinct variation in the opening pattern and coverage area between different center-US frequencies.

[0075] According to some embodiments, as exemplified herein, comparative analysis of the fluorescent markers revealed a consistent opening area in the X axis, and the strongest foldincrease in mean intensity was observed for EB, followed by Dextran 150 kDa, which was the closest in size to the siRNA-Cy5-LNP (~70 nm).

[0076] According to some embodiments, as exemplified herein, the delivery performance of mRNA-LNPs may be assessed using bioluminescent mRNA-LUC-LNP and subsequent luciferase expression measurement 24 hours post-treatment. The results demonstrated effective delivery exclusively in the FUS-mediated BBBO group with 12-fold delivery enhancement compared to the non-treatment control.

[0077] According to some embodiments, as exemplified herein, BBBO experiments were conducted in a GBM mouse model established by stereotactic injection of 005 glioma cells. Unlike conventional GBM established cell lines such as U87, that tend to grow as encapsulated tumors, 005 is a genetically engineered, immunocompetent, murine glioma model that is highly infiltrative and better mimics the molecular and histopathological features of patient-derived mesenchymal GBM. It develops in syngeneic mice, allowing for evaluation within the context of a functional immune system, and exhibits limited baseline BBB permeability at early stages, making it suitable for testing BBBO. This model displays realistic tumor-stroma interactions, invasion, and therapy resistance, features that are often lacking in human cell line-based xenografts. Given these advantages, 005 provides a more physiologically relevant and translational platform to assess the effects of FUS + MB on LNP delivery across the BBB. As exemplified herein, at the stage when the tumors were treated, the BBB remained largely intact under baseline conditions, and FUS-mediated BBBO was required to induce extravasation, similar to the observations in healthy brains. Experiments for Cy5-siRNA-LNP delivery to the GBM tumors showed a 6.7-fold increase in fluorescence signal compared to controls. In terms of tumor delivery, confocal microscopy showed that LNPs were distributed throughout the tumor and taken up both by the 005 glioma cells and by cells within the tumor microenvironment.

[0078] Reference is made to Fig. 1A, which shows a schematic illustration of overview of experimental setting for non-invasive brain delivery of lipid nanoparticles (LNPs), by ultrasound-mediated BBB opening (BBBO), according to some embodiments. As shown in Fig. 1 A, the BBBO is induced using administered microbubbles (MBs) and application of low- intensity Focused ultrasound (FUS), targeted to brain region (e.g., the right hemisphere (RH)). The subject may be systemically injected with LNPs, prior to, after or concomitantly with application of US. Shown in Fig. 1 A, several examples of substances / particles of varying sizes, ranging from less than about IkDa (Evans Blue (EB)), about 4-150kDa substances (Dextrans), about 70-100nm LNPs (e.g., LNPs harboring siRNA, having an average diameter of about 70nm, LNPs harboring mRNA, having an average diameter of about lOOnm). In order to assess BBBO, brain regions may be imaged. According to some embodiments, the microbubbles are lipid bubbles, having an external lipid shell. In some embodiments, the shell may include such components as, but not limited to: disteroylphosphatidylcholine (DSPC), 2-dibehenoyl-sn-glycero-3-phosphocholine (C22), l,2-dipalmitoyl-sn-glycero-3 -phosphate (DPPA), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine-N-

[0079] [methoxy(poly ethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K) and 1,2- distearoylsnglycero-3-phosphoethanolamine-N-[biotinyl(poly ethylene glycol) 2000] (DSPE- PEG2000-Biotin). In some embodiments, the bubbles include a fluid core. In some embodiments, the fluid is gas. In some embodiments, the gas is selected from: perfluorobutane (C4F10), octafluoropropane C3F8, perfluorocarbons, sulfur hexafluoride, air and nitrogen.

[0080] In some embodiments, the microbubbles have an external lipid shell. In some embodiments, the lipid shell may include phospholipids. In some embodiments, the lipid shell of the microbubbles may include: (2.5 mg per ImL) disteroylphosphatidylcholine (DSPC), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K). In some exemplary embodiments, for formation of targeted MBs (TMB) the lipids may further include l,2-distearoylsnglycero-3-phosphoethanolamine- N-[biotinyl(polyethylene glycol) 2000] (DSPE-PEG2000-Biotin). In some embodiments, a molar ratio between the lipids may be 90: 10, or 90:5:5.

[0081] According to some embodiments, the MBs may have a diameter in the range of about 300-3000nm. In some embodiments, the MBs may have an average diameter in the range of about 400-2500nm. In some embodiments, the MBs may have an average diameter in the range of about 500-2000nm. In some embodiments, the MBs may have an average diameter in the range of about 600-1800nm. In some embodiments, the MBs may have an average diameter in the range of about 700-1500nm. In some embodiments, the MBs may have an average diameter in the range of about 750-1200nm. In some embodiments, the MBs may have an average diameter in of about 750nm. In some embodiments, the MBs may have an average diameter of over 300 nm, over about 500nm, over about 750nm, over about 800nm. Each possibility is a separate embodiment.

[0082] According to some embodiments, the microbubbles may exhibit a spherical morphology.

[0083] According to some embodiments, the microbubbles may further include a targeting moiety on an external region thereof. In some embodiments the targeting moiety may be on the shell of the bubbles. In some embodiments, the targeting moiety may be a cell-type specific targeting moiety. In some embodiments, the targeting moiety may be a cell-type specific antibody.

[0084] According to some embodiments, the amount / concentration / number of the microbubbles may be determined according to the target tissue, size of tissue, type of tumor, size of tumor, location of the tumor, and the like. In some embodiments, the amount of MBs administered may be about IxlO5MBs. In some embodiments, the amount of MBs administered may be about IxlO6MBs. In some embodiments, the amount of MBs administered may be about 5xl06MBs. In some embodiments, the amount of MBs administered may be about 6.6xl06MBs. In some embodiments, the amount of MBs administered may be at least about IxlO7MBs. In some embodiments, the amount of MBs administered may be at least about IxlO8MBs. In some embodiments, the amount of MBs administered may be at least about 1X109MBS. In some embodiments, the amount of MBs administered may be at least about IxlO10MBs. In some embodiments, the concentration of

[0085] MBs administered may be about IxlO11MBs / 20pl.

[0086] According to some embodiments, the lipid nanoparticles (LNPs) of the invention may encapsulate various types of nucleic acid molecules. The term “nucleic acids” refers broadly to naturally occurring or synthetic polynucleotides having natural or modified stretches of nucleotides. In some embodiments, the nucleic acids are capable of encoding or modulating gene expression.

[0087] In some embodiments, the nucleic acid molecules may include DNA molecules. In some embodiments, the nucleic acid molecules may include RNA molecules. In some embodiments, a mixture of DNA and / or RNA molecules may be used.

[0088] In some embodiments, a plurality of nucleic acids may be encapsulated within the LNP. In some embodiments, various types of nucleic acids (e.g., different types of RNAs) may be encapsulated within the LNP. In some embodiments, nucleic acids having different nucleotide sequence may be encapsulated within the LNP. In some embodiments, a composition of LNPs may include an homogenous or heterogenous distribution of LNPs, with respect of size, load and / or composition. In some embodiments, a composition may include LNPs harboring one type of nucleic acid. In some embodiments, a composition may include LNPs harboring different types of nucleic acids. In some embodiments, a composition may include LNPs harboring nucleic acids having the same or similar nucleotide sequence. In some embodiments, a composition may include LNPs harboring nucleic acids having the different nucleotide sequences.

[0089] In some embodiments, the RNA molecules may include such molecules as, but not limited to: small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), shRNA, CRISPR-associated RNAs, guide RNAs, ribonucleoprotein complexes, and the like, or any combinations thereof. In some embodiments, the RNA may include Modified RNA species, in which RNA molecules may include one or more nucleotides having chemical modifications, such as pseudouridine, Nl-methyl-pseudouridine, 5-methylcytidine, 2’-O- methyl, 2’-O-methoxyethyl, locked nucleic acids (LNAs) phosphorothioate linkages or others to enhance stability, reduce immunogenicity and / or improve translation efficiency.

[0090] According to some embodiments, small interfering RNA (siRNA) relates to doublestranded RNA molecules, typically 19-27 base pairs in length, capable of inducing RNA interference and silencing specific gene targets. According to some embodiments, Messenger RNA (mRNA) refers to single-stranded RNA molecules encoding a polypeptide or protein of interest, capable of translation in a host cell. According to some embodiments, MicroRNA (miRNA) refers to small, non-coding RNA molecules that can regulate post-transcriptional gene expression. According to some embodiments, CRISPR guide RNA (gRNA) refers to RNA molecules directing CRISPR-associated proteins, such as Cas9, to specific genomic loci for gene editing.

[0091] According to some embodiments, the DNA molecules may include, for example, but not limited to, plasmid DNA, complementary DNA (cDNA), antisense DNA oligonucleotides, gene-editing constructs (including, for example, DNA templates), or any combinations thereof. Each possibility is a separate embodiment. In some embodiments, the DNA may include one or more modified nucleotides.

[0092] In some embodiments, the nucleic acid molecules may have a therapeutic effect in or at the vicinity of the target brain tissue. In some embodiments, the effect may include inhibition of gene expression of specific genes in the target tissue, inducing expression of genes / proteins in the target tissue, encoding / expressing genes of interest in the target tissue, and the like.

[0093] According to some embodiments, the nucleic acid molecules delivered by the lipid nanoparticles are designed to modulate expression of specific genes that may be associated with brain cancers, neurodegenerative diseases, genetic disorders and / or immune modulation. Such genes may include, for example, but not limited to: oncogenes, tumor suppressors, proteins involved in DNA repair, and genes implicated in neuronal survival, synaptic function, or neuroinflammation. The selection of the nucleic acid modality, such as small interfering RNA (siRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), CRISPR-based guide RNAs, and the like, may be adjusted to the therapeutic objective, such as gene silencing, gene replacement, or genome editing.

[0094] According to some embodiments, the nucleic acid may target oncogenes associated with glioblastoma and other brain tumors, including, but not limited to: EGFR and mutant forms thereof, PDGFRA, MYC, CCND1, MET, and the like. The nucleic acids may be siRNA, antisense oligonucleotides (ASOs), or CRISPR guide RNAs designed to reduce expression of such oncogenes, or mRNAs encoding decoy receptors or dominant-negative variants.

[0095] According to some embodiments, the nucleic acid may target tumor suppressor genes or pathways altered in glioblastoma, including, for example, but not limited to: TP53, PTEN, RBI, and the like, wherein the nucleic acids may be mRNA to restore functional protein expression, CRISPR-based editing tools to correct mutations, and the like.

[0096] According to some embodiments, the nucleic acid may target DNA repair pathway genes, including, for example, MGMT or PARP1, to modulate repair activity and sensitize tumors to chemotherapy or radiotherapy.

[0097] According to some embodiments, the nucleic acid may target genes associated with Alzheimer’s disease, including, for example, APP, PSEN1, PSEN2, MAPT (Tau), APOE4, and the like. The nucleic acids may include siRNA, ASOs, CRISPR guide RNAs, or mRNAs encoding protective protein variants.

[0098] According to some embodiments, the nucleic acid may target genes associated with Parkinson’s disease, including, for example, SNCA (alpha-synuclein), LRRK2, PARK2, GBA1, and the like, wherein the nucleic acids may silence toxic protein accumulation or restore deficient enzyme function.

[0099] According to some embodiments, the nucleic acid may target genes associated with amyotrophic lateral sclerosis (ALS), including, for example, SOD1, C9orf72, TARDBP (TDP- 43), and FUS, wherein the nucleic acids may silence toxic gain-of-function alleles or deliver mRNAs encoding functional proteins.

[0100] According to some embodiments, the nucleic acid may target genes associated with genetic neurodev elopmental disorders, including, for example, SMN1 for spinal muscular atrophy, MECP2 for Rett syndrome, and FMRI for Fragile X syndrome. According to some embodiments, the nucleic acid may encode immune-stimulatory cytokines, including, for example, IL-12, IL-15, and GM-CSF, to promote anti-tumor immune responses in the brain.

[0101] According to some embodiments, the nucleic acid may target genes associated with immune checkpoint molecules, including, for example, PD-1, PD-L1, and CTLA-4, using for example, siRNA, ASOs, gene editing (e.g., CRISPR), and the like, to overcome immune evasion in brain tumors.

[0102] According to some embodiments, the LNPs may have a particle size in the range of about 50-150nm, for example, about 55-140nm, for example, about 60-130nm, for example, about 65-120 nm. For example, the LNPs may have an average size of about 60-80nm. For example, the LNPs may have an average size of about 70nm. For example, the LNPs may have an average size of about 90-1 lOnm. For example, the LNPs may have an average size of about lOOnm. In some embodiments, the particle size may be adjusted according to the nucleic acid payload of the particles.

[0103] In some embodiments, the LNPs may have an encapsulation efficiency greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%. In some exemplary embodiments, the nucleic acid loading in the LNPs may be about 0.05-2 mg / mL, corresponding to an encapsulation efficiency of at least 70%.

[0104] According to some embodiments, the LNPs may have a lipid shell and a core carrying the nucleic acids. In some embodiments, the ionizable lipid component of the LNPs may be selected from SM-102, Lipid 14, DLin-MC3-DMA, or functional derivatives thereof. According to some embodiments, the LNPs may include one or more additional components, including, for example, cholesterol, a helper phospholipid, such as phosphocholine (PC) based lipid, such as, but not limited to: DSPC (l,2-distearoyl-sn-glycero-3 -phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (l,2-dimyristoyl-sn-glycero-3- phosphocholine), DOPC (l,2-dioleoyl-sn-glycero-3 -phosphocholine); and / or phosphoethanolamine (PE) based lipid such as, but not limited to: DOPE (1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine) or DSPE (distearoyl-sn-glycero-3-phosphoethanolamine), a PEG-lipid, and the like, or any combinations thereof. Each possibility is a separate embodiment.

[0105] According to some embodiments, the lipid nanoparticles may include an ionizable lipid, cholesterol, a helper phospholipid, and a PEG-lipid. In some embodiments, the molar composition of the LNP may include the following ranges: Ionizable lipid: about 30-60 mol%, (for example, about 40-50 mol%), Cholesterol about 20-45 mol%, (for example, about 30-40 mol%), Helper phospholipid (e.g., DSPC) about 5-15 mol%, (for example, about 8-12 mol%), PEG-lipid about 0.5-5 mol%, (for example, about 1-3 mol%).

[0106] According to some embodiments, the ionizable lipid may be selected from SM-102, Lipid 14, DLin-MC3-DMA, or derivatives thereof, a lipid having a tertiary amine headgroup with a pKa of 6.0-7.4, or any combinations thereof. Each possibility is a separate embodiment.

[0107] According to some embodiments, the nucleic acid-to-lipid weight ratio may be between about 1 :5 and about 1 :20, for example, about 1 :6 or 1: 10.5.

[0108] According to some embodiments, the lipid nanoparticles (LNPs) have a zeta potential adjusted to optimize blood-brain barrier passage and cellular uptake in brain tissue. The zeta potential may be between -20 mV and +20 mV, such that the nanoparticles remain colloidally stable in circulation while avoiding aggregation with serum proteins. In some embodiments, the zeta potential of the LNPs may be near neutral, for example between -10 mV and +10 mV at physiological pH, to minimize non-specific interactions and reduce systemic toxicity. In some embodiments, the LNPs may have a slightly positive zeta potential, for example between +5 mV and +15 mV, to enhance electrostatic interaction with negatively charged cell membranes and improve uptake into endothelial and neuronal cells following BBB opening. In some embodiments, the LNPs may have a slightly negative zeta potential, for example between -5 mV and -15 mV, to improve circulation half-life by reducing rapid clearance by the reticuloendothelial system (RES). In some embodiments, the zeta potential of the LNPs may be tuned by altering the ratio of ionizable lipid to helper lipid, or by modifying the PEG- lipid composition and chain length.

[0109] According to some embodiments, the nucleic acid payload may include single or multiple species of RNA and / or DNA molecules, optionally including siRNA, mRNA, miRNA, antisense DNA oligonucleotides, plasmid DNA, or CRISPR gRNAs, and the like, or combinations thereof.

[0110] According to some embodiments, the LNPs may optionally be formulated with stabilizers, cryoprotectants, or excipients, such as sucrose, trehalose, mannitol, or glycine, to improve storage stability and lyophilization properties. According to some embodiments, the LNPs may be surface-modified with targeting ligands / moieties, such as antibodies, peptides, aptamers, or small molecules (e.g., transferrin or RGD peptides), to enhance tissue or tumor selectivity.

[0111] According to some embodiments, the LNPs may be prepared by microfluidic mixing, ethanol injection, or high-pressure homogenization, with process parameters adjusted to achieve desired particle size and poly dispersity index (PDI <0.2).

[0112] According to some embodiments, the LNPs may include pH-sensitive ionizable lipids that facilitate endosomal escape, thereby improving cytoplasmic delivery of nucleic acids following BBB crossing.

[0113] According to some embodiments, the LNPs may be adapted for controlled release by incorporation of biodegradable lipids or stimuli -responsive components (e.g., disulfide linkages sensitive to redox conditions).

[0114] According to some embodiments, the LNPs may be provided in aqueous buffer (e.g., citrate buffer, pH 4-7) suitable for intravenous injection, optionally packaged in a single-use vial, multi-dose vial, or co-packaged kit with microbubbles.

[0115] In some embodiments, the lipid nanoparticles may be formulated at a concentration of about 0.1-10 mg / mL total lipid. In some embodiments, the LNPs may be formulated at 0.5-5 mg / mL total lipid.

[0116] In some embodiments, the LNPs may be administered at a dose of about 0.01 mg / kg and 10 mg / kg of nucleic acid. In some embodiments, the dose may be about 0.05 mg / kg and 5 mg / kg of nucleic acid. In some embodiments, the dose may be about 0.1 mg / kg and 1 mg / kg of nucleic acid, such as about 0.5 mg / kg.

[0117] In some embodiments, the LNPs may be administered as a single dose followed by ultrasound application. In some embodiments, the LNPs may be administered in multiple treatment cycles, for example once weekly, bi-weekly, or monthly. In some embodiments, repeated dosing may be performed over a treatment period of 2 weeks to 6 months.

[0118] In some embodiments, the LNPs may be administered in a volume of about 1-10 mL intravenously. In some embodiments, the LNPs may be administered in a volume of about 5- 100 pL by intrathecal, intracerebroventricular, or intranasal routes.

[0119] According to some embodiments, the FUS may be applied at a frequency below 1 MHz, e.g., at about 850 kHz, at about 250kHz. The ultrasound may be delivered at a peak negative pressure (PNP) of about 150-220 kPa, for example, at about 180 kPa, and at a duty cycle of about 0.05-0.5 %, e.g., 0.1%. According to some embodiments, as exemplified herein below, these parameters were found to produce consistent BBB opening while avoiding vascular damage such as microhemorrhage. At higher pressures (e.g., above 400 kPa), microvascular damage was observed, whereas lower pressures (e.g., 124 kPa) were insufficient to induce BBB opening.

[0120] According to some embodiments, the low-energy US may have a center frequency of 1MHz or less, 950kHz or less, 850kHz or less 800kHz or less, 500kHz or less, 200kHz or less, 100 kHz or less. In some embodiments, the US may have a peak negative pressure (PNP) of 350 kPa or less, 300 kPa or less, 280 kPa or less, 250 kPa or less, 200 kPa or less, e.g., about 180 kPa.

[0121] According to some embodiments, the therapeutic ultrasound frequency may be selected based on target size, depth, and skull characteristics, such that frequencies effective in small animals (e.g., -850 kHz in mice) can translate to lower frequencies for human use (e.g., <250 kHz) to maintain penetration at the same delivery performance.

[0122] According to some embodiments, in the mouse model, frequencies of about 850 kHz may perform best among the tested frequencies, while 250 kHz is comparatively less effective is terms of focal shape with respect to the mouse brain (as shown, for example, in Figs. 2A-G). Accordingly, in some embodiments, for human applications, lower ultrasound frequencies (e.g., on the order of <250 kHz) may be preferred due to scaling and skull acoustics. In some embodiments, what is optimal in mice (for example, 850kHz) may translate to shift downward for human subjects (for example, 250kHz).

[0123] According to some embodiments, the ultrasound may be delivered using a spherically focused single-element transducer, or other clinically relevant transducer arrays, including hemispherical phased arrays. According to some exemplary embodiments, the US system may include a spherical single-element FUS transducer. The transducer may be operated at 849 kHz with a peak negative pressure of 180 kPa. In some embodiments, the transducer may be operated at about 250 kHz with a peak negative pressure of about 180 kPa.

[0124] According to some embodiments, the application of US may be performed at a time period after administration of the MBs. In some embodiments, the time period may be at least 5 seconds, at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes. Each possibility is a separate embodiment.

[0125] According to some embodiments, there is provided a pharmaceutical composition which includes lipid nanoparticles encapsulating nucleic acid (e.g., RNA); and microbubbles suitable for ultrasound activation. In some embodiments, the composition may be formulated for intravenous injection, optionally in a single vial or co-packaged form.

[0126] According to some embodiments, the methods, systems and compositions disclosed herein may be applicable to the treatment of brain related or neurological disorders requiring delivery of nucleic acid therapeutics across the BBB, including but not limited to glioblastoma and other brain cancers, Alzheimer’s disease (AD), Parkinson’s disease (PD), psychiatric disorders, genetic diseases of the CNS, and the like, or any combinations thereof. Each possibility is a separate embodiment.

[0127] According to some embodiments, a brain cancer may include, but not limited to: Glioblastoma multiforme (GBM), Astrocytoma (tumors arising from astrocytic glial cells), Oligodendroglioma (tumors originating from oligodendrocytes), Ependymoma (tumors derived from ependymal cells of the ventricular system), Medulloblastoma (a malignant pediatric brain tumor arising in the cerebellum), and the like, or any combinations thereof. Each possibility is a separate embodiment.

[0128] According to some embodiments, glioblastoma represents a preferred embodiment because it frequently retains an intact BBB in early stages, limiting the penetration of large therapeutics.

[0129] According to some embodiments, the condition is glioblastoma (GBM) brain cancer. In some embodiments, the afflicted subjects bear glioblastoma tumors that retain an intact BBB in early stages.

[0130] According to some embodiments, various treatment regimens may be used. In some embodiments, the treatment regime may include a one-time (single) treatment (e.g., gene editing with CRISPR or AAV replacement therapy). In some embodiments, the treatment regime may include repeated administrations (e.g., mRNA therapies, chemotherapeutics, or RNAi-based treatments).

[0131] According to some embodiments, the composition including lipid nanoparticles (LNPs) encapsulating nucleic acids and microbubbles is administered prior to application of focused ultrasound (FUS), such that the nanoparticles circulate in the vasculature during BBB opening. According to some embodiments, the composition may be administered during the application of focused ultrasound, whereby LNPs and microbubbles are present in the bloodstream at the time of sonication, maximizing co-localization with transient BBB opening.

[0132] According to some embodiments, the composition may be administered immediately after the application of focused ultrasound, taking advantage of the BBB remaining permeable for a period of several hours post-treatment, thereby permitting efficient nanoparticle extravasation.

[0133] According to some embodiments, microbubbles may be administered concurrently with focused ultrasound while lipid nanoparticles are administered sequentially before or after ultrasound exposure.

[0134] According to some embodiments, lipid nanoparticles and microbubbles may be coformulated or co-administered intravenously, followed by application of focused ultrasound within a time window of 0-5 minutes.

[0135] According to some embodiments, repeated cycles of BBB opening and LNP administration may be performed, such that treatment may be carried out daily, weekly, biweekly, or monthly, depending on the therapeutic indication.

[0136] According to some embodiments, the method may include multiple ultrasound exposures delivered sequentially to cover larger brain regions or tumor volumes, wherein each exposure is synchronized with systemic circulation of LNPs and microbubbles.

[0137] According to some embodiments, the method may include pre-treatment with a diagnostic or reporter LNP formulation (e.g., luciferase-encoding LNPs) to confirm BBB opening and delivery efficiency, followed by subsequent treatment with therapeutic RNA- or DNA-loaded LNPs under the same ultrasound parameters.

[0138] According to some embodiments, the treatment regime may be adapted for singleadministration therapies, such as delivery of CRISPR-Cas9 or gene replacement constructs, or for repeated-administration therapies, such as delivery of mRNA therapeutics, RNAi, or chemotherapy adjuvants.

[0139] According to some embodiments, the lipid nanoparticles and / or microbubbles may be administered to the subject by any suitable route that enables systemic or local delivery prior to or during focused ultrasound exposure. Suitable routes of administration include, but are not limited to: intravenous administration, intra-arterial administration, intraperitoneal administration, subcutaneous administration, oral administration, intranasal or inhalation administration, and direct central nervous system administration (including intrathecal or intracerebroventricular inj ection).

[0140] According to some embodiments, the route of administration may be selected based on the therapeutic indication, the pharmacokinetics of the lipid nanoparticle formulation, or the desired localization of delivery within the brain or tumor tissue.

[0141] According to some embodiments, the lipid nanoparticles and microbubbles may be administered via the same or different routes. For example, lipid nanoparticles may be administered intravenously while microbubbles are administered intra-arterially.

[0142] According to some embodiments, the lipid nanoparticles and microbubbles may be coformulated in a pharmaceutical composition suitable for intravenous administration, optionally provided as a single vial or dual-vial kit.

[0143] According to some embodiments, the ultrasound system may include a focused ultrasound transducer configured to deliver acoustic energy at a frequency below 1 MHz, preferably 850 kHz.

[0144] In some embodiments, the system may further include a phased-array transducer capable of steering and shaping the ultrasound beam electronically, thereby targeting multiple regions of the brain without mechanical repositioning.

[0145] In some embodiments, the ultrasound system may include a cavitation detection module, such as passive cavitation detectors, to monitor microbubble activity and optimize treatment parameters in real time.

[0146] In some embodiments, the system may include acoustic feedback and control circuitry that can automatically adjust ultrasound power or duty cycle to maintain safe operation and prevent vascular damage.

[0147] In some embodiments, the ultrasound system may include or be associated with MRI or ultrasound imaging module for treatment planning, targeting, and monitoring of BBB opening.

[0148] In some embodiments, the system may include a therapeutic console with programmable control software allowing the operator to select treatment regimens (e.g., before / during / after nanoparticle administration). In some embodiments, the ultrasound system may further include a neuronavigation or stereotactic positioning unit to precisely align the ultrasound focus with a target brain region or tumor.

[0149] In some embodiments, the system may include a cooling or coupling unit, such as a degassed water bath or acoustic coupling membrane, to ensure efficient energy transfer and thermal safety.

[0150] In some embodiments, the ultrasound system may further include a microbubble infusion system, optionally synchronized with ultrasound pulses, to deliver microbubbles at controlled doses and timings.

[0151] In some embodiments, the system may include signal processing and data acquisition modules to record treatment parameters and cavitation signatures, thereby providing traceability for clinical use.

[0152] In one embodiments, the system may include patient safety interlocks, such as automatic shutdown in case of excessive cavitation or overheating.

[0153] In some embodiments, the system may be integrated into a clinical treatment workstation, optionally networked with hospital imaging systems, allowing real-time visualization, feedback, and patient record integration.

[0154] According to some embodiments, the system may further include a motor and / or a motorized platform.

[0155] According to some embodiments, the system may further include a user interface, a monitor, a controller, a communication unit, and the like.

[0156] In some embodiments, the processor is remotely based. In some embodiments, the processor is physically associated with the system.

[0157] According to some embodiments, there is provided a method of treating a neurological disorder in a subject, the method includes the steps of administering to the subject lipid nanoparticles encapsulating a therapeutic nucleic acid; administering microbubbles; and applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa, whereby the blood-brain barrier is transiently opened and the lipid nanoparticles are delivered into the brain tissue (e.g., parenchyma). According to some embodiments, the neurological disorder may be selected from Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS) or multiple sclerosis. Each possibility is a separate embodiment.

[0158] According to some embodiments, the neurological disorder may include a genetic disorder selected from spinal muscular atrophy, Rett syndrome, or Fragile X syndrome.

[0159] According to some embodiments, wherein the nucleic acid includes siRNA, mRNA, microRNA, plasmid DNA, CRISPR-associated nucleic acids, and the like, or any combinations thereof.

[0160] According to some embodiments, there is provided a composition which includes the LNPS disclosed herein, encapsulating a nucleic acid; and microbubbles, for use in a method of treating glioblastoma, wherein the treatment includes applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between about 150 and 220 kPa to transiently open the blood-brain barrier and facilitate the delivery of the LNPs into or in the vicinity of the tumor tissue.

[0161] According to some embodiments, there is provided a composition including LNPs as disclosed herein, encapsulating a nucleic acid; and microbubbles for use in the treatment of a neurological disorder, wherein the treatment includes applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa to transiently open the blood-brain barrier and deliver the LNPs into the brain tissue (e.g., parenchyma).

[0162] According to some embodiments, there is provided a method of treating glioblastoma in a subject in need thereof, the method includes: administering to the subject lipid nanoparticles encapsulating a nucleic acid; administering microbubbles; and applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa, whereby the blood-brain barrier is transiently opened and the lipid nanoparticles are delivered into or in the vicinity of tumor tissue.

[0163] According to some embodiments, there is provided a composition which includes the LNPS disclosed herein, encapsulating a nucleic acid; and microbubbles, for use in a method of treating glioblastoma, wherein the treatment includes applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between about 150 and 220 kPa to transiently open the blood-brain barrier and facilitate the delivery of the LNPs into or in the vicinity of the tumor tissue. According to some embodiments, the nucleic acid may include siRNA, mRNA, or antisense oligonucleotides targeting oncogenes.

[0164] According to some embodiments, the treatment may further include repeating the administration and ultrasound exposure in multiple cycles.

[0165] According to some embodiments, the glioblastoma treatment may further include coadministration of a chemotherapeutic agent, checkpoint inhibitor, or radiotherapy.

[0166] According to some embodiments, there is provided a method of treating a neurological disorder in a subject, the method includes the steps of administering to the subject the LNPs encapsulating the therapeutic nucleic acid; administering microbubbles; and applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa, whereby the blood-brain barrier is transiently opened and the lipid nanoparticles are delivered into the brain tissue (e.g., parenchyma).

[0167] According to some embodiments, there is provided a composition including LNPs as disclosed herein, encapsulating a therapeutic nucleic acid; and microbubbles for use in the treatment of a neurological disorder, wherein the treatment includes applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa to transiently open the blood-brain barrier and deliver the LNPs into the brain tissue (e.g., parenchyma).

[0168] According to some embodiments, the neurological disorder may be selected from Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS) or multiple sclerosis. Each possibility is a separate embodiment.

[0169] According to some embodiments, the neurological disorder may include a genetic disorder selected from spinal muscular atrophy, Rett syndrome, or Fragile X syndrome.

[0170] According to some embodiments, the nucleic acid includes siRNA, mRNA, microRNA, plasmid DNA, CRISPR-associated nucleic acids, and the like, or any combinations thereof.

[0171] According to some embodiments, there is provided a kit which includes: a first container comprising lipid nanoparticles encapsulating a nucleic acid; and a second container comprising microbubbles; for use in a method of transiently and reversibly opening the blood brain barrier (BBB) of a subject, wherein the method comprises administering the kit components to the subject and applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa. According to some embodiments, there is provided a kit which includes: a first container comprising lipid nanoparticles encapsulating a nucleic acid; and a second container comprising microbubbles; for use in a method of treating brain cancer (such as GBM), or other neurological disorders, wherein the method comprises administering the kit components to the subject and applying focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa.

[0172] According to some embodiments, there is provided a pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid and microbubbles, in the manufacture of a medicament for transiently and reversibly opening the blood-brain barrier and facilitating delivery of the nucleic acid into brain tissue of a subject, wherein the medicament is for administration in combination with focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa.

[0173] According to some embodiments, there are provided lipid nanoparticles encapsulating a nucleic acid and microbubbles in the manufacture of a medicament for the treatment of a brain-related condition, wherein the medicament is for administration in combination with focused ultrasound at a frequency below 1 MHz and a peak negative pressure between 150 and 220 kPa, such that the blood-brain barrier is transiently and reversibly opened and the nucleic acid is capable of being delivered into brain tissue.

[0174] According to some embodiments of the kit, the nucleic acid may be selected from siRNA, mRNA, microRNA, antisense oligonucleotides, plasmid DNA, cDNA, or CRISPR- associated nucleic acids.

[0175] According to some embodiments of the kit, the microbubbles include a phospholipid shell and a gas core.

[0176] According to some embodiments of the kit, the lipid nanoparticles and microbubbles are formulated for intravenous administration.

[0177] According to some embodiments of the kit, the first and second containers are provided in a co-packaged form.

[0178] According to some embodiments of the kit, the kit further includes written instructions for administration in combination with focused ultrasound to achieve BBB opening and / or treating a brain related conditions. According to some embodiments of the kit, the lipid nanoparticles and microbubbles are co-formulated in a single container.

[0179] According to some embodiments of the kit, the kit may further include excipients, stabilizers, or cryoprotectants, such as sucrose, trehalose, mannitol, or glycine.

[0180] According to some embodiments, there is provided a method which includes: administering one or more biocompatible microbubbles to an organism; applying low-frequency focused ultrasound to excite the one or more biocompatible microbubbles, thereby generating one or more openings in the blood-brain barrier of the organism; and introducing one or more therapeutic compounds through the one or more openings, wherein the one or more therapeutic compounds includes one or more lipid nanoparticles harboring nucleic acids.

[0181] According to some embodiments, the MBs may include one or more gas bubbles having a size of between 50 nm to 10 micrometers. In some embodiments, the MBs have a lipid shell surrounding a gas core.

[0182] According to some embodiments, the low-frequency focused ultrasound has a center frequency in a range of between 50 kHz and 1000 kHz. According to some embodiments, the center frequency is in range of between 80 kHz and 850 kHz.

[0183] According to some embodiments, applying the low-frequency focused ultrasound comprises applying a peak negative pressure in a range of about 50 kPa to about 500 kPa.

[0184] According to some embodiments, the low-frequency focused ultrasound has a center frequency of about 850 kHz, and wherein the peak negative pressure is in a range of about 125 kPa to 135 kPa.

[0185] According to some embodiments, the nucleic acids may have a size of between 0.4kDa and 500 nm. In some embodiments, the nucleic acids include RNA molecules. In some embodiments, the RNA may include, but not limited to: short interfering ribonucleic acid sequences (siRNA), messenger ribonucleic acid sequences (mRNA), single guide RNA (sgRNA), and the like, or any combinations thereof.

[0186] According to some embodiments, the nucleic acids are therapeutic compounds, cable of treating or affecting a condition, such as, brain cancers, glioblastoma, neurological disorders, neurodegenerative diseases, Alzheimer’s disease, Parkinson’s disease, psychiatric disorders, effects of chemotherapy -based cancer treatments, effects of antibody -based cancer treatments, effects of gene therapies, and the like, or any combinations thereof.

[0187] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0188] EXAMPLES

[0189] Materials and Methods

[0190] Microbubble preparation

[0191] MBs composed of a phospholipid shell and a Perfluorobutane (C4F10) gas core, were prepared using the thin film hydration method (Ilovitsh, T. et al. Enhanced microbubble contrast agent oscillation following 250 kHz insonation. Sci Rep 8, 16347 (2018)). Two lipids of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; 850365C) and 1,2-distearoyl-sn- glycero-3- phosphoethanolamine-N-[methoxy (polyethylene- glycol)-2000] (ammonium salt) (DSPE-PEG2K; 880129C) (Sigma Aldrich, St Louis, MO, USA) were mixed at a molar ratio of 90: 10. A buffer mixture (10% glycerol, 10% propylene and 80% saline (pH 7.4) were added to the lipid film and sonicated at 62°C until full transparency. The resulting 2.5 mg / ml MB precursor solution was aliquoted into 1 ml in each vial and saturated with Perfluorobutane gas (C4F10, Cas no. 355-25-9, F2 Chemicals LTD, UK) to remove air. Upon use, the solution was activated by mechanical shaking with a VialMix shaker (Bristol-Myers Squibb Medical Imaging Inc., MA, USA) and centrifuge to purify MBs with radii smaller than 0.5 pm. Size selection was applied to remove MBs with radii larger than 5 pm. The MBs size and concentration were measured with a particle counter system (AccuSizer® FX-Nano, Particle Sizing Systems, Entegris, MA, USA) and showed median diameter of 1.5 pm and a typical concentration of ~5xl09MB / ml. The MBs were used within three hours of their activation.

[0192] RNA-LNP preparation and characterization

[0193] Tracer RNA-LNPs were prepared as previously described (Naidu, G. S. et al., A Combinatorial Library of Lipid Nanoparticles for Cell Type-Specific mRNA Delivery. Advanced Science 10, 2301929 (2023)). Briefly, one volume of lipid mixture (Ionizable lipid SM-102, DSPC, Cholesterol, PEG-DMG at 50: 10:38.5: 1.5 molar ratio) in ethanol combined with mRNA (1 :6 molar ratio RNA to ionizable lipid, either 50% siRNA-Cy5 or mRNA-LUC) in a citrate buffer, pH 4.5 were injected into a NanoAssemblr microfluidic mixing device (Precision Nanosystems Inc., Canada) at a combined flow rate of 12 mL min-1. The resulting LNPs were dialyzed twice using 0.5x phosphate buffered saline (PBS) (Dulbecco's PBS w / o CA and MG) (pH 7.4) for 16 h and 4h to remove ethanol. Cholesterol, DSPC (1,2-distearoyl- sn-glycero-3 -phosphocholine), polyethylene glycol (PEG)-DMG (1,2-dimyristoyl-rac- glycerol) were purchased from Avanti Polar Lipids Inc (Alabaster, AL, USA). Heptadecan-9- yl 8- ((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102 or Lipid 14) were synthesized in-house as previously described. mRNA sequences were purchased from TriLink (San Diego, CA, USA), and synthesized with complete Nl-methyl-pseudouridine nucleotide substitution. siRNA-Cy5 was purchased from Integrated DNA Technologies, Inc. The resulting LNP sizes were characterized by dynamic light scattering (DLS) (Zetasizer Ultra, Malvern, Panalytical, Westborough, MA, UK). Zeta potential was determined using the Zetasizer S system (Malvern, Worcestershire, UK). Encapsulation efficiency was determined using the RiboGreen essay (Thermo Fisher Scientific, Waltham, MA, USA). Concentration and size distribution were performed on diluted samples (1 :5,000 in PBS) using NanoSight (NTA, NS300, Malvern, UK).

[0194] Focused Ultrasound (FUS) setup

[0195] The FUS setup was composed of spherically focused single-element transducer (Hl 15, Sonic Concepts, Bothell, WA, USA) operated by a transducer power output system (TPO-200, Sonic Concepts) that was located at the bottom of a water tank facing upwards. The transducer had a diameter of 64 mm and a focal distance of 45 mm. It supported center frequencies of 80, 250, and 850 kHz via custom matching networks (purchased from Sonic Concept). The PNPs for each center frequency were calibrated using a needle hydrophone (NH0500, Precision Acoustics, UK). For the in vivo experiments, the mice were positioned on top of an agarose spacer. This spacer was prepared by dissolving agarose powder (A10752, Alfa Aesar, MA, USA) in distilled water to achieve a 1.5% concentration, followed by heating to completely dissolve the agar powder and remove gas bubbles. The solution was poured into a mold of 5 cm x 5 cm x 1 cm (length x width x height) and cooled at room temperature. The spacer was placed on top of the water tank as a mouse bed. To precisely target the RH, the setup was equipped with a vertically fixed laser pointer to co-align with the transducer’s focal spot (Fig. 1A). PCD experiment

[0196] The spectra of echoes from oscillating MB were passively recorded using a focused 1 MHz PCD single-element transducer (IMO102, 0.25” diameter, 1” focal distance, Valpey Fisher) during their interaction with 850 kHz ultrasound pulses. For each transmitted PNP (ranging from 140 to 650 kPa), four independent measurements were acquired. The same MB solution (5 x 109 MB / pL) used in the in vivo experiments was diluted 1 :50 in PBS in a 1 mL syringe and manually injected via a 27G needle into PTFE tubing (BTPE-20 (.38x1.09mm), Instech Laboratories Inc., Plymouth Meeting, PA, USA), positioned at the FUS transducer’s focal spot using a custom-made holder. The PCD transducer was aligned at a 45° angle to the tube at its focal distance. Received echoes were displayed on a digital oscilloscope (MDO3024, Tektronix, Beaverton, OR, USA) and saved for post-processing in MATLAB. Signal analysis was performed as previously described (Plaksin, M. et al. Magnetic resonance imaging analysis predicts nanoparticle concentration delivered to the brain parenchyma. Commun Biol 5, (2022)). The area under the curve (AUC) was calculated for the second harmonic (850 kHz ± 8.5 kHz), subharmonic (425 kHz ± 6.375 kHz), and broadband noise (283.3 kHz ± 5.67 kHz), each indicative of nonlinear MB responses and inertial cavitation onset. For each PNP, control signals were recorded by injecting degassed PBS into the tubing. Final results are presented after subtracting the PBS reference from the MB spectra.

[0197] In-vivo BBBO experiments

[0198] Eight to twelve- week-old female C57BL / j6 mice, weighing between 19-23 grams (Harlan, Jerusalem, Israel), were used for the in-vivo FUS-mediated BBBO experiments. All animal procedures were approved by the Institutional Animal Ethics Committee at Tel Aviv University and carried out in accordance with established guidelines.

[0199] Animal preparation: Mice preparation for the BBBO procedure was as following: all mice were anesthetized with 2% isoflurane using a low-flow vaporizer system (120 ml / min, SomnoFlo, Kent Scientific, Connecticut, USA). Their heads were fully shaved with a machine, and any remaining hair was removed using hair removal cream (Veet, Reckitt Benckiser, France), that was applied for 40 seconds and then removed with a water-soaked pad. The region of interest in the RH was marked by a dot with a marker to assist in the positioning of the mice.

[0200] BBBO in healthy brains: For the BBBO procedure, mice were systemically injected with 2 x 107MBs per 20 g of body weight, in 50 pl of degassed PBS (Dulbecco's PBS w / o CA and MG). US gel was applied on top of the agarose spacer, and the mouse's head was positioned supine on top of the gel. The FUS treatment was operated within 60 seconds from MBs injection in varied parameters depending on the tested center-frequency. FUS treatments in 850 kHz center-frequency comprised of 1 ms bursts and RPF of 1 Hz (duty cycle of 0.1%), testing nine PNPs: 650, 540, 470, 400, 370, 330, 280, 220, 180 and 124 kPa (n=18 in total). In 80 kHz center-frequency, 3.25 ms bursts and RPF of 1 Hz were operated (duty cycle of 0.1%) and the tested PNPs were: 180, 120, 90, 75 (n=8). The PNPs were optimized for 850 kHz and 80 kHz, by gradually decreasing the PNP until safe BBBO was observed, without signs of microhemorrhage in histology. For 250 kHz, the PNP was set at 200 kPa with the same PRF (1Hz). Immediately after the FUS treatment, mice were systemically injected with one out of six different molecules as described next.

[0201] Evans Blue (EB) delivery: EB (E2129, Sigma Aldrich) dye solution of 2% in PBS at 4 ml / kg and was systemically injected and allowed to circulate for a duration of 28 minutes before mice were sacrificed. Once identified, the optimized BBBO parameters (850 kHz, 180 kPa, 1 ms bursts, duty cycle of 0.1%) were used in all subsequent experiments that were conducted using this protocol.

[0202] Dextrans delivery: For experiments with dextran molecules, three distinct molecular weights (4 kDa, 70 kDa, and 150 kDa), each labeled with a fluorescent moiety were used (Antonia Red-lysine-dextran [ARLD4, ARLD70], TdB Labs AB, Uppsala, Sweden, and 150 kDa FITC-Dextran [68042-46-8], Sigma-Aldrich). To maintain consistency, each dextran dose was 1 mg in lOOpl and circulation time was 10 minutes before scarification. Mice were divided into three groups (n=5 in each): treated group injected with 1 mg of red-labeled 70 kDa dextran; a second treated group injected with a mixture of red-labeled 4 kDa and green-labeled 150 kDa dextrans, and a control group injected with a mixture of all three dextrans.

[0203] LNPs delivery: 3 types of LNPs were fabricated. First, LNPs incorporating two types of ionizable lipids were evaluated: SM-102 and Lipid 14. Both encapsulated mRNA-LUC for in-vivo bioluminescent imaging using IVIS. In addition, SM-102 based LNPs were fabricated composing 50% siRNA-Cy5 for testing using fluorescent microscopy. For siRNA-Cy5-LNP, a fixed dose of Img / kg was systemically injected, and brains were harvested 2.5 hours posttreatment. A total of 15 (n=3 each) mice were divided into four groups treated at descending pressures (400, 330, 220, 180 kPa) and NTC. In all experiments with mRNA-LUC-LNPs, a fixed dose of Img / kg dose was systemically injected, and mice were imaged 24 hours post BBBO. Two experiments were conducted. The first was to confirm successful uptake of SM- 102 mRNA-LUC-LNP in healthy brains. For this, a total of 21 mice, all injected with SM-102 mRNA-LUC, were divided into four groups: LNPs only (n=6), MBs + LNPs (n=3), FUS + LNPs (n=4), and MBs + FUS (180 kPa) + LNPs (n=8). 24 hours post treatment, prior to the IVIS imaging the mice were injected intraperitoneally with XenoLight d-luciferin (15 mg / kg) (122799, PerkinElmer Inc.). Post-sacrifice, brains and livers were extracted and imaged for LUC signal. The bioluminescence analysis was performed using the Living Image software comparing the samples total flux [p / s]. In the second experiment, a comparative study was conducted between LNPs formulated with two different ionizable lipids. A total of 30 mice were included: the treatment groups included MB + FUS + Lipid 14 LNP (n = 6) and MB + FUS + SM-102 LNP (n =12), while the control groups included Lipid 14 LNP only (n = 6) and SM-102 LNP only (n = 6). The same IVIS imaging protocol and bioluminescence quantification procedures were applied across all groups.

[0204] At the end point of each experiment (EB / Dextrans / LNPs), the brains were collected (without perfusion) and positioned on top of a Tragacanth Gum paste, which had been prepared by mixing Tragacanth Gum powder (G1128-100G, Sigma-Aldrich) in distilled water at a concentration of 15% (w / v). Subsequently, the samples were flashed-frozen in 2-methylbutane (Sigma-Aldrich) using liquid nitrogen and stored in a -80°C refrigerator until cryo-sectioning to 20 pm slices.

[0205] 005 glioma orthotopic model

[0206] Mouse-derived glioma cell line (005 glioma, GFP+, LUC+), were established using lentiviral transduction of H-Ras and activated Akt in Cre-GFAP / p53+ / - C57BL / 6 mice (Marumoto, T. et al. Development of a novel mouse glioma model using lentiviral vectors. Nat Med 15, 110-116 (2009)). Maintained in stem cell medium, specifically DMEM / F : 12 medium supplemented with 1% Glutamax (100X), 1% penicillin-streptomycin, B27 supplement (Invitrogen), N2 supplement (Invitrogen), heparin (50 pg / mL), EGF (20 ng / mL), and FGF2 (20 ng / mL), the 005 glioma cells were cultured as spheres and split every 3-4 days using TrypLE Express dissociation reagent (Gibco Corp, 12604-013, Grand Island, NY, USA) when reaching 90% confluency. A total of 48 eight-week-old female C57BL / 6J01aHsd mice (Envigo, Jerusalem, Israel) were anesthetized using isoflurane, positioned in the Kopf Stereotaxic Alignment System, and inoculated with 3* 105GBM 005-GFP-luciferase cells in a 1.5-pl volume using automatic syringe pump in a rate of 0.3 pl / min. Injections were made to the right frontal lobe: ~1.5 mm lateral, 2 mm caudal from bregma, and at a depth of 2.3 mm. Tumor inoculation and growth monitoring was performed by bioluminescence imaging (IVIS Spectrum, PerkinElmer Inc.) every 5 days post tumor cell implantation until the experiment between days 17-21. XenoLight d-luciferin was inj ected at 15 mg / kg intraperitoneally and mice were imaged within 10 to 30 mins post injection. Bioluminescence analysis was conducted using the Living Image software (PerkinElmer Inc.) comparing the subject's total flux14.

[0207] BBBO for siRNA-Cy5-LNP delivery in 005 glioma orthotopic model

[0208] The BBBO experiment in GBM mouse model was performed between days 17-21 post tumor inoculation. To assess the integrity of the BBB in the GBM tumor model, an EB delivery experiment was conducted, comparing EB alone to MB + FUS + EB. The experimental protocol was identical to that used in the EB delivery study in healthy mice. Next, LNP delivery experiments were conducted following a similar protocol to that used for siRNA-Cy5-LNP delivery in healthy mice. The optimized BBBO parameters were 850 kHz, 180 kPa, 1 ms bursts, duty cycle of 0.1%; siRNA-Cy5-LNP was systemically injected at a fixed dose of Img / kg, and brains were harvested 2.5 hours post-treatment for microscopy imaging. For standard 20x fluorescence microscopy, the brains were flash-frozen in 2-methylbutane (Sigma-Aldrich) cooled with liquid nitrogen and stored at -80 °C until cryo-sectioning into 20 pm slices. For confocal microscopy, mice were perfused with phosphate-buffered saline (0.01 M), followed by 4% paraformaldehyde. The brains were then harvested and stored at 2 °C until further use.

[0209] Microscopy imaging and Quantitative analysis

[0210] Frozen brains imaging: Un-perfused brains were cryo-sectioned to 20-pm-thick coronal in a -20° C cryostat microtome (CM1950, Leica Biosystems). The sections were placed on standard microscope slides and kept in a slide box at -20° C until use. Upon imaging, the brain slides were thawed to room temperature and imaged within 1 hour to avoid dye diffusion. All full brain images in this study were obtained using a hybrid automated microscope (Revolution, Echo, San Diego, USA). The imaging process involved stitching 20 x 30 tiles, each measuring 0.432 mm x 0.36 mm, to create a full slice scan. These scans were conducted at 20x optical magnification. For fluorescent images the following excitation wavelengths and exposure times were used: DAPI (365 nm, 90 ms), GFP (410 nm, 460 ms), Cy5 (690 nm, 790 ms), and Evans Blue (690 nm, 790 ms). Measurement of the width of the opening and the full brain distances in the X-axis and the Z-axis were conducted using the microscope software (Figs. 3B, 4B and 5C). Segmentation of the opening area with EB as a function of pressure in the representation of an ellipsoid was calculated using an ellipse function in MATLAB (version 2018a, MathWorks, Natick, MA, USA) (Fig. 2F). The values were normalized to average mouse brain size. Quantification of opening size area, microhemorrhages and markers intensity were conducted using ImageJ® software (National Institutes of Health, Bethesda, MD). Fullbrain fluorescence microscopy images were first imported into ImageJ. Markers' intensity was calculated by selecting an opening area at the same size across all brain images (Fig. 4C, 5B,D). Quantification of BBBO-induced hemorrhage was conducted on full-brain brightfield microscopy images (without H&E staining) using the IHC toolbox in ImageJ®. To reduce background noise, each hemorrhage center was defined as 200 pixels2, and the IHC toolbox was trained to recognize relevant shades of brown from total brain area (Fig. 3H).

[0211] Formalin fixated brains: Brains were coronally sectioned into 40pm slices using a HM450 Microtome (Thermo Fisher Scientific). Floating sections were mounted on standard microscope slides and stained with DAPI. Confocal microscopy images were acquired with confocal microscope (Revolution, Echo, San Diego, USA) using UPLXAPO x60 oil objective (NA 1.42) and subsequently merged using ImageJ® software (Fig. 8C). 3D visualization of a FUS + MB + LNP treated tumor was generated by Leice LAS X V3.3 Software.

[0212] Histology

[0213] The safety of the BBBO treatments was assessed by a standard Hematoxylin (Leica 3801542) and Eosin (Leica 3801602) (H&E) staining of the 20-pm-thick frozen brain sections and scan x20 using the brightfield channel (Fig. 2D-E). DAPI Staining; Tissue slices were mounted onto glass slides and cover slipped after the application of three drops of Mounting Medium with DAPI (Fluoroshield, abl04139, Abeam). The slides were allowed to develop for 15 minutes before further analysis.

[0214] Statistical analysis

[0215] Prism 10.1.2 (GraphPad Software) was employed for the statistical analysis. A two- sided Student’s t-test was utilized to compare two experimental groups. In experiments involving multiple groups, differences among multiple populations and sub-populations were assessed using One-Way and Two-Way ANOVA with Tukey’s multiple comparisons. A value of p<0.05 was considered statistically significant. Differences are presented on graphs in the following abbreviations: blank, for not significant, * for p< 0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001.

[0216] 1 Focused ultrasound center¬ in vivo experiments were conducted using a custom setup, where the mouse was positioned supine, and a laser indicator was used to aid in targeting the right hemisphere (RH) (Fig. 2A). Initial experiments were aimed at determining the optimal center-frequency by monitoring EB extravasation patterns at 850, 250, and 80 kHz in healthy mice brains. For each center-frequency, initial PNP values were chosen based on previous studies or by using the PNP values established at other frequencies as a starting point for testing at lower frequencies. Among the tested frequencies, the 850 kHz center-frequency emerged as optimal for targeted BBBO in the RH, covering an area of ~3.5 x 7 mm2 (Fig. 2B and 2E). The 250 kHz centerfrequency resulted in a heterogeneous pattern concentrated only at the brain's edges (Fig. 2C and 2F). The 80 kHz center-frequency resulted in a strong, wide focal opening spot, but also caused microhemorrhage at 90 kPa, and only a very mild opening at 75 kPa (Fig. 2D and 2G). Based on these results, 850 kHz center-frequency was found optimal in mice brains and selected for further in vivo experiments aimed at the delivery of larger fluorescent particles.

[0217] 2: Passive cavitation detection

[0218] After identifying the center frequency of 850 kHz, the next step was to characterize the MB acoustic response using passive cavitation detection (PCD) at this frequency. This was done by quantifying the amplitude of the second harmonic (2fo), broadband noise, and subharmonic components as a function of the applied PNP. The second harmonic response increased with rising PNP. No signal was observed at the lowest PNP of 140 kPa, and a gradual rise was seen across the tested range, reaching peak amplitude at 650 kPa. This trend reflects increasing MB oscillation amplitude. Broadband noise, indicative of inertial cavitation onset, was negligible at lower pressures and began to rise at approximately 200-250 kPa, with a more pronounced increase beyond 400 kPa, suggesting PNP-dependent destabilization and collapse of MBs under high acoustic drive. Subharmonic emissions, a marker of MB nonlinear oscillation, were generally low across the entire pressure range, with a slight upward trend observed at higher PNPs.

[0219] Example 3: EB extravasation assessment following FUS-mediated BBBO at 850 kHz

[0220] In vivo optimization experiments for assessing the safe range of PNPs and extravasation assessment were then performed. The PNPs were progressively decreased from 650 to 180 kPa until a safe BBBO was achieved, with no microhemorrhage observed in histological analysis. BBBO was visible in the brain’s RH via prone and coronal cuts (Fig. 3A). To compare the size of the BBBO as a function of PNP, the width and height of the opening along the X and Z axes were measured out of the fluorescence images of the coronal brain slices (Fig. 3B and 3D). The results show a consistent opening along the Z axis with an average of 6.74 ± 0.13 mm, independent of the applied PNP, while a significant gradual decrease was observed along the X axis as the PNP decreased from 650 to 180 kPa (5.46 mm ± 0.39 vs 3.49 mm ± 0.29, p<0.0001; One-way ANOVA with Tukey's multiple comparison). Alternatively, these empirical results can be plotted as an ellipsoid representing the BBBO in each axis as a function of the PNP (Fig. 3C). A lower PNP of 124 kPa was also tested, yet no BBBO was observed (Fig. 3H). Histological assessment of the presence of blood in each brain section as a function of PNP calculated as percent out of total brain area was used as a measure to evaluate microhemorrhage following treatment, with the amount of blood in control (EB only) sections serving as a reference to healthy brain. Presence of blood in brain histology sections decreased as a function of PNP. At 180 kPa, the values were similar to those of the control, indicating an absence of microhemorrhage and the safety of treatment at this PNP (not significant, One-way ANOVA with Tukey's multiple comparison) (Figs. 3E-3G).

[0221] It is noted that is terms of the empirical dimensions of the BBBO within the brains at 850 kHz, the ultrasound focus is inherently asymmetric, with a narrower focal width along the X-axis and a substantially longer depth of focus along the Z-axis, forming an ellipsoidal shape (Fig. 3C). The focal width in the X direction is smaller than the diameter of the mouse brain, and thus the measured BBBO width in X reflects the ultrasound focus and its modulation with increasing PNP. In contrast, the focal depth in the Z direction exceeds the anatomical dimensions of the mouse brain (~7 mm), leading to an opening observed throughout the entire Z-axis (Fig. 3D). Therefore, the measured BBBO extent in Z reflects the anatomical limitation of the mouse brain rather than the true focal length of the ultrasound beam.

[0222] 4: Fluorescent Dextrans brain

[0223] After successfully delivering the dye Evans Blue (EB), having a size of less than <1 kDa), the same FUS parameters were used to study the delivery of larger dextrans with sizes of 4, 70, and 150 kDa. The results are presented in Figs. 4A-C. The 4 and 70 kDa were Antonia-Red Dextran, which fluoresces in red, while 150 kDa was FITC-Dextran that fluoresces in green (Fig. 4A). Brain slices were imaged using a fluorescent microscope, where the opening size and fluorescence intensity were quantified. Comparison of the opening width along the Z-axis revealed consistent results across all particle types: EB, 4 kDa, 150 kDa and siRNA-Cy5-LNP (not significant), with a small height reduction in the 70 kDa group compared with EB (values were: 6.55 ± 0.36, 6.30 ± 0.31, 6.38 ± 0.33, 6.17 ± 0.35 and 5.6 ± 0.54, respectively). In the X axis, opening with EB (3.49 ± 0.30) was similar to 70 kDa (3.52 ± 0.15), and stronger than 4 and 150 that overlapped (3.05 ± 0.16) (*** for p<0.001; One-way ANOVA with Tukey's multiple comparison) (Fig. 4B). Comparative analysis (Two-Way ANOVA with Tukey's multiple comparison) of fluorescence intensity in the BBBO area compared to control revealed that EB exhibited the strongest intensity among the particles that were tested (Fig. 4C). 4 and 150 kDa dextran presented a similar intensity (not significant), and a stronger intensity over 70 kDa dextran (*** p<0.001) (with values of: EB: 245.73 ± 6.53 a.u., Dextran 150 kDa: 204.78 ± 28.04 a.u, Dextran 4 kDa: 190.5 ± 19.06 a.u. and Dextran 70 kDa: 133.72 ± 37.1 a.u.). In each group, treated brains exhibited significantly higher marker intensity compared to their control counterparts (**** p<0.0001) (from left to right: EB: 245.73 ± 6.53 a.u. vs. 13.48 ± 2.38, Dextran 150 kDa: 204.78 ± 28.04 a.u. vs 12.52 ± 4.13, Dextran 4 kDa: 190.5 ± 19.06 a.u. vs 27.42 ± 10.18 and Dextran 70 kDa: 133.72 ± 37.1 a.u. vs 13.65 ± 4.66 a.u.). Notably, the 4 kDa Dextran also showed a diffusion pattern towards the lateral parts of the brain slices, in contrast to the more localized EB distribution represented.

[0224] The ability to deliver LNPs was consequently tested. LNPs were constructed using the benchmark ionizable cationic lipid SM-1029,10. siRNA loaded LNPs were produced at an average size of ~70 nm and a zeta potential of -0.426mV, while mRNA loaded LNPs were produced at an average size of -100 nm and a zeta potential of -0.142mV. Both LNPs had an encapsulation efficiency of above 95% (as shown in Fig. 5H). The first type of LNP used was a non-template control siRNA-LNP conjugated to Cy5. The Cy5-siRNA-LNP brain delivery was assessed in the treated group (MB + FUS + LNP) compared to the control group (LNP only) by measuring fluorescence intensity. Since these particles are significantly larger than the dextrans, higher PNP of 400 kPa was initially used, and gradually lowered to the minimum PNP at which a clear opening was achieved without microhemorrhage (Fig. 5A). At the highest PNP of 400 kPa, the opening was accompanied by microhemorrhage, but at 180 kPa, which is the PNP used for the dextran delivery, particle delivery with similar fluorescence intensity to that of the higher PNP was achieved, but without histological damage and full mice recovery. First, a comparative analysis of the mean intensity revealed similar fluorescence signal in the BBBO region for all the PNPs that were tested (not significant), with a significantly increased intensity compared to LNP only control (Fig. 5B) (** p<0.01, **** p<0.0001; One-Way ANOVA with Tukey's multiple comparison). When calculating the opening width in the X axis, measurements were: 4.03 ± 0.42 at 400 kPa, 3.428 ± 0.245 at 220 kPa, and 3.25 ± 0.11 at 180 kPa (Fig. 5C). Importantly, reducing the pressure until 180 kPa did not compromise particles delivery (not significant), and at the same time achieved increased safety with no clinical or histological damage. This PNP enabled the successful delivered a variety of dextrans of different sizes to the brain (Fig. 4C, and Fig. 5D). A direct comparison of all the particles indicates that EB has the highest fluorescence intensity, with the delivery being 18.2 times that of its control. Following that, 150 kDa with a 16.4-fold, siRNA-Cy5-LNP with a 10-fold, 70 kDa with a 9.8-fold and 4 kDa dextran with a 7-fold increase in signal (Fig. 5E) (* p<0.05; One-Way ANOVA with Tukey's multiple comparison).

[0225] After delivering siRNA-LNP, LNPs containing mRNA encoding the luciferase protein were fabricated by encapsulation of mRNA-luciferase sequence with SM-102 ionizable lipid based LNPs, with a mean diameter of 100 nm (shown in Fig. 5H). These particles facilitate the expression of the luciferase protein and enable the bioluminescent detection of cells that were successfully transfected in the brain. Luciferase expression was evaluated using in vivo imaging system (IVIS) by determining the total flux in whole brains, 24 hours post FUS+MB+LNP treatment (Fig. 5F). The mice group treated with MB + FUS + LNP versus the three control groups (FUS + LNP, MB + LNP, and LNP only) showed a significant increase in the total flux (** p<0.01; One-way ANOVA with Tukey's multiple comparison), with a 12- fold increase compared to the LNP-only group (Fig. 5G). Livers were used as positive controls and had no significant difference between the groups.

[0226] Example 6: Direct comparison of brain delivery using two mRNA-LUC-LNP formulations

[0227] To compare brain delivery of an additional LNP type using the same MB + FUS settings, the delivery of Lipid 14-LNP was tested (Figs. 6A-F). This is a ionizable cationic lipid used in several FDA-approved formulations (Fig. 6B). These luciferase mRNA loaded LNPs were produced at an average size of about 65 nm and a zeta potential of 3.83 mV. Direct brain delivery experiments compared the delivery efficiency of SM-102 LNP and Lipid 14-LNP following MB + FUS treatment, using identical concentrations and FUS parameters. Bioluminescence imaging of the brains 24 hours post-treatment demonstrated successful delivery, with significantly higher luciferase expression in both SM-102 and Lipid 14 LNP- treated groups compared to LNP-only controls (Fig. 6C and Fig. 6E) p < 0.01; Two-way ANOVA with Tukey's multiple comparison). Lipid 14 LNPs exhibited the highest brain expression post treatment, with a ~1.7-fold increase compared to SM-102 LNPs (* p < 0.05; Two-way ANOVA with Tukey's multiple comparison), indicating enhanced delivery efficiency with this formulation. In the liver, a statistically significant increase in luciferase expression was observed for Lipid 14 in the MB + FUS + LNP group compared to its LNP- only control. This trend was not observed in the SM-102-treated group (Fig. 6D and Fig. 6F) (p < 0.05; ; Two-way ANOVA with Tukey's multiple comparison).

[0228] 7: EB extravasation in the 005 glioma tumor model

[0229] After establishing the capability to deliver LNPs to the brains of healthy mice, the delivery to brain tumors was evaluated using a syngeneic GBM mouse model derived from 005 glioma cells. This model closely recapitulates the molecular and histopathological features of human mesenchymal glioblastoma and retains an intact BBB in early tumor stages, making it a suitable platform for assessing FUS-mediated BBB opening. To confirm the baseline permeability of the model, EB extravasation was assessed with and without FUS-mediated BBBO (Figs. 7A-F). Microscopy images of brain slices, imaged using identical parameters, show GFP-labeled 005 glioma tumors in the green channel and EB brain accumulation in the red channel. In the absence of FUS (No FUS), EB signal is minimal, indicating that at this stage of tumor development, the BBB remains largely intact under baseline conditions. In contrast, in brains treated with FUS and MB, prominent EB extravasation is observed in the red channel, both within the tumor and in the surrounding ultrasound focal region. This behavior was observed across different tumor stages and sizes.

[0230] Example 8: Delivery of LNPs into GBM brain tumor

[0231] Subsequently, the ability to deliver the SM-102 LNPs was tested in the 005 glioma tumor model using the same parameters. BBBO was targeted to the RH of GBM bearing mice. Fluorescence microscopy confirmed the delivery of siRNA-Cy5-LNP into the tumors (Fig. 8A). Comparison of mean intensity between the treated MB + FUS + LNP (n=4) and FUS + LNP (n=3) control groups revealed a 6.7-fold increase in LNP fluorescence signal within the tumor region in treated brains compared to the controls (78.9 ± 28.0 a.u. vs 11.76 ± 3.29 a.u., *** p<0.001; Unpaired students t-test) (Fig. 8B). Next, to further characterize LNP distribution within the tumor following BBBO, an additional experiment was conducted in 9 GBM-bearing mice. Confocal microscopy (*60 magnification) confirmed widespread distribution of siRNA- Cy5-LNP throughout the tumor and their uptake by both 005 glioma cells and cells in the tumor microenvironment. Co-localization of the Cy5 signal (red channel) with the GFP signal (green channel) confirmed LNP uptake by 005 glioma cells. In the DAPI signal (blue channel), which marks all cell nuclei within the tumor, the presence of LNP in regions lacking GFP indicated internalization by non-GFP cells within the tumor microenvironment. Representative images from two different treated mice illustrate these findings in the FUS + MB panels (Fig. 8C). In contrast, in the LNP-only control tumors (no FUS), minimal levels of Cy5 fluorescence were observed in the tumor region. 3D visualization of LNP internalization within a treated tumor further supported these findings.

[0232] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

Claims

1. CLAIMSWhat we claim is:

1. A method for transiently opening blood-brain barrier (BBB) of a subject and facilitating introduction of a nucleic acid into brain tissue of the subject, the method comprising: administering to the subject lipid nanoparticles (LNPs) encapsulating the nucleic acid; administering microbubbles; and applying focused ultrasound at a frequency below about 1 MHz and a peak negative pressure between 150 and 220 kPa; wherein the blood-brain barrier is transiently and reversibly opened, thereby enabling the lipid nanoparticles to enter the brain tissue and release the nucleic acid.

2. The method of claim 1, wherein the focused ultrasound is applied at a frequency of about 850 kHz or less.

3. The method of claim 1 or 2, wherein the nucleic acid is a therapeutic nucleic acid, selected from mRNA, siRNA, miRNA, antisense oligonucleotides, plasmid DNA, cDNA, or CRISPR-associated nucleic acids.

4. The method of any one of claims 1-3, wherein the nucleic acid is configured to: reduce or inhibit expression of a gene of interest in a brain tissue, increase expression of a gene of interest in a brain tissue, or express a gene of interest in a brain tissue.

5. The method of any one of claims 1-4, wherein the lipid nanoparticles comprise an ionizable lipid, cholesterol, a helper phospholipid, and a PEG-lipid.

6. The method of any one of claims 1-5, wherein the lipid nanoparticles comprise: an ionizable lipid in an amount of 30-60 mol%, cholesterol in an amount of 20-45 mol%, a helper phospholipid in an amount of 5-15 mol%, and a PEG-lipid in an amount of 0.5-5 mol%.

7. The method of any one of claims 5-6, wherein the ionizable lipid is selected from DLin- MC3-DMA, SM-102, Lipid- 14, derivatives thereof, or any combinations thereof.

8. The method of any one of claims 5-7, wherein the helper phospholipid comprises DSPC, DOPE, DOPC, DMPC, DSPE and / or DPPC.

9. The method of any one of claims 5-8, wherein the PEG-lipid comprises DMG- PEG2000, DSPE-PEG2000, and / or PEG-ceramide.

10. The method of any one of claims 1-9, wherein the lipid nanoparticles have a surface charge (zeta potential) of between about -10 mV and about +10 mV at physiological pH.

11. The method of any one of claims 1-10, wherein the lipid nanoparticles have an average particle size of about 65-120 nm.

12. The method according to any one of claims 1-11, wherein the microbubbles have an average diameter in the range of about 250-2000nm.

13. The method according to any one of claims 1-12, wherein the microbubbles comprise a phospholipid shell and a perfluorocarbon gas core.

14. The method of any one of claims 1-13, wherein the nucleic acid is capable of being introduced into neurons, astrocytes, endothelial cells and / or tumor cells in the brain.

15. The method of any one of claims 1-14, wherein the blood-brain barrier opening persists for between at least about 1 and 6 hours.

16. The method of any one of claims 1-15, wherein the LNPs are administered before, concomitantly with, or after administration of the microbubbles.

17. The method of any one of claims 1-16, wherein the NLPs are administered before, during, or after ultrasound application.

18. The method of any one of claims 1-17, wherein the LNPs and the microbubbles are formulated in a single or separate compositions.

19. The method of any one of claims 1-18, wherein the LNPs and / or the microbubbles are administered systemically.

20. The method of any one of claims 1-19, wherein the method is repeated in multiple treatment cycles.

21. The method of any one of claims 1-20, for treating brain cancer in a subject in need thereof.

22. The method of claim 21, wherein the cancer is Glioblastoma (GBM).

23. The method of any one of claims 1-22, wherein the nucleic acid targets a gene selected from EGFR, EGFRvIII, TP53, PTEN, APP, SNCA, HTT, SOD1, SMN1, MECP2, CCND1, CCND2, CCND3, FMRI, or any combinations thereof.

24. A combination comprising a composition comprising lipid nanoparticles (LNPs) encapsulating a nucleic acid, and a composition comprising microbubbles, for use in a method of transiently and reversibly opening blood-brain barrier (BBBO) of a subject and enabling introduction of the nucleic acid into brain tissue of the subject, wherein the method comprises administering the combination to the subject and applying focused ultrasound at a frequency below about 1 MHz and a peak negative pressure between 150 and 220 kPa.

25. The combination for use of claim 24, wherein the focused ultrasound is applied at a frequency of about 850 kHz or less.

26. The combination for use of claims 24-25, wherein the nucleic acid is a therapeutic nucleic acid, selected from mRNA, siRNA, miRNA, antisense oligonucleotides, plasmid DNA, cDNA, or CRISPR-associated nucleic acids.

27. The combination for use of any one of claims 24-26, wherein the nucleic acid is configured to: reduce or inhibit expression of a gene of interest in a brain tissue, increase expression of a gene of interest in a brain tissue, or express a gene of interest in a brain tissue.

28. The combination for use of any one of claims 24-27, wherein the lipid nanoparticles comprise an ionizable lipid, cholesterol, a helper phospholipid, and a PEG-lipid.

29. The combination of claim 24-28, wherein the lipid nanoparticles comprise: an ionizable lipid in an amount of about 30-60 mol%, cholesterol in an amount of about 20-45 mol%, a helper phospholipid in an amount of about 5-15 mol%, and a PEG-lipid in an amount of about 0.5-5 mol%.

30. The combination for use of any one of claims 28-29, wherein the ionizable lipid is selected from DLin-MC3-DMA, SM-102, Lipid-14, derivatives thereof, or any combinations thereof.

31. The combination for use of any one of claims 28-30, wherein the helper phospholipid comprises DSPC, DOPE and / or DPPC.

32. The combination for use of any one of claims 28-31, wherein the PEG-lipid comprises DMG-PEG2000, DSPE-PEG2000, and / or PEG-ceramide.

33. The combination for use of any one of claims 24-32, wherein the lipid nanoparticles have a surface charge (zeta potential) of between about -10 mV and about +10 mV at physiological pH.

34. The combination for use of any one of claims 24-33, wherein the lipid nanoparticles have an average particle size of about 65-120 nm.

35. The combination for use of any one of claims 24-34, wherein the microbubbles have an average diameter in the range of about 250-2000nm.

36. The combination for use of any one of claims 24-35, wherein the microbubbles comprise a phospholipid shell and a perfluorocarbon gas core.

37. The combination for use of any one of claims 24-36, wherein the nucleic acid is capable of being introduced into neurons, astrocytes, endothelial cells and / or tumor cells in the brain.

38. The combination for use of any one of claims 24-37, wherein the blood-brain barrier opening persists for between at least about 1 and 6 hours.

39. The combination for use of any one of claims 24-38, wherein the LNP composition is administered before, concomitantly with, or after administration of the microbubbles composition.

40. The combination for use of any one of claims 24-39, wherein the LNP composition is administered before, during, or after ultrasound application.

41. The combination for use of any one of claims 24-40, wherein the LNP composition and the microbubbles composition are formulated in a single or separate compositions.

42. The combination for use of any one of claims 24-41, for systemic administration.

43. The combination for use of any one of claims 24-42, for treating a brain cancer in a subject in need thereof.

44. The combination for use of claim 43, wherein the cancer is Glioblastoma (GBM).

45. The combination for use of any one of claims 24-44, wherein the nucleic acid targets a gene selected from EGFR, EGFRvIII, TP53, PTEN, APP, SNCA, HTT, SOD1, SMN1, CCND1, CCND2, CCND3, MECP2, FMRI, or any combinations thereof46. A system for transiently opening blood-brain barrier (BBB) of a subject and facilitating introduction of a nucleic acid into brain tissue of the subject, the system comprising a focused ultrasound transmitter configured to emit ultrasound (US) at a frequency of below about 1 MHz and a peak negative pressure between 150 and 220 kPa, towards the brain tissue of the subject, wherein the subject has been administered with microbubbles and lipid nanoparticles (LNPs) encapsulating nucleic acid molecules; wherein said US causes the microbubbles to induce BBBO and facilitate the introduction of the nucleic acid molecules into the brain tissue.

47. The system of claim 46, wherein the nucleic acid is a therapeutic nucleic acid, selected from mRNA, siRNA, miRNA, antisense oligonucleotides, plasmid DNA, cDNA, or CRISPR-associated nucleic acids.

48. The system of any one of claims 46-47, wherein the nucleic acid is configured to: reduce or inhibit expression of a gene of interest in a brain tissue, increase expression of a gene of interest in a brain tissue, or express a gene of interest in a brain tissue.

49. The system of any one of claims 46-48, wherein the lipid nanoparticles comprise an ionizable lipid, cholesterol, a helper phospholipid, and a PEG-lipid.

50. The system according to any one of claims 46-49, wherein the microbubbles have an average diameter in the range of about 250-2000nm.

51. The system according to any one of claims 46-50, wherein the microbubbles comprise a phospholipid shell and a perfluorocarbon gas core.

52. The system of any one of claims 46-51, wherein the blood-brain barrier opening persists for between at least about 1 and 6 hours.

53. The method of any one of claims 46-52, wherein the LNPs are administered before, concomitantly with, or after administration of the microbubbles.

54. The method of any one of claims 46-53, wherein the LNPs are administered before, during, or after ultrasound application.

55. The method of any one of claims 46-54, wherein the LNPs and the microbubbles are formulated in a single or separate compositions.

56. The method of any one of claims 46-55, wherein the LNPs and / or the microbubbles are administered systemically.

57. The system of any one of claims 46-56, for use in treating a brain cancer in a subject in need thereof.

58. The system of claim 57, wherein the cancer is Glioblastoma (GBM).

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