Material and method of nucleic acid delivery crossing the blood‑brain barrier and other blood-tissue barriers

Abstract

Described herein are methods directed to delivering therapeutic agents across the blood-brain barrier (BBB) or other tissue barriers in subjects requiring treatment or care. The method involves obtaining a small molecule comprising a Janus Base with Amino Acid (JBAA) and mixing it in a solution with a therapeutic agent and processing the mixture with sonication to form a nanoparticle (NP) encapsulating the therapeutic agent. The NP is then administered to the subject, facilitating the crossing of the BBB or other tissue barriers to deliver the therapeutic agent directly to target cells. This approach enhances the delivery efficiency of therapeutic agents to specific sites within the body, potentially improving treatment outcomes for conditions requiring targeted delivery across biological barriers.

Description

MATERIAL AND METHOD OF NUCLEIC ACID DELIVERY CROSSING THE BLOOD-BRAIN BARRIER AND OTHER BLOOD-TISSUE BARRIERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent No.: 63 / 550,594, filed 06-February-2024, the entire ‘disclosure of which is incorporated by reference as if fully set forth herein in its entirety.FIELD OF THE INVENTION

[0002] The embodiments of the present invention relate to enhanced methods and compositions for delivery of therapeutic agents to subjects in need thereof, in particular, delivery across tissue barriers such as the blood-brain-barrier (BBB) or other barrier(s) within the subject’s body.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under 75N95022C00029 awarded by the National Institutes of Health. The government has certain rights in the invention.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0004] Not applicable (N / A).BACKGROUND OF THE INVENTION

[0005] The blood-brain barrier (BBB) is a highly selective permeability barrier that separates the circulating blood from the brain and extracellular fluid in the central nervous system (CNS). It is crucial for maintaining the brain's microenvironment, protecting it from toxins and pathogens, and regulating the transport of essential nutrients and molecules. However, this protective function also poses a significant challenge for the delivery of therapeutic agents to treat neurological disorders in the CNS. Many potential therapeutic agents and treatments for brain diseases, such as Alzheimer's, Parkinson's, and brain tumors, are unable to cross the BBB in effective concentrations, limiting their therapeutic efficacy.

[0006] The BBB is composed of endothelial cells that are tightly joined together, forming anearly impermeable barrier. These cells are supported by astrocytes and pericytes, which contribute to the barrier's integrity and function. The tight junctions between endothelial cells restrict the passage of substances, allowing only small, lipophilic molecules and certain gases to diffuse passively across the barrier. Larger molecules and hydrophilic substances require specific transport mechanisms, such as carrier-mediated transport or receptor-mediated transcytosis, to cross the BBB. This selective permeability is essential for protecting the brain from harmful substances while allowing the passage of necessary nutrients and signaling molecules.

[0007] Traditional methods to bypass the BBB, such as direct injection into the CNS (e.g., brain) or chemical modification of drugs to enhance permeability, often come with significant risks and limitations. These include potential damage to brain tissue, limited distribution of the drug, and the need for invasive procedures. Direct injection, for instance, can lead to localized damage and inflammation, while chemical modification of drugs may alter their pharmacokinetics and pharmacodynamics, potentially reducing their efficacy or increasing toxicity. Additionally, these methods may not provide uniform distribution of the therapeutic agent throughout the brain, limiting their effectiveness in treating widespread neurological conditions.

[0008] As a result, there is a pressing need for innovative strategies that can safely and effectively deliver therapeutic agents across the BBB. What is urgently needed are technologies that can deliver therapeutic agents across sensitive barriers within subjects without compromising quality or safety.BRIEF SUMMARY OF THE INVENTION

[0009] The following presents a simplified summary of the innovation in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

[0010] Traditional methods for delivering therapeutic agents across the blood-brain barrier (BBB) have faced significant challenges due to the selective permeability of the BBB, which restricts the passage of most drugs. Various strategies have been employed to overcome this barrier, including the use of lipid-based nanoparticles, liposomes, and polymeric nanoparticles. These carriers are designed to encapsulate therapeutic agents and facilitate their transport across the BBB. However, these approaches often suffer from limitations such as low drug loading capacity, instability in the bloodstream, and potential toxicity, which can hinder their clinicalapplication.

[0011] Another approach involves the use of receptor-mediated transcytosis, where therapeutic agents are conjugated to ligands that bind to specific receptors on the BBB. This method leverages the natural transport mechanisms of the BBB to ferry drugs across. While promising, this technique requires precise targeting and can be limited by the availability and expression levels of receptors on the BBB, as well as the potential for immune responses against the conjugated ligands.

[0012] Recent advancements have explored the use of cell-penetrating peptides and other small molecules to enhance drug delivery across tissue barriers. These molecules can facilitate the transport of therapeutic agents by transiently disrupting the barrier or by enhancing cellular uptake. Despite these innovations, challenges such as ensuring the stability of the therapeutic agent during transport and achieving efficient release at the target site remain.

[0013] Nanotechnology has emerged herein as promising approach, offering the potential to engineer nanoparticles that can encapsulate drugs and facilitate their transport across the BBB. Nanoparticles can be designed to exploit various transport mechanisms, such as receptor- mediated transcytosis, to cross the BBB. By targeting specific receptors on the endothelial cells of the BBB, nanoparticles can be selectively transported into the brain, enhancing the delivery and efficacy of treatments for CNS disorders while minimizing side effects and invasiveness. In some examples, nanoparticles can traverse one or more spaces between cells.

[0014] Overall, the development of effective methods for delivering therapeutic agents across the BBB is a critical area of research with significant implications for the treatment of CNS disorders. Herein, by leveraging advances in nanotechnology and other innovative approaches, the limitations of the past are overcome.

[0015] Example embodiments of the present invention can provide methods for prevention, diagnosis, and treatment of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, epilepsy, brain tumor, meningitis, encephalitis, cerebral palsy, Rett syndrome, Fragile X syndrome, Down syndrome, autism spectrum disorder, schizophrenia, depression, anxiety, bipolar disorder, addiction, attention deficit hyperactivity disorder, migraine, chronic pain, neuropathic pain, or a combination thereof.

[0016] As an additional brief summary, for example, to provide discussion points for a brief summary, some features of the technology disclosed herein can be briefly summarized (ordiscussed) by the following list of features, any of which can be inter-combined with any other feature, detail, embodiment, discussion point, aspect, or example disclosed herein:

[0017] Feature 1 : A method for delivering a therapeutic agent across the blood-brain-barrier (BBB) or other tissue barrier of a subject in need thereof, the method comprising the steps of: (1) obtaining a small molecule comprising a Janus Base with Amino Acid (JBAA); (2) mixing the JBAA solution with the therapeutic agent and followed by processing the mixture with sonication, whereby a nanoparticle named nanopiece (NP) forms with the therapeutic agent inside or partially inside the NP; (3) administering the NP to the subject, whereby the NP subsequently crosses the BBB or other tissue barrier in the subject to deliver the therapeutic agent to a cell in the subject.

[0018] Feature 2: The method of feature 1 , wherein the JBAA forms a shape of a rosette nanotube co-assembling with and surrounding the therapeutic agent with non-covalent bonds and / or a non-covalent assembly.

[0019] Feature 3: The method of feature 1, wherein the therapeutic agent comprises a nucleic acid, a nucleotide, DNA, RNA (mRNA, rRNA, tRNA, siRNA, microRNA, IncRNA), and / or one or more oligonucleotides including native and modified forms.

[0020] Feature 4: The method of feature 1 , wherein the JBAA solution comprises a pH in a range from not less than 3.0 to not more than 4.2.

[0021] Feature 5: The method of feature 1 , wherein the therapeutic agent comprises a small molecule, a peptide, a protein, a nucleic acid, or a combination thereof.

[0022] Feature 6: The method of feature 1, wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier.

[0023] Feature 7: The method of feature 1, wherein a zeta value of the nanoparticle is not less than 2.

[0024] Feature 8: The method of feature 1 , wherein an administration of the therapeutic agent to the subject without the JBAA will result in the therapeutic agent not crossing the BBB in the subject in a therapeutically effective amount.

[0025] Feature 9: The method of feature 1 , wherein the JBAA nanoparticle has a diameter of about 1 nm to about 1000 nm.

[0026] Feature 10: The method of feature 1 , wherein the other tissue barrier comprises ablood-retina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or the glomerular filtration barrier in the kidney.

[0027] Feature 11: A JBAA nanoparticle for delivering a therapeutic agent across a bloodbrain-barrier or other tissue barrier, the JBAA nanoparticle comprising: a Janus Base with Amino Acid (JBAA) molecule; and a therapeutic agent, wherein the therapeutic agent is inside or partially inside the JBAA nanoparticle, and wherein the JBAA nanoparticle is capable of crossing the blood-brain-barrier or other tissue barrier to deliver the therapeutic agent to a target cell.

[0028] Feature 12: The JBAA nanoparticle of feature 11, wherein the JBAA molecule is a small molecule comprising a Janus Base with Amino Acid (JBAA).

[0029] Feature 13: The JBAA nanoparticle of feature 11, wherein the therapeutic agent is selected from the group consisting of a small molecule drug, a peptide, a protein, a nucleic acid, and combinations thereof.

[0030] Feature 14: The JBAA nanoparticle of feature 11 , wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier.

[0031] Feature 15: The JBAA nanoparticle of feature 11, having a diameter of about 1 nm to about 1000 nm.

[0032] Feature 16: The JBAA nanoparticle of feature 11, formulated in a pharmaceutically acceptable carrier for administration to a subject.

[0033] Feature 17: The JBAA nanoparticle of feature 16, wherein the pharmaceutically acceptable carrier is suitable for at least one of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intraventricular, intracranial, intranasal, intraarticular or intratracheal administration.

[0034] Feature 18: The nanoparticle of feature 11 , wherein the other tissue barrier comprises a blood-retina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, blood-synovium barrier, blood-tumor barrier, and / or the glomerular filtration barrier in the kidney.

[0035] Feature 19: The JBAA nanoparticle of feature 11, wherein the subject is a mammal.

[0036] Feature 20: The JBAA nanoparticle of feature 11, wherein the therapeutic agent is delivered to the target tissue and cell to provide a therapeutic effect.

[0037] While the summary examples disclosed above provide some introduction to embodiments of the invention, other implementations are also contemplated, described, and recited herein. These and other features and advantages will be apparent from a reading of the following detailed description, the Examples, and a review of the associated drawings. It is to be understood that both the foregoing general description and the following detailed description are explanatory only and are not restrictive of aspects as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] For the purpose of illustration, certain discernable embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, data, dimensions, and illustrations shown. In the drawings:

[0039] FIG. 1 shows an example schematic of nanopieces crossing the BBB by targeting amino acid (AA) transporters (CAT1 , Gene name, SLC7A1).

[0040] FIG. 2A and FIG. 2B show schematics of nanopieces endocytosis (FIG. 2A) and phagocytosis (FIG. 2B) by targeting amino acid transporter SLC7A1.

[0041] FIG. 3A and FIG. 3B shows example properties of nanotube scaffold assisted siRNA nanopieces assembly (FIG 3A) and DNA nanopieces assembly (SA-DNA) (FIG. 3B).

[0042] FIG. 4 shows example fluorescent microscopy of human HEK 293T cells transfected with Nanopieces formulation of CMV / GFP (green fluorescence protein) plasmid DNA for 72 hours.

[0043] FIG. 5 shows example transfection efficiency of human HEK293T cells transfected with Nanopieces formulation of CMV / GFP plasmid DNA for 24, 48, and 72 hours. Cntl: Control (mock transfection only). ****p<0.001.

[0044] FIG. 6 shows an example of how sonication degrades DNA in a dosage dependent manner while vortexing does not; electrophoresis of CMV / GFP plasmid DNA processed by ultrasound and vortex at different amplitudes for different time periods. These data identified the time period and magnitude of NP sonication processing conditions without degrading the DNA cargo.

[0045] FIG. 7A shows a first example plasmid DNA tested for non-viral gene therapy by Nanopieces delivery. FIG. 7B shows a second example plasmid DNA tested for non-viral gene therapy by Nanopieces delivery. FIG. 7C shows a third example plasmid DNA tested for non-viralgene therapy by Nanopieces delivery. FIG. 7D shows a fourth example plasmid DNA tested for non-viral gene therapy by Nanopieces delivery.

[0046] FIG. 8 shows Zeta potentials of the four example plasmids (see FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D) in Nanopieces formulations.

[0047] FIG. 9 shows microscopy of human HEK293T cells transfected with Nanopieces formulation of pCMV-GFP plasmid DNA for 24, 48, and 72 hours.

[0048] FIG. 10 shows example transfection efficiency of human HEK293T cells transfected with Lipofectamine, NPs formulation of pCMV-GFP plasmid DNA (NP_pCMV_GFP), NPs formulation of pmiRFP plasmid DNA (NP_pmiiRFP) and naked plasmid DNA only (pDNA only).

[0049] FIG. 11 shows example transfection rates of three NPs formulated plasmids in human HEK293T cells after transfection for 48 hours.

[0050] FIG. 12 shows example transfections of GFP plasmid DNA (pDNA) in C8-D1A astrocyte cell line by Lipofectamine (center) or Nanopieces (right).

[0051] FIG. 13 shows microscopy of human cells transfected with Nanopieces formulation of GFP plasmid DNA in the absence (OnM) or in the presence of LAT1 inhibitor (KYT053, 10nM, 100nM, and WOOnM).

[0052] FIG. 14A shows transfection efficiency of human cells transfected with NPs formulation of GFP plasmid DNA in the absence (OnM) or presence of LAT1 inhibitor for 24, 48, and 72 hours. FIG. 14B shows an example small-molecule LAT1 inhibitor, JPH.

[0053] FIG. 15A shows transfection rates (%) of pDNA formulated with Lipofectamine (Lipo_pDNA), Nanopieces (NP_pDNA), and nothing (pDNA only) in CHO-K1 cells that lack CAT- 1 expression. FIG. 15B shows transfection rates (arbitrary units) of NP_pDNA in wild-type HEK293T cells that express SLC7A1 (Wild Type) and SLC7A1 knock-out HEK293T cells (SLC7A1-KO).

[0054] FIG. 16A shows examples of low transfection rates of pDNA formulated with Nanopieces (NP_pDNA-GFP), and Control (pDNA only) in rat glioma C6 cells. FIG. 16B shows examples of SLC7A1 mRNA levels in HEK293-T cells that are susceptible to NPs gene transfection and in C6 and CHO-K1 cells that are resistant to NPs transfection.

[0055] FIG. 17A shows transfection of EGFP plasmid DNA (pDNA) in HEK293T cells by Lipofectamine or Nanopieces. EGFP mRNA levels normalized to 18S RNA after Lipofectamine transfection. FIG. 17B shows an example from FIG. 17A after Nanopieces transfection. FIG. 17Cshows a comparison of EGFP mRNA levels after Lipo and NPs transfection; n = 3, P<0.05.

[0056] FIG. 18A shows examples of transfection of EGFP plasmid DNA (pDNA) in C8-D1A cells by Lipofectamine or Nanopieces. FIG. 18B shows data of FIG. 18A after Nanopieces transfection. FIG. 18C shows a comparison of EGFP mRNA levels after Lipo and NPs transfection, n = 3, P<0.05.

[0057] FIG. 19A shows examples of transfection of EGFP plasmid DNA (pDNA) in C8-D1A cells by Lipofectamine or Nanopieces. FIG. 19B shows data of FIG. 19A after Nanopieces transfection (B). FIG. 19C shows a comparison of EGFP mRNA levels per cell after Lipo and NPs transfection, n = 3, P<0.05.

[0058] FIG. 20 shows an example summary of SLC7A1 distribution in human tissues (upper left), in human brain (upper right), in male and female (see bodies at lower panels). From Human Protein Atlas.

[0059] FIG. 21 shows an example summary of SLC7A1 mRNA distribution in human brain tissues (lighter 14 bars shown at left, or yellow bars), and in other human tissues as depicted by different colors or shades of grey. From Human Protein Atlas.

[0060] FIG. 22 shows an example SLC7A1 distribution in human brain tissues. From Human Protein Atlas.

[0061] FIG. 23 shows example SLC7A1 distribution in mouse brain tissues. From Human Protein Atlas.

[0062] FIG. 24 shows example MALAT1 RNA levels in seven regions of mouse brain two weeks after in vivo NPs / ASO delivery via IV.

[0063] FIG. 25 shows an example diagram of how after IV injection of eGFP plasmid DNA (pDNA) in Nanopieces formulation for three days, mice were sacrificed before the brain was dissected into three regions as indicated.

[0064] FIG. 26 shows (in left panel): dark field fluorescent microscopy detected fluorescent microglial cells in the mid-brain with a diagram at right.

[0065] FIG. 27A shows examples of real-time quantitative RT-PCR analysis of eGFP mRNA levels in the front brain. FIG. 27B shows data from the mid-brain (B), and FIG. 27C shows data from the back brain (C). **p<0.01 , ns, p>0.05.

[0066] FIG. 28 shows single cell RNAseq analysis in the Human Protein Atlas indicates that SLC7A1 is highly expressed in microglial cells (center arrow) among different types of CNS cellsin the human brain. From Human Protein Atlas.

[0067] FIG. 29A shows macrophage-specific expression of IV delivered NPs / GFPpDNA in esophagus--H&E histology of mouse esophagus with large, multi-nucleated, and axe-shaped macrophages on the surface of esophagus tissue undergoing remodeling. FIG. 29B shows dark field microscopy demonstrates green florescence (or change in grey shade) derived from GFPpDNA expression in macrophages. FIG. 29C shows real-time quantitative RT-PCR analysis indicates the significant increase of eGFP mRNA levels in the esophagus of NPs / GFPpDNA injected mice (treated) than the mock injected mice (control) three days after IV injection.

[0068] FIG. 30A shows examples of how NPs target liver resident-macrophage Kupffer cells for delivery and expression of GFPpDNA. FIG. 30B shows dark field fluorescence microscopy showing GFP fluorescence in the Kupffer cells along the sinusoids and between hepatocytes, coinciding with the distribution patterns of brown stained Kupffer cells in H&E section in FIG. 30A.

[0069] FIG. 31 shows real-time quantitative RT-PCR analysis indicates the significant increase of eGFP mRNA levels in the liver of NPs / GFPpDNA injected mice (treated) than the mock injected mice (control) three days after IV injection.

[0070] FIG. 32A illustrates how NPs can be used for immunotherapy targeting macrophages or tissue-resident macrophages (microglial cells, and Kupffer cells etc.). FIG. 32B illustrates how regulating M1 and M2 polarization is critical for treatment of cancer, autoimmune diseases, and age-related degenerative diseases including Alzheimer's Disease.

[0071] FIG. 33 illustrates dual chemical labeling of fluorescently tagged Nanopieces (NPs) (nanotube and siRNA) for time course and metabolism studies.

[0072] FIG. 34A shows siRNA injection alone via IV, brain fluorescent image was taken after 24 hrs. FIG. 34B shows NPs injection via IV. FIG. 34C shows NPs injection via IV for 72 hrs. FIG. 34D shows NPs injection via IV after a week.

[0073] FIG. 35A shows data for blood (lighter grey or red bars) and FIG. 35B shows data for urine (darker grey or blue bars) were collected after NPs were injected via IV for 24hrs, 72hrs, and 1 week.

[0074] FIG. 36A shows gross appearance and tissue areas of a mouse brain. FIG. 36B shows fluorescent molecular tomography of a mouse brain after IV injection of fluorescently labeled NPs for 72 hrs. FIG. 36C shows blood vessel distribution on the surface of the mouse brain (red arteries and blue veins).

[0075] FIG. 37A shows examples of mouse brain parts and containing areas. FIG. 37B shows eGFP mRNA levels in Front, Middle, and Back regions of the mouse brain 48 hrs after NPs-eGFP plasmid DNA IV injection. FIG. 37C shows 18S RNA levels in Front, Middle, and Back regions of the mouse brain 48 hrs after NPs-eGFP plasmid DNA IV injection.

[0076] FIG. 38A shows green fluorescence (or change in grey shade after green removal during image processing) protein fluorescence signals brain tissue distribution example data for two-month-old young mice48 hrs after NPs-eGFP plasmid DNA IV injection. FIG. 38B shows green fluorescence protein fluorescence signals brain tissue distribution example data for ten- month-old older mice 48 hrs after NPs-eGFP plasmid DNA IV injection. FIG. 39A shows green fluorescence protein fluorescence signals brain tissue distribution example data for the hippocampus 48 hrs after NPs-eGFP plasmid DNA IV injection. FIG. 39B shows green fluorescence protein fluorescence signals brain tissue distribution example data for the cortex. FIG. 40A shows immunohistochemistry of green fluorescence protein brain tissue distribution analysis of thalamus (T) on the left and hippocampus (HC) on the right. FIG. 40B shows immunohistochemistry of green fluorescence protein brain tissue distribution analysis of the cortex. FIG. 40C shows immunohistochemistry of green fluorescence protein brain tissue distribution analysis of the cerebellum.

[0077] FIG. 41 shows a proposed mechanism for tunneling nanotubes (TNTs) mediated partial cell fusion (left) and permanent cell fusion (right). FIG. 42A shows NPs receptor SLC7A1 mRNA distribution. FIG. 42B shows mTOR mRNA distribution. FIG. 43 shows super-imposing SLC7A1 and mTOR mRNA distribution areas.

[0078] FIG. 44 shows SEQ ID NO: 1 , a sequence of antisense oligonucleotide used to knock down Malat

[0079] FIG. 45 shows an example diagram of self-assembly of Nanopieces with the ASO cargo.

[0080] FIG. 46A and FIG. 46B show mouse protocols (and ID numbers) for experiments.

[0081] FIG. 47A shows a saggittal cut bisects the hemispheres of the brain, from which the hippocampus was isolated. FIG. 47B shows the right hemisphere was divided via three equispaced cuts along the coronal axis.

[0082] FIG. 48 illustrates how brain dissection of miR-146a knockout mice was performed based on major anatomical regions.

[0083] FIG. 49 shows example primer sequences used for qPCR analysis, namely, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0084] FIG. 50A shows the extent of MALAT1 knockdown for olfactory lobe after IV injection of NPs-MALATI ASO in miR-146a- / - mice. FIG. 50B shows the extent of MALAT1 knockdown for cerebral hemisphere 1 after IV injection of NPs-MALAT1 ASO in miR-146a- / - mice. FIG. 50C shows the extent of MALAT1 knockdown for cerebral hemisphere 2 after IV injection of NPs- MALATI ASO in miR-146a- / - mice. FIG. 50D shows the extent of MALAT1 knockdown for cerebral hemisphere 3 after IV injection of NPs-MALAT1 ASO in miR-146a- / - mice. FIG. 50E shows the extent of MALAT1 knockdown for the superior and inferior colliculus after IV injection of NPs-MALAT1 ASO in miR-146a- / - mice. FIG. 50F shows the extent of MALAT1 knockdown for the cerebellum after IV injection of NPs-MALAT1 ASO in miR-146a- / - mice. FIG. 50G shows the extent of MALAT1 knockdown for the spinal cord after IV injection of NPs-MALATI ASO in miR- 146a- / - mice. * p<0.05, **p<0.01 , ***p<0.001.

[0085] FIG. 51 A shows the extent of MALAT1 knockdown for olfactory lobe two weeks after IV injection of NPs-MALATI ASO in miR-146a- / - mice. FIG. 51 B shows the extent of MALAT1 knockdown for cerebral hemisphere 1 two weeks after IV injection of NPs-MALAT1 ASO in miR- 146a-Z- mice. FIG. 51C shows the extent of MALAT1 knockdown for cerebral hemisphere 2 two weeks after IV injection of NPs-MALAT1 ASO in miR-146a- / - mice. FIG. 51 D shows the extent of MALAT 1 knockdown for cerebral hemisphere 3 two weeks after IV injection of NPs-MAU\T 1 ASO in miR-146a- / - mice. FIG. 51E shows the extent of MALAT1 knockdown for the superior and inferior colliculus two weeks after IV injection of NPs-MALAT1 ASO in miR-146a-Z- mice. FIG. 51 F shows the extent of MALAT1 knockdown for the cerebellum two weeks after IV injection of NPs- MALATI ASO in miR-146a- / - mice. FIG. 51 G shows the extent of MALAT1 knockdown for the spinal cord two weeks after IV injection of NPs-MALAT1 ASO in miR-146a- / - mice. * p<0.05, **p<0.01 , ***p<0.001.

[0086] FIGs. 52A-52I each show Malatl knockdown in the non-brain regions of miR-146a- / - mice over the timespans 3 days, 1 week, and 2 weeks (mean ± SEM; 3, 4, 5 mice per group, respectively) each for different organs labeled above each plot. * p<0.05, **p<0.01, ***p<0.001. FIG. 52A shows data for heart. FIG. 52B provides data for lung. FIG. 52C is data for stomach. FIG. 52D shows data for kidney. FIG. 52E provides data for liver. FIG. 52F is spleen data. FIG. 52H provides the plot for small intestine. FIG. 52I is data / plot for transversus abdominusmuscle.

[0087] FIGs. 53A-53I show Malatl knockdown in the non-brain regions of miR-146a- / - mice two weeks after injection compared to wild-type C57BL / 6 mice (mean±SEM, 5 and 3 mice per group, respectively) each for different organs labeled above each plot. * p<0.05, **p<0.01 , ***p<0.001. FIG. 53A shows data for heart. FIG. 53B provides data for lung. FIG. 53C is data for stomach. FIG. 53D shows data for kidney. FIG. 53E provides data for liver. FIG. 53F is spleen data. FIG. 53H provides the plot for small intestine. FIG. 53I is data / plot for transversus abdominus muscle..

[0088] It should be understood that while different illustrations are sometimes used in some of the figures above to describe different embodiments and different aspects of the technology, any aspect from any figure can be inter-combined with an aspect from any other figures. Any example disclosed herein can be inter-combined with any other. All trademarks, images, likenesses, words, and depictions in the drawings and the disclosure are plainly in fair use and are provided solely for the purposes of illustration of the invention in view of an urgent need to prevent injuries and to treat subjects as further discussed in more detail below.DETAILED DESCRIPTION OF THE INVENTION

[0089] The subject innovation is now described, in some examples with reference to the drawings, wherein examples can used to refer to the aspects of the breadth of concepts of the invention. In the following description, for purposes of explanation, specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.DEFINITIONS

[0090] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention can be determined by the claims. 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 invention belongs. If there is an apparentdiscrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0091] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0092] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0093] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0094] As used herein, a “range” may be provided. A statement may include “in the range from about A to about B”. All points from A to B are subsumed by the range, and all those points can define preferred ranges. Within said range, any range subsumed therein means any range that is within the stated range. Endpoints within the range can define a new range. For example, the following are all subsumed within the range of about 10 to about 50. 10 to 20; 15 to 35; 23 to 40; or 50 to 31; or any other range or set of ranges within the stated range. As such, within the range any set of endpoints subsumed therein can be used as an exemplary range.

[0095] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Any method described herein can be claimed and / or described as a composition and vice versa.

[0096] The term "consisting of" as it is known in the practice refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0097] As used herein the term "consisting essentially of' refers to those elements requiredfor a given embodiment The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. In specific examples, “consisting essentially of” can be explained herein for each example or can be defined broadly, for example, by stating that an administration to a subject (in a method herein) does not include any other active pharmaceutical ingredient or therapeutic agent in addition to the one specified. In another example, the term “consisting essentially of can be utilized to indicate a nanocarrier and a therapeutic agent with no other ingredients that are listed in a claim and yet including any other ingredients that are not specifically listed. That is, due to the life-saving advantages the technology herein presents, the term “consisting essentially of” can be utilized to exclude any additional ingredient that can be discussed (or listed); for example, a claim can be directed to a J BAA nanoparticle for delivering a therapeutic agent across a blood- brain-barrier or other tissue barrier, the J BAA nanoparticle consisting essentially of: (1) a Janus Base with Amino Acid (JBAA) molecule, (2) a therapeutic agent, and wherein the therapeutic agent is inside or partially inside the JBAA nanoparticle, and wherein the JBAA nanoparticle is capable of crossing the blood-brain-barrier or other tissue barrier to deliver the therapeutic agent to a target cell and this same claim can include a “proviso”, list or condition within that any ingredient ( / .e., that can be discussed or listed) is not included in the composition while retaining the condition that any other ingredient that is not listed can be included with the composition. This stipulation is intended to save lives by application of this technology. Thus, the technology is not to be subjected to limitations that could be normally applied to strict specific definitions and / or examples required for “consisting essentially of” (e.g., the need to define specific examples in this specification) so that increased lives are saved via applications of the Invention’s claims.

[0098] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference. The term “feature” and the term “detail” can be interchanged with a “claim”. Any list of features, details, examples, embodiments, and / or aspects herein can be placed into a “claim”.

[0099] As used herein, the term "subject" refers to a mammal, bird, or the like, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), racing mammals, laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex. In another example, the term “subject” can refer to a connective tissue culture, and the methods disclosed herein, while claimed towards subjects,contemplate use in the laboratory in synthetic tissue(s). As used herein, a female cell can refer to a cell with 2X chromosomes; a male cell can refer to a cell with 1X and 1Y chromosome.

[0100] As used herein, the terms "effective amount" and “therapeutically effective amount” include an amount sufficient to modulate a treatment or prevent or ameliorate a manifestation of disease or medical condition, such as a connective tissue condition or a risk of a connective tissue injury. Such a condition (or risk) may not be readily discernable and may take years, statistical analysis, and / or machine learning to determine a prevention, treatment, or amelioration. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the condition and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0101] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, manage, modulate, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect (undesirable characteristic) or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized ( / .e., not worsening) state of a condition or decay, delay or slowing of a progression and / or risk of injury, and an increased lifespan / enjoyment as compared to that expected in the absence of treatment.

[0102] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. The therapeutic agent or drug may refer to a formulation, composition, or agent. The formulation can be changed to a fresh formulation during administration. This includes that the therapeutic agent or drug is administered such thatit is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long- acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1 , 2, 3, 5, 7 or 10 years, or more.

[0103] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by any technique known in the art that is subsequently applied to a subject, topical application, absorption, injection, ingestion, transfusion, implantation or transplantation. In an example embodiment, compositions are applied as a tablet or drug in capsule. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subdermal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. It is known in the art that therapeutic agents can be rapidly deployed through the skin and directly into joint / ligaments by use of DMSO (dimethyl sulfoxide) as a carrier solvent applied (with the therapeutic agent) to the skin near to or surrounding a joint. While DMSO is rarely used anymore for these purposes because of its nature as a universal solvent and its tendency to carry any residual chemicals present on the skin into the bloodstream (along with the intended agent), the technology contemplates such uses. In one contemplated embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tissue, lymph node, or site of treatment. In another example, administration is provided in the form of a natural product, vitamin, supplement, food, aerosol, inhalation, vapor, or drink. Formulations disclosed herein can be ready made or require mixing just before administration.

[0104] The words “JBAA nanoparticle”, “nanoparticle”, “nanopiece”, and the letters “NP” are used interchangeably herein. Any of the methods disclosed herein can be carried out in part or completely by including a dietary change, a food, natural product, precursor, or prodrug of a therapeutic agent. As used herein, a precursor or a prodrug is intended to encompass compounds or therapeutic agents which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., a compound for any of the present claims or features). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments,the prodrug is converted by an enzymatic activity of the host subject. For example, esters or carbonates (e.g., esters or carbonates of alcohols or of carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the small-molecule chemical structures selected from this disclosure can be replaced with the corresponding suitable prodrug, for example, wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. A common method of making a precursor / prodrug that can be used herein is to use a carrier / nanocarrier (e.g., mesoporous silica particles). The precursor / prodrug can be released from a carrier to form the active therapeutic agent. A precursor or prodrug can be metabolized to the active parent compound (therapeutic agent) in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid). No argument can be made that the term “prodrug” is not enabled herein based on an assertion that actual prodrugs were not made and tested.

[0105] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0106] The terms: “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a markeror symptom, a “increase” is a statistically significant increase in such level.

[0107] “Janus Base with Amino Acid" refers to a synthetic molecule designed to covalently link the structure of a DNA base pair with an amino acid, where one side is a standard covalently linked DNA base pair like adenine and thymine or cytosine and guanine, and the other side is an amino acid side chain, creating a distinct "Janus-faced" structure with two different functionalities on a single molecule; this design is used in nanomaterials like "Janus Base Nanotubes" for targeted drug delivery or other biomedical applications due to the ability to interact with both nucleic acids and biological systems through the amino acid component. As used herein, the term: “small molecule” refers to a molecule that has a molecular weight < 1000. As used herein, the term: “large molecule” refers to a molecule that has a molecular weight > 1000, and the term includes biologies such as the examples of oligonucleotides, peptides, antibodies, linkers, oligosaccharides, polymers, DNA chains, and RNA chains. The term: “therapeutic agent” may refer to small molecule, element, large molecule, biologic, formulation, composition, agent, or a combination thereof.PHARMACEUTICAL COMPOSITIONS

[0108] The compositions and methods of the present invention may be utilized to prevent a need for other treatment, to provide benefit when other treatment(s) fail, or to treat an individual in need thereof. In some embodiments, the individual is suspected of needing treatment. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. A compound can represent a combination therapy herein. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration ( / .e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g.,a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0109] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0110] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0111] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible compositions employed in pharmaceutical formulations.

[0112] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally, for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue; absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermal administration (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. Inhalation can include inhalation of a liquid (droplets or aerosol). Inhalation can include a micronized powder adhered to carrier particles or can be without carrier particles. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541 ,231 , 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0113] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0114] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound (or combination therapy) of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0115] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules,or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0116] To prepare solid dosage forms for oral administration (capsules, including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0117] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0118] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example,hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the abovedescribed excipients.

[0119] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0120] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0121] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0122] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0123] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0124] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0125] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0126] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0127] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0128] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0129] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0130] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0131] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0132] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non- degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0133] Actual dosage levels of the active ingredients in the pharmaceutical compositions maybe varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0134] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0135] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher, et al., (1996).

[0136] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0137] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0138] The subject or patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; bird such as poultry; and pets in general.

[0139] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0140] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1 H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2- naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4- acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts. In some embodiments, an N-H in elraglusib can become charged and a corresponding salt formed at or near the charge.

[0141] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization,inherent in the solvent of preparation or crystallization, or adventitious to such solvent The invention contemplates polymorphs, cocrystals, and amorphous forms of all substances discussed herein. As discussed above, solvates and / or hydrates can be formed by, for example, a slow evaporation whereby water and / or solvent remain hydrogen bonded with OH groups in the molecule. The formation of a sol vate / hyd rate can be quickly confirmed after the evaporation by using attenuated total reflectance Fourier transform infra-red spectroscopy (ATR-IR) wherein the solid solvate / hydrate is directly placed on the instrument and the subsequent IR spectrum is compared to the IR spectrum of the solid non-solvate, non-hydrate. Any of the constructs herein can be utilized with solvation, targeting moieties, hydration, and / or with lipid formulations. No reasonable argument can be made that a solvate and / or hydrate is not enabled herein because specific chemical compositions were not made. It is known in the art that an ATR-IR can be utilized to rapidly make a solvate and / or hydrate.

[0142] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0143] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0144] As discussed above, unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy; The Encyclopedia of Molecular Cell Biology and Molecular Medicine; Molecular Biology and Biotechnology: a Comprehensive Desk Reference; Immunology; Janeway's Immunobiology; Lewin's Genes XI; Molecular Cloning: A Laboratory Manual.; Basic Methods in Molecular Biology;Laboratory Methods in Enzymology; Current Protocols in Molecular Biology (CPMB); Current Protocols in Protein Science (CPPS); and Current Protocols in Immunology (CPI).

[0145] In the embodiments discussed and in any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0146] It is important to note that the terms “tissue barrier”, “other tissue barrier”, “blood-tissue barrier” and any terms referring to a barrier within a subject refer to any barrier within a subject that can prevent entry of a therapeutic agent into a cell or prevent a therapeutic agent from contacting a cell thus the technology can be utilized to overcome any pharmacological barrier within a subject in the spirit of the Invention (namely to save lives) and this disclosure is not to be construed as to limit the Invention by the semantics or words utilized herein. For example, it is known to an ordinary person that the COVID- 19 pandemic (also known as the coronavirus pandemic and COVID pandemic), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), required, to save human lives, a fast development of an mRNA vaccine with a delivery formulation and it is known to a person of skill that the technology disclosed herein could be utilized in the same scenario. Furthermore, the technology disclosed herein can improve expression (e.g., of nucleic acids in a human) efficiencies even though this clinical data is on the horizon ( / .e., prophetic). Other terms are defined herein within the description of the various aspects of the invention or are used as would be understood by an ordinary person.MATERIAL AND METHOD OF NUCLEIC ACID DELIVERY CROSSING THE BLOOD-BRAIN BARRIER AND OTHER BLOOD-TISSUE BARRIERS:

[0147] Disclosed now below are increasing levels of details for materials and methods of nucleic acid delivery, for example, in crossing the blood-brain barrier and other blood-tissue barriers. Any of the details discussed below can be inter-combined with features, aspects, embodiments, and / or examples disclosed above. In an example introduction to details, FIG. 1 shows an example schematic of nanopieces crossing the BBB by utilizing the cationic amino acid transporter CAT1 (SLC7A1). In this schematic, FIG. 1 is modified from Zaragosa, Transport of Amino Acids Across the Blood Brain Barrier, doi: 103389 / fphys.2020.00973; wherein NPs are nanopieces, JBAK is Janus Base with amine and lysine (K), and NA is representing nucleic acid. In FIG. 1, CAT1 / y+ are facilitative Na+ independent cationic amino acid (Lys, Arg, Orn)transporters in the BBB. Amino acids (AAs) are transported by systems L and y+ from blood to ECs and then into the brain. These two systems are located at both sides of the cell membrane. However other systems will also be present but exclusively at the luminal side of the BBB. The location of these transporters and their main substrates is depicted in the example of FIG. 1.

[0148] FIG. 2A and FIG. 2B show schematics of nanopieces endocytosis (FIG. 2A) and phagocytosis (FIG. 2B) by targeting amino acid transporter SLC7A1. In FIG. 2A is a schematic diagram showing the proposed mechanism for cell entry of the smaller-sized siRNA NPs and the larger-sized pDNA NPs is shown in FIG. 2B. While the smaller siRNA NPs enter cells via receptor mediated endocytosis (FIG. 2A), the larger pDNA NPs enter cells via phagocytosis (FIG. 2B). Since phagocytosis is limited to macrophage, immune cells, and other specialized cells, NPs gene delivery may be utilized for immunotherapy and / or targeted delivery to these specialized cells.

[0149] The blood-brain barrier is formed by the brain capillary endothelium, which acts effectively to protect brain tissue from circulating pathogens and other potentially toxic substances. However, it excludes 100% of large-molecule neurotherapeutics including biologicals and more than 98% of all small-molecule drugs from the brain (Pardridge, NeuroRx 2005). Overcoming the difficulty of delivering therapeutic agents to the brain presents a major challenge to treatment of most brain disorders including Alzheimer’s disease. Direct injections of the CNS, including intrathecal and intracerebroventricular injections, are highly invasive (and carry high risk). The only way drugs or genes can be distributed widely in the brain is the transvascular route following injection into the blood stream or other transport routes connected with the blood stream. However, this transvascular route requires the drug’s ability to undergo transport across the BBB. Viral delivery is the most used means to deliver nucleic acid therapeutics (e.g. gene therapy and editing) and oligonucleotides (e.g. RNAi) across the BBB. However, it has shortcomings or pose safety concerns including cell-type targeting, biodistribution, immunogenicity, loading capacity and cost.

[0150] We developed herein novel methods for delivering cargos including nucleic acid and oligonucleotides across the BBB using rosette nanotubes-mediated nanoparticle self-assembly termed Nanopieces (NPs). NPs consist of non-covalent assembly of cargos with biomimetic small molecules of Janus Base with Amino Acid (JBAA). It is safe, cost effective and has tunable cargo loading capacity. However, it was not known whether and how NPs can carry cargos to cross the BBB in a safe and non-toxic manner. The current invention discloses the new technology with nanomaterial, method and formulations that enable NPs to cross the BBB by transcytosis of capillary endothelial cells that form the blood-tissue barrier including the BBB. The inventiondescribes specific assembly conditions of J BAA with cargos, under which NPs can target specific membrane receptor of capillary endothelial cells. A specific example is presented for this enabling technology. NPs derived from the J BAA with lysine are able to carry nucleic acid cargos including plasmid DNA and oligonucleotides across the BBB by utilizing SLC7A1(CAT1), a cation amino acid transporter including lysine in the solute carrier (SLC) membrane receptor family. The described technology enables endocytosis and / or phagocytosis of NPs to allow shuttling of cargos across the BBB to reach the protected inner CNS tissues in the brain. The cargos may include DNA, RNA (mRNA, rRNA, tRNA, siRNA, microRNA, IncRNA), and oligonucleotides including native and modified forms. The cargos can be in single, double, and multiple strands, with linear, circular and other secondary configurations, for diagnostics and therapy. NPs achieve enhanced CNS bioavailability throughout the brain without immunogenicity, inflammation, and safety concerns of viral or other non-viral vectors. Since NPs have tunable cargo loading capacity, it can be potentially adapted for delivering cargos of varying sizes. Thus, NPs have the potential of becoming a platform technology for gene therapy delivery after commercialization.

[0151] The invention described herein can be used for delivering therapeutics and diagnostics across the blood-tissue barriers including the BBB. The nucleic acid delivery technology infiltrating the BBB can be used for the purposes of gene expression, gene targeting, gene regulation, gene therapy, gene deletion or attenuation, gene editing, gene modification, RNA editing, RNA knockdown, RNA interference, RNA expression, RNA stimulation or attenuation, RNA modification, RNA degradation, RNA targeting, protein targeting, protein expression, protein modification, protein degradation, and protein modulation. Such delivery material and methods can be used to target specific cell populations, lineages and differentiation (activation) stages, both inside the brain and in the peripheral tissues and organs. In addition to BBB, such method and material can also be used for cargo delivery across other blood tissue barriers including blood-retina, blood-placenta, blood-testis, blood-thymus, blood-bile, and the glomerular filtration barrier in the kidney.

[0152] The central idea is that the optimal size, shape, and charge of NPs are critical to enable its infiltration across the BBB by utilizing the amino acid transporter in the endothelial cells, endocytosis into the CNS cells that express the transporter in the plasma membrane, and release the cargos inside the cells, thereby achieving significant therapeutic effects on modulating gene expression.

[0153] We characterized various parameters of the interaction of nucleic acid (or oligonucleotides) and Nanopieces thereby optimizing their incorporation conditions and deliveryefficiency across the BBB and into the CNS cells. We determined the optimal nanomaterial formulation for gene delivery and sustained release. We performed in vitro efficacy studies in neuronal cell lines to obtain the optimal formulation combining at least two critical parameters: 1) NT pH value and 2) the NT / DNA (or Oligo) ratio.

[0154] We characterized the optimal size, shape, and charge of NP for delivering oligonucleotides cargos including anti-sense oligos (ASO), siRNA, microRNA, and other single or double strand, RNA or DNA no more than 200 nucleotides (or base pairs). An example of the morphology and chemical properties of Oligo-NP is shown in FIG. 3A. FIG. 3A shows example properties of oligonucleotide nanopieces assembly (Oligo-NP).

[0155] We assessed pharmacokinetics and bioavailability of NP gene delivery in the mouse brain. We provided rigorous evidence that NP gene delivery is BBB penetrant by demonstrating successful NPs gene delivery in mouse brain via intravenous (IV) administration and by obtaining brain delivery and safety data in vivo.

[0156] We determined the optimal Nanopieces formulation condition of the plasmid DNA encoding CMV / GFP DNA. Since the formulation condition is DNA sequence independent, such formulation condition is suitable for plasmid DNA or RNA of larger than 200 nt or bp, either linear or circular.

[0157] We determined the optimal pH of NT / DNA assembly: 50pL 1mg / ml_ JBAK NTs were assembled with 2 pg GFPpDNA (4.48kb) to form NPs under a series of pH conditions. The pH of JBAK NT solution studied were 3.0, 3.4, 3.6, 3.8, or 4.2 (NOTE: NPs cannot be formed at pH<3.0 or pH>4.2.). Then, the assembled NPs were measured via zeta potential for overall charge, DLS for size, TEM for morphology and UV-vis for packing. The optimal pH condition was selected in terms of forming nano-rod shape with strong zeta-potential. We determined that the optimal pH condition for NP assembly into nano-rod shape with strong zeta-potential is pH 3.2-3.4.

[0158] We determined the optimal ratio for NT / DNA assembly: After we obtained the optimal pH condition for NP assembly, we tested formation of NPs with different ratios of JBAK NT vs. GFPpDNA (4.48kb). 10, 15, 20, 30, and 50 pL 1mg / mL JBAK NTs was assembled with 2.0 ug DNA under the optimal pH in JBAK NT solution. Then, the assembled NPs were measured via zeta potential for overall charge, DLS for size, TEM for morphology and UV-vis for packing. We determined that the optimal ratio of JBAK NT vs. pDNA for NP assembly into nano-rod shape with strong zeta-potential is 50pg JBAK for 2 pg GFPpDNA.

[0159] We called the NT / DNA assembly process Scaffold-Assisted DNA NanopiecesAssembly (SA-DNA). An example of the morphology and chemical properties of SA-DNA is shown in FIG. 3B. FIG. 3B shows example properties of nanotube scaffold assisted DNA nanopieces assembly (SA-DNA).

[0160] Data and descriptions are developed and provided for Developing Nanopieces Formulations to Transfect Cells for Gene Therapy, for Specific Nanopieces Formulations that are Required for Cell Transfection of Plasmid DNAs of Different Lengths, for NPs gene transfection targeting cells expressing high levels of cation amino acid transporter CAT-1 encoded by the SLC7A1 gene, for Validation of SLC7A1-mediated NPs transfection in vitro and in the CNS in vivo, for NPs targeting immune cells including microgial cells and macrophages in the brain and peripheral tissues, and also for NPs Gene Delivery and PK in the brain of normal mice and Alzheimer’s Disease (AD) mouse models.

[0161] We determined the transfection efficiency of optimized NP formulation: We transfected HEK293T cells, one of the most popular cell lines for transfection (e.g., CAR-T), with optimized NP formulations. Lipofectamine transfection of plasmid DNA was used as control. Fluorescent microscopy demonstrated that the Nanopieces transfection efficiency is (35%+12%) for pCMV / GFP DNA, as shown in FIG. 4 (of HEK293T cells). FIG. 4 shows example microscopy of human 293T cells transfected with Nanopieces formulation of CMV / GFP plasmid DNA for 72 hours. Left, phase contrast cell morphology, Middle, GFP fluorescence (green is light grey due to color removal in patent applications); the right panel shows a merge image.

[0162] Microscopy and flow cytometry demonstrated the time course of transfection, and that transfection for 72 hrs was required for achieving optimal transfection efficiency of Nanopieces (FIG. 5). FIG. 5 shows example transfection efficiency of human 293T cells transfected with Nanopieces formulation of CMV / GFP plasmid DNA for 24, 48, and 72 hours. n=3. ****p<0.0001.

[0163] We determined the conditions for processing plasmid DNA Nanopieces (NP pDNA) with ultrasound without breaking covalent bonds within DNA. To do this, we tested sonication at different intensities (Amplitude 1 to 10) for different time periods (1 to 5 minutes). As shown in FIG. 6, processing at Amp 10 for any time periods would break plasmid DNA into fragments. When we processed the plasmid DNA at Amp 4, while processing for 1 minute did not break DNA, processing for 2.5 to 5 minutes did. In addition, vortexing DNA for 1 to 5 minutes did not break DNA. Therefore, our standard protocol of pDNA NP processing included sonication at Amp4 for 1 minute to process the noncovalent bond of JBAK nanotubes without breaking the covalent bonds of DNA. It was followed by mixing the pDNA NP by vortexing. FIG. 6 shows an example of how sonication degrades DNA in a dosage dependent manner while vortexing does not;electrophoresis of CMV / GFP plasmid DNA processed by ultrasound and vortex at different amplitudes for different time periods. The final Nanopieces formulation sonication condition which does not break plasmid DNA into fragments is highlighted in a red ( / .e., darker grey) box.

[0164] The length of the GFP gene with CMV promoter plasmid DNA is about 4.5 kb. An AAV vector has a cloning limitation of DNA cargo of less than 4.6 kb. Thus, NPs can serve as a non- viral alternative delivery vehicle for gene therapy across the BBB. We expect Nanopieces to carry and transfect a plasmid DNA longer than 4.6 kb with longer NPs. AAV, the current gold standard for gene therapy, cannot carry such a larger plasmid DNA.

[0165] Specific Nanopieces Formulations Are Required for Cell Transfection of Plasmid DNAs of Different Lengths. We not only obtained the optimal non-viral Nanopieces (NPs) formulation conditions for plasmid DNAs up to 4.5kb, but also tested a series of NPs formulation conditions for plasmid DNAs larger than 4.5kb. As described in the above section, we obtained the optimal NPs formulation containing two critical parameters: 1) Nanotube (NT) pH value and 2) the NT / DNA ratio, to achieve successful gene expression in vitro.

[0166] Since the NPs formulation condition is DNA sequence independent, such formulation is suitable for any plasmid DNAs larger than 200bp but smaller than 4.5kb. These plasmid DNAs include pCMV-GFP (vector: 3.45kb, insert: 1.03kb, total pDNA length: 4.48kb) and pCMV-APOE2 (vector: 3.45kb, insert: 0.954kb, total pDNA length: 4.404kb). For the ease of observation under the microscope and quantification of transfection rates, we chose to use pCMV-GFP in our testing.

[0167] We determined the optimal nanomaterial formulation for NPs delivery of genes larger than 4.6kb. For the in vitro work, the resulted NP is tested for their transfection efficiency in HEK283 cells and an astrocyte cell line isolated from mouse CNS. The data of these experiments are described as follows.

[0168] Four DNA plasmids of different lengths were tested: FIG. 7A shows pCMV-GFP (4.48kb); FIG. 7B shows pmiRFP670 (4.96kb); FIG. 7C shows pCMV3-C-OFPSpark-Kpn1-Xba1, or pAPOE-RFP for short (7.64kb), and FIG. 7D shows pCMV3-C-GFPSpark, or pAPOE-GFP for short (7.68kb). All four plasmids share several (non-limiting examples) common features. First, their transcription ability is the same since all of them have the same promoter (CMV). Second, the sizes of the encoded proteins (APOE, GFP, and RFP) are very similar, since all the cDNA insert sizes are between 954-1030 bp. Third, the observation and quantification methods are the same since all of them can be visualized with fluorescent markers. These commonalities eliminated various factors that might influence transfection outcomes. The only major differencewas the size of the plasmid DNA. While the first construct (pCMV-GFP) is less than 4.6kb, the second construct (pmiRFP670) is larger than 4.6kb but smaller than 5kb. The third (pAPOE-RFP) and fourth (pAPOE-GFP) are more than 7.5kb, much larger than 4.6kb (FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D).

[0169] In general, FIG. 7A shows a first example plasmid DNA tested for non-viral gene therapy by Nanopieces delivery. FIG. 7B shows a second example plasmid DNA tested for non- viral gene therapy by Nanopieces delivery. FIG. 7C shows a third example plasmid DNA tested for non-viral gene therapy by Nanopieces delivery. FIG. 7D shows a fourth example plasmid DNA tested for non-viral gene therapy by Nanopieces delivery.

[0170] To determine the gene delivery difference between the first plasmid (pCMV-GFP) and the second plasmid (pmiRFP670), we formulated both plasmids with the optimized NPs formulation condition as described in the last section above. After NPs formulation, the zeta potential of the first construct reached more than 6. However, the zeta value of the second construct was only around 2 (FIG. 8). It indicated that a larger sized plasmid might require formulation improvement to reach the optimal chemical surface property. To test this idea, we optimized NPs formulation of the two larger plasmids including the third construct (pAPOE-RFP, 7.64kb) and the fourth construct (pAPOE-GFP, 7.68kb). The optimized formulation was obtained by testing two series of critical parameters: 1) Nanotube (NT) pH value and 2) the NT / DNA ratio. We were able to obtain the optimal NPs formulation conditions for the third and fourth plasmids that enabled their zeta values to reach the acceptable range (more than 2) (FIG. 8).

[0171] FIG. 8 shows Zeta potentials of the four example plasmids (see FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D) in Nanopieces formulation.

[0172] Next, we tested transfection efficiency by transfecting the DNA plasmids of different length in NPs formulation into HEK293T cells, one of the most popular cell lines for transfection (e.g., CAR-T). Lipofectamine transfection of plasmid DNA was used as a transfection control. For the plasmid DNA encoding CMV-GFP DNA (4.48kb), NPs formulation enabled DNA transfection in a time dependent manner (FIG. 9). It reached robust transfection after 48- and 72-hours transfection (FIG. 9).

[0173] FIG. 9 shows microscopy of human 293T cells transfected with Nanopieces formulation of pCMV-GFP plasmid DNA for 24, 48, and 72 hours. Left, GFP fluorescence, Middle, phase contrast cell morphology in brightfield, Right, Merge.

[0174] The NPs transfection rate was at the same level as that of the commercialLipofectamine, i.e., lipid nanoparticle (see FIG. 10). Thus, we achieved excellent transfection rates with optimized NPs formulation conditions for DNAs.

[0175] FIG. 10 shows example transfection efficiency of human 293T cells transfected with Lipofectamine, NPs formulation of pCMV-GFP plasmid DNA (NP_pCMV_GFP), NPs formulation of pmiRFP plasmid DNA (NP_pmiiRFP) and naked plasmid DNA only (pDNA only). The transfection rates were quantified after transfection for 48 hours. n=3.

[0176] We tested two series of parameters including the NT pH value and the NT / DNA ratio to reach the optimal zeta potentials for the two large plasmids pAPOE-RFP (7.64kb) and pAPOE- GFP (7.68kb) (FIG. 8). Thus, it is possible to formulate large DNA to reach ideal chemical surface conditions for NPs. However, the two large plasmids in NPs formulation only achieved less than 1 % transfection rate compared to the more than 30% transfection rate of the smaller plasmid pCMV-GFP (FIG. 11). All these plasmids had similar zeta potentials (FIG. 8). Thus, both zeta potential and the size of plasmid are critical parameters to influence the outcome of transfection. FIG. 11 shows example transfection rates of three NPs formulated plasmids in human 293T cells after transfection for 48 hours. n=3.

[0177] Example problems and potential solutions: an example problem is what prevents a plasmid DNA larger than 4.5kb from achieving a robust transfection rate despite a good zeta potential? It is interesting that AAV viral transfection has a similar DNA cargo length limit of 4.6kb while lipid nanoparticles (LNP) do not. While LNP enters the plasma and nuclear membrane through fusing lipid bilayers, NPs and AAV enter cells via receptors. It is possible that while lipid fusion does not have a cargo limit, receptor mediated cell and / or nucleus entry may have a size limit. It was thought that the cargo size limit of AAV was due to the size limit of the spherical viral particle. NPs do not have such size limit because of its nanorod shape and tunable lengths. NPs can pack a larger DNA to form a longer nanorod for delivery. The excellent zeta values of the NPs of large plasmids supports this hypothesis. Despite the successful NPs formulation, gene expression level of large DNA was still low. It suggests that DNA cargo size is an important parameter for receptor-mediated non-viral gene delivery as well. Even if NPs transfection has a similar DNA cargo size limit as AAV, it still has advantages since it is non-viral gene delivery, which is a safer, easier and cheaper technology.

[0178] We showed that Nanopieces successfully transfected plasmid DNA in HEK293T cells. We also discovered that Nanopieces (NPs) transfection of DNA did not work in all cell types, indicating NPs transfection is cell specific. The astrocyte cell line tested was C8-D1A, purchased from ATCC. It is an astrocyte cell line generated from mouse cerebellum. While lipofectamine(Lipo) transfection of plasmid DNA resulted in more than 20% transfection rate in C8-D1A cells after 72h transfection, Nanopieces (NPs) transfection resulted in only 0.25% transfection rate (FIG. 12). This indicated that NPs transfection mechanism is different from that of Lipid Nanoparticles (LNP), which did not have cell specificity.

[0179] FIG. 12 shows example transfections of GFP plasmid DNA (pDNA) in C8-D1A astrocyte cell line by Lipofectamine or Nanopieces. Left panel: Microscopic images of cell transfection. Top panels: Green fluorescence (or change in grey after color removal) of cells transfected with pDNA by Lipo or NP. Bottom panels: Phase contrast images of cell morphology of C8-D1A. Right panels: Lipofectamine transfection efficiency (%), and Nanopieces transfection efficiency (%) in C8-D1A. n = 3.

[0180] We determined NPs gene expression in C8-D1A cells, which had a lower transfection rate than HEK293T cells. In contrast, the Lipo transfection rates were the same between C8-D1A and HEK293T cells. While Lipo transfection increased EGFP mRNA for more than 500,000-fold (FIG. 18A), NPs transfection increased more than 100-fold (FIG. 18B). Thus, the gene expression levels of Lipo transfection were 3-magnitudes higher than NPs transfection in C8-D1A cells (FIG. 18C).

[0181] FIG. 18A shows examples of transfection of EGFP plasmid DNA (pDNA) in C8-D1A cells by Lipofectamine or Nanopieces. EGFP mRNA levels normalized to 18S RNA after Lipofectamine transfection (A). FIG. 18B shows data of FIG. 18A after Nanopieces transfection. FIG. 18C shows a comparison of EGFP mRNA levels after Lipo and NPs transfection, n = 3, P<0.05.

[0182] Since Lipo transfection was mediated by lipid membrane fusion while NPs transfection was mediated by cell receptor, the low gene expression levels of NPs transfection could be due to a smaller number of C8-D1 A cells expressing the cellular receptor or C8-D1 A cells having lower amount of receptor in each cell. Our analysis indicated that only 0.25% C8-D1A cells expressing the recombinant EGFP after NPs transfection (FIG. 19B), while around 20% C8-D1A cells were positive after Lipo transfection (FIG. 19A). Further analysis indicated that, for each positively transfected cell, the gene expression level was fifty times less in NPs than Lipo transfected cells (FIG. 19C).

[0183] FIG. 19A shows examples of transfection of EGFP plasmid DNA (pDNA) in C8-D1A cells by Lipofectamine or Nanopieces. Transfection efficiency after Lipofectamine transfection (A). FIG. 19B shows data of FIG. 19A after Nanopieces transfection (B). FIG. 19C shows acomparison of EGFP mRNA levels per cell after Lipo and NPs transfection, n = 3, P<0.05.

[0184] In contrast, the NP transfection rate was more than 20% in HEK293T cells after 72h transfection (FIG. 14A), which was identical to the Lipo transfection rate (see FIG. 10). This suggests that NP transfection has cell specificity while Lipo transfection does not. Lipofectamine transfects cells through fusion with the lipid bilayer of the plasma membrane, thus lacking cell specificity. On the other hand, NP transfection may be mediated by the interaction between lysine amino acid residues on the surface of NPs with a cell membrane receptor. This putative receptor- mediated endocytosis would result in NPs transfection in the cells that express the NPs receptor, but not in the cells that do not express the NPs receptor. It is known that there are five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Thus, it is possible that while one cell line, such as C8-D1A, expresses a low level of the NP receptor, other types of astrocytes may express high levels of the NPs receptor, leading to the cell specific gene delivery by NPs.

[0185] NPs gene transfection targets cells expressing high levels of cation amino acid transporter CAT-1 encoded by the SLC7A1 gene. To identify the cell receptor for NPs, we hypothesized that the cell receptor for NPs is a lysine amino acid transporter. There are two major classes of amino acid transporters capable of transporting lysine. The first is L-amino acid transporter (LAT1 , or SLC7A5) and the second is cationic amino acid transporter 1 (CAT-1 , or SLC7A1). There are strong candidates because both have been shown to transport essential amino acids to the neuronal cells in the CNS by crossing the blood brain barrier.

[0186] To test whether LAT1 is a NPs receptor, we inhibited LAT1 in 293T cells by adding a LAT 1 specific inhibitor KYT053 in a series of concentrations. The LAT 1 inhibitor did not inhibit NP transfection since the fluorescence resulted from pDNA transfection did not decrease when the LAT1 inhibitor concentration was increased (FIG. 13). Furthermore, the NPs transfection rate was not decreased by LAT1 inhibition for all three time periods tested (FIG. 14A). These data suggested that LAT1 is not a NPs receptor. FIG. 14B shows an example small-molecule LAT1 inhibitor, JPH 203.

[0187] FIG. 13 shows microscopy of human 293T cells transfected with Nanopieces formulation of GFP plasmid DNA in the absence (OnM) or in the presence of LAT1 inhibitor (KYT053, 10nM, 100nM, and 1000nM). Top panels, GFP fluorescence of pDNA transfection by NPs, Bottom panels, phase contrast cell morphology. Images were taken 48 hours post transfection.

[0188] FIG. 14A shows transfection efficiency of human 293T cells transfected with NPs formulation of GFP plasmid DNA in the absence (OnM) or presence of LAT1 inhibitor for 24, 48, and 72 hours. n=3. * p<0.0001.

[0189] To test whether CAT-1 is a NPs receptor, we transfected the plasmid DNA in CHO-K1 cells, which lack CAT-1 expression. Lipofectamine transfected pDNA in CHO-K1 cells in a timedependent manner (FIG. 15A, Lipo-pDNA). It reached more than 20% transfection rate after 72h transfection, the same as its transfection rate in 293T cells and C8-D1A cells (compare FIG. 12). This confirmed that lipofectamine transfection is not cell specific. However, Nanopieces failed to transfect pDNA into CHO-K1 cells that lack CAT-1 expression (FIG. 15A, NP_pDNA). It strongly suggests that NPs transfection of cells requires CAT-1 expression. To further test the requirement of CAT-1 for NPs transfection, we tested NP transfection in wildtype 293T cells and the SLC7A1- KO 293T cells in which the CAT-1 gene has been knocked out by CRISPR-CAS9 gene editing. NPs transfected wildtype 293T cells in a time-dependent manner (FIG. 15B, Wild Type), as we demonstrated previously (FIG. 14A). However, NPs transfection was greatly and significantly reduced in SLC7A1-KO 293T cells (FIG. 15B, SLC7A1-KO). Thus, SLC7A1 is necessary for NPs transfection in cells. These data demonstrated that CAT-1 (SLC7A1) is a cell receptor for Nanopieces transfection.

[0190] FIG. 15A shows transfection rates (%) of pDNA formulated with Lipofectamine (Lipo_pDNA), Nanopieces (NP_pDNA), and nothing (pDNA only) in CHO-K1 cells that lack CAT- 1 expression. Post-transfection period: 24, 48, and 72 hours. n=3, **** p<0.000001. FIG. 15B shows transfection rates (arbitrary units) of NP_pDNA in wild-type 293T cells that express SLC7A1 (Wild Type) and SLC7A1 knock-out 293T cells (SLC7A1-KO). Post-transfection period: 24, 48, and 72 hours. n=3, **** p<0.00001.

[0191] NPs failed to transfect certain mouse astrocyte such as C8-D1A at a high efficiency (FIG. 12). There is a heterogeneity of astrocytes in the mouse CNS with each type of astrocyte having a different transcriptome. In addition, mouse astrocytes may be different from those of human, which is the ultimate target of the therapeutic under development. It is not possible to test whether NPs can transfect every type of human and mouse CNS cells, since most of these cell lines are not available for in vitro testing. For example, the C8-D1 A cell line was derived from only one cell in mouse cerebellum, while different types of astrocytes are widely distributed in different regions of the brain including cortex and hippocampus. To understand the cell specificity for NP transfection, we identified the specific receptor for NPs cell transfection. We identified CAT-1 , encoded by the SLC7A1 gene, as the NPs receptor for cell transfection. CAT-1 is a cationic aminoacid transporter responsible for transporting lysine, arginine, and histidine into the cells and crossing the blood brain barrier. SLC7A1 is highly expressed in the BBB and neuronal cells including astrocytes in the CNS. This strongly suggests a cell transfection mechanism of NPs in which CAT-1 interacts with the lysine residues on the surface of NPs, thereby enabling NPs to cross the BBB and enter the neuronal cells. Furthermore, CAT-1 has been shown to be a cell receptor for leukemia virus. Thus, CAT-1 can facilitate entry of a nucleic acid containing nanoparticle into cells.

[0192] We hypothesize that the failure of NPs to transfect some cells was due to the low level of expression of SLC7A1 . This hypothesis is tested here using a rat fibroblast glioma cell line C6, which is resistant to NPs DNA transfection (FIG. 16A). The pattern of the low NPs transfection rates in C6 cells mimics those in CHO-K1 cells that lack SLC7A1 expression (FIG. 15A). To address whether SLC7A1 is down-regulated in C6 cells, we generated SLC7A1 specific primers to detect its expression by real-time RT-PCR reactions. It shows that, while 293-T cells express abundant SLC7A1 , C6 cells only express 30% and CHO-K1 express 0% of SLC7A1 mRNA. Thus, NPs DNA transfection correlates with the abundance of SLC7A1 in the cell. The implication of this finding is that we can predict whether certain cells are susceptible to NPs gene therapy based on their SLC7A1 expression profile. NPs gene therapy can be targeted specifically to the cells that express SLC7A1(CAT-1).

[0193] FIG. 16A shows examples of low transfection rates of pDNA formulated with Nanopieces (NP_pDNA-GFP), and Control (pDNA only) in C6 cells that lack the high expression of NPs receptor SLC7A1. Post-transfection period: 24, 48, and 72 hours. n=3. FIG. 16B shows examples of SLC7A1 mRNA levels in HEK293-T cells that are susceptible to NPs gene transfection and in C6 and CHO-K1 cells that are resistant to NPs transfection. mRNA levels are quantified by real-time RT-PCR. n=3, **** p<0.001. ** p<0.01.

[0194] Validation of SLC7A1 -mediated NPs transfection in vitro and in the CNS in vivo. as discussed above, we showed that Nanopieces (NPs) successfully transfected plasmid DNA (pDNA) in HEK293T cells at a more than 20% transfection rate. This transfection rate was identical to the pDNA transfection rate of lipofectamine (Lipo). Thus, NP transfection of pDNA was equivalent to that of lipid nanoparticle (LNP), the gold standard for non-viral gene delivery to cells. However, NPs transfected the mouse cerebellum astrocyte cell line C8-D1A at a lower transfection rate (0.25%) while Lipo transfection rate of pDNA remained the same (20%) in C8- D1A cells. This observation strongly suggested that NPs transfection had cell specificity while Lipo transfection did not. Such transfection specificity, if true, can be used to target specific cellsor tissues for gene delivery. The mechanistic study to reveal the cell and tissue specificity of NPs gene delivery is described as now follows. Gene expression efficiency by NPs transfection was 26 times higher than Liposome transfection in HEK293T cells that express high levels of SLC7A1. For example, FIG. 17A shows transfection of EGFP plasmid DNA (pDNA) in HEK293T cells by Lipofectamine or Nanopieces. EGFP mRNA levels normalized to 18S RNA after Lipofectamine transfection. FIG. 17B shows an example from FIG. 17A after Nanopieces transfection. FIG. 17C shows a comparison of EGFP mRNA levels after Lipo and NPs transfection; n = 3, P<0.05.

[0195] As described previously, the transcription rate of EGFP pDNA was the same between NPs and Lipo in HEK293T cells, which is the most used cells for manufacturing recombinant protein in vitro. To determine the gene expression levels after transfection, we quantified EGFP mRNA levels using real-time RT-PCR (FIG. 17A, FIG. 17B, FIG. 17C). While Lipo transfection increased EGFP mRNA for more than 4000-fold (FIG. 17A), NPs transfection increased more than 100,000-fold (FIG. 17B). Thus, NPs gene expression efficiency was 26 times higher than LNP (FIG. 17C), the gold standard of non-viral transfection and production of recombinant proteins in cell culture.

[0196] These data strongly suggest that a smaller number of C8-D1A cells expressed the NPs receptor, which resulted in a lower transfection rate than HEK293T cells. In addition, each C8-D1A susceptible to NPs transfection expressed the NPs receptor at a lower level, which led to a lower gene expression level per cell. Thus, analyses of NPs receptor distribution pattern and abundance could be the key to understanding the tissue and cell specific properties for NPs gene delivery. Such analyses and their implications are described as follows. NPs receptor SLC7A1 distribution patterns in brain and other tissues in vivo is illustrated in FIG. 20. FIG. 20 shows an example summary of SLC7A1 distribution in human tissues (upper left), in human brain (upper right), in male and female (see lower panels). Red (or darker grey) shade represents SLC7A1 expression areas. From the Human Protein Atlas (ProteinAtlas.org).

[0197] As presented above, we made a discovery that NPs cell transfection is mediated by a cell receptor SLC7A1 . SLC7A1 is a high affinity, low-capacity permease involved in the transport of the cationic amino acids (arginine, lysine, and ornithine) in non-hepatic tissues. NPs would be recognized by a cation amino acid transporter, since NPs were full of lysine on its surface. Lysine may serve as a contact group for SLC7A1 on the cell surface to grab NPs for the entry of the NPs nanoparticle, thereby carrying nucleic acid into the cells. This is very much like the spike protein on the corona virus to bind its cellular receptor ACE2, which mediates the viral infection of human cells. Indeed, SLC7A1 has been identified to be a cellular receptor for retrovirus including bovineleukemia virus, which is capable of infecting human cells, especially human immune cells and epithelial cells. Thus, NPs enter cells like viruses in a receptor-mediated fashion, carrying nucleic acid cargos (RNA or DNA) to infect cells. Unlike viruses, NPs cannot self-replicate, which is advantageous for its safety during drug delivery.

[0198] Analyses based on The Human Protein Atlas indicated that the most abundant tissues expressing NPs receptor SLC7A1 are brain and skin (see FIG. 20). This is true for both male and females (FIG. 20, grey and darker grey or red shaded body areas). Within the human brain, SLC7A1 is expressed in every tissue with low region specificity (FIG. 21 , lightest bars at left or yellow bars, and FIG. 22). This suggests that NPs can be used to target brain for gene delivery since its receptor SLC7A1 is highly expressed in brain tissues. In addition to brain, SLC7A1 is also highly expressed in esophagus, pancreas, and skin (FIG. 21), suggesting that NPs can be used for targeting these tissues outside of brain as well. FIG. 21 shows an example summary of SLC7A1 mRNA distribution in human brain tissues (lighter 14 bars at left, or yellow bars), and in other human tissues as depicted by different colors. From the Human Protein Atlas (ProteinAtlas.org).

[0199] We encountered a problem that NPs failed to transfect a mouse cerebellum astrocyte cell line C8-D1A at a high efficiency (FIG. 18A, FIG. 18B, FIG. 18C; and FIG. 19A, FIG. 19B, FIG. 19C). This was surprising, since the NPs receptor SLC7A1 was expressed in all human brain tissues including cerebellum with low tissue specificity (see FIG. 20, FIG. 21 , and FIG. 22).

[0200] FIG. 22 shows an example SLC7A1 distribution in human brain tissues. Left, different human brain tissues are indicated by different colors. Right, SLC7A1 mRNA levels in corresponding brain regions. From the Human Protein Atlas (ProteinAtlas.org).

[0201] One possible explanation could be that SLC7A1 distribution patterns in mouse brain were different from human brain. Indeed, RNA analysis of mouse brain tissues indicated that SLC7A1 was expressed at the lowest level in mouse cerebellum among all brain tissues (FIG. 23, dark yellow bar or cerebellum bar).

[0202] FIG. 23 shows example SLC7A1 distribution in mouse brain tissues. Left, different mouse brain tissues expressing SLC7A1 are indicated by red shade (or a change in grey due to color removal in application processing). Right, SLC7A1 mRNA levels in corresponding brain regions. From the Human Protein Atlas (ProteinAtlas.org).

[0203] To determine whether the low expression of NPs receptor in cerebellum contributed to the deficient NPs delivery outcome in the region, we delivered an anti-sense oligonucleotide(ASO) of a long noncoding RNA MALAT-1 , which was ubiquitously expressed in all CNS cells, with NPs via IV (e.g., intra-venous) in mice (FIG. 24). Two weeks after tail vein injection, mouse brain was partitioned into seven regions including cerebellum (FIG. 24). RNA was extracted and MALAT-1 RNA levels were quantified by real-time RT-PCR. MALAT-1 was knocked down significantly in every brain region except cerebellum (FIG. 24, see “CB”). This observation was consistent with the hypothesis that SLC7A1 was expressed at a low level in mouse cerebellum, which contributed to the low NPs ASO delivery.

[0204] FIG. 24 shows example MALAT1 RNA levels in seven regions of mouse brain after in vivo NPs / ASO delivery via IV for two weeks. All values were adjusted against 18S RNA control. Blank: Injection of solution without NPs / ASO. CH: cerebral hemisphere. In FIG. 24, “OL” is olfactory lobe; “CH1” is cerebral hemisphere 1; “CH2” is cerebral hemisphere 2; “CH3” is cerebral hemisphere 3; “SIC” is superior and inferior colliculus; “CB” is data for the cerebellum; and “SC” is data for the spinal cord.

[0205] The GFPpDNA was used in our experiments to identify GFP positive cells in the brain and other tissues upon IV injection. If the green fluorescent cells were detected in the NPs IV injected mice but not in the mock (no NPs) injected mice, it would indicate that not only NPs deliver GFPpDNA into the tissue in vivo, but also the delivered gene was transcribed into mRNA and translated into protein, which emits green fluorescence in the body. In addition to in vivo fluorescent imaging, we also performed real-time RT-PCR to quantify GFP mRNA levels in NPs delivered tissues. Since mice do not contain or express endogenous GFP gene, the detected GFP mRNA and protein in mice are solely from NPs delivery of GFPpDNA through IV injection.

[0206] NPs target immune cells including microglial cells and macrophages in the brain and peripheral tissues: We test different formulations to identify the ideal formulation for NPs / DNA delivery in vivo. We accomplished this goal by formulating and testing the NPs formulation to achieve successful delivery of NPs / DNA via IV in mice. The detailed experiment, data, and conclusion are presented as follows.

[0207] NPs specifically target microglial cells for gene delivery in the brain: We performed NPs / GFPpDNA delivery in two-month-old female mice (n=8). The administration route was intravenous (IV). Three days after IV injection, mice were sacrificed, and brain tissues were dissected into three regions (FIG. 25) for examining GFP florescence at cellular levels, while RNA of the other half of the brain was extracted and quantified for the expression levels of GFP mRNA by real-time quantitative RT-PCR.

[0208] FIG. 25 shows an example diagram of how after IV injection of eGFP plasmid DNA (pDNA) in Nanopieces formulation for three days, mice were sacrificed before the brain was dissected into three regions as indicated. Half of the brain was sectioned, and the other half was extracted for total RNA isolation transfection, n = 8.

[0209] Florescent microscopy demonstrated that GFP florescence was detected in microglial cells in the mid-brain (FIG. 26), but not in mock injected mice (no NPs injection).

[0210] FIG. 26 shows (in left panel): dark field fluorescent microscopy detected fluorescent microglial cells in the mid-brain. Note the presence of fluorescent GFP (as light grey) in the cytoplasm next to the nucleus; FIG. 26 shows in the right panel: microglial cell morphology, which is distinct from neuronal cells and astrocytes.

[0211] Real-time PCR analysis indicated that GFP mRNA levels were significantly increased in the mid-brain but not in the front or back brain (FIG. 27A, FIG. 27B, FIG. 27C).

[0212] FIG. 27A shows examples of real-time quantitative RT-PCR analysis of eGFP mRNA levels in the front brain. FIG. 27B shows data from the mid-brain (B), and FIG. 27C shows data from the back brain (C). The successful NPs delivery of GFPpDNA resulted in the increase of GFP mRNA in the mid-brain. Control: mice were injected with mock injection solution without NPs in IV. Treated: mice were injected with NPs / eGFPpDNA formulation in IV. RNA was isolated from the brain three days after IV injection, n - 3. **P<0.01. ns: p>0.05.

[0213] We observed GFP gene expression in microglial cells. This indicates that NPs gene delivery targeted microglial cells in the brain. To understand the cell and molecular basis of the microglial cell specificity for NPs delivery, we examined the expression of NPs receptor SLC7A1 in different types of CNS cells in the human brain. Human Protein Atlas indicates that, among neuronal and glial cells in the CNS, SLC7A1 is highly expressed in microglial cells specifically (FIG. 28). This suggests that the NPs receptor SLC7A1 facilitates NPs delivery to microglial cells in the brain.

[0214] FIG. 28 shows single cell RNAseq analysis in the Human Protein Atlas indicates that SLC7A1 is highly expressed in microglial cells (center arrow) among different types of CNS cells in the human brain. Lighter grey bars (yellow bars) at the arrow indicate the express levels of SLC7A1 in the neuronal cells in the CNS. Brown bars (darker grey bars at right) indicate SLC7A1 expression levels in different glial cell populations in the CNS. From the Human Protein Atlas (ProteinAtlas.org).

[0215] NPs specifically target macrophages for gene delivery via IV: in addition to the braintissues, SLC7A1 is highly expressed in esophagus (e.g., see FIG. 21). Therefore, we examined esophagus tissue sections of the NPs IV-injected mice to determine the type of cells susceptible to NPs gene delivery. Strong GFP florescence is observed in large and multinucleated macrophages on the surface of esophagus (FIG. 29A, FIG. 29B). The axe-shaped macrophages are seen inserted into and engulfing the submucosa tissue for tissue remodeling (FIG. 29A). Multiple GFP-positive macrophages were observed in esophagus. Real-time quantitative RT- PCR analysis confirmed the significant upregulation of GFPmRNA in the esophagus of NPs / GFPpDNA IV injected mice than mock injected mice (FIG. 29C). We did not observe any types of GFP positive cells except macrophages in esophagus. This suggests that NPs target gene delivery to macrophages specifically. We also observed targeted macrophage-specific gene delivery of NPs in other tissues such as stomach and skeletal muscle in addition to esophagus. These observations suggest that NPs target macrophages for gene delivery systematically.

[0216] FIG. 29A shows macrophage-specific expression of IV delivered NPs / GFPpDNA in esophagus — H&E histology of mouse esophagus with large, multi-nucleated, and axe-shaped macrophages on the surface of esophagus tissue undergoing remodeling. FIG. 29B shows dark field microscopy demonstrates green florescence (or change in grey) derived from GFPpDNA expression in macrophages. FIG. 29C shows real-time quantitative RT-PCR analysis indicates the significant increase of eGFP mRNA levels in the esophagus of NPs / GFPpDNA injected mice (treated) than the mock injected mice (control) three days after IV injection. n=3. *p<0.05.

[0217] NPs target tissue-resident macrophage for gene delivery: Since microglial cells are resident macrophages in the brain, we investigated whether other tissue-resident macrophages are targeted by NPs for gene delivery. Kupffer cells are tissue-resident macrophages in the liver. We observed GFP florescence signals around hepatocytes along sinusoids in the liver. However, hepatocytes were negative for GFP by themselves (FIG. 30A, FIG. 30B). Comparison between Kupffer cells staining (FIG. 30A) and GFP florescence patterns (FIG. 30B) indicate the two patterns overlap. It suggests that NPs target Kupffer cells for gene delivery and expression in the liver. Real-time quantitative RT-PCR analysis indicated that the GFP mRNA levels were increased more than 120-fold in the liver of the NPs / GFPpDNA IV injected mice (FIG. 31). Taken together, these data confirm the successful gene delivery targeting tissue-resident macrophages in the liver.

[0218] FIG. 30A shows examples of how NPs target liver resident-macrophage Kupffer cells for delivery and expression of GFPpDNA. FIG. 30A shows histology of the liver showing hepatocytes (purple cells with nuclei), sinusoids between hepatocytes, and brown stain labeledKupffer cells along the sinusoids and between hepatocytes. FIG. 30B shows dark field fluorescence microscopy showing GFP fluorescence in the Kupffer cells along the sinusoids and between hepatocytes, coinciding with the distribution patterns of brown stained Kupffer cells in H&E section in FIG. 30A. FIG. 31 shows real-time quantitative RT-PCR analysis indicates the significant increase of eGFP mRNA levels in the liver of NPs / GFPpDNA injected mice (treated) than the mock injected mice (control) three days after IV injection. n=3. ***p<0.001.

[0219] Our data presented here demonstrated that NPs / pDNA delivery target macrophages and tissue-resident macrophages including microglial cells in the CNS for gene delivery. However, our previous data of NPs / siRNA delivery indicated that NPs / siRNA were delivered to other types of non-immune cells too. To answer this question, we increased the size of NPs / siRNA from its regular size of smaller than 200nm (FIG. 2A) to a larger size of more than 500nm, which matches with the size of NPs / pDNA (FIG. 2B). The larger sized NPs / siRNA target macrophages specifically, like NPs / pDNA. Such larger sized NPs / TNFa-siRNA targeted macrophages specifically, thereby knocking down TNF-a mRNA levels in the inflamed joint of rheumatoid arthritis mouse model for more than 95% (data not shown). Thus, we propose the NPs cell targeting mechanisms as follows. There are two types of cell entry mechanisms for NPs. For the smaller sized NPs (<200nm), they enter the cell via receptor-mediated endocytosis (FIG. 2A). It requires SLC7A1 only, thereby targeting macrophages and other types of cells expressing SLC7A1. For the larger sized NPs (>500nm), they enter the cell via phagocytosis (FIG. 2B). In addition to SLC7A1 , it may also require SLC7A2. SLC7A2, especially SLC7A2B, has been shown to play a critical role in activating macrophage and regulating macrophage polarization into M1 and M2 (FIG. 32A). While M1 is pro-inflammatory macrophage responsible for killing cells, M2 promotes cell proliferation and facilitates tissue repair (FIG. 32B). While macrophages in autoimmune diseases are pro-inflammatory M1 , the tumor associated macrophages (TAM) are M2. Thus, inducing M1 polarization is a key approach to immunotherapy of cancer while inducing M2 polarization is a key feature of anti-inflammatory therapeutics for treating autoimmune diseases.

[0220] FIG. 2A shows the schematic diagram showing the proposed mechanism for cell entry of the smaller-sized siRNA NPs (Left panel, A), and FIG. 2B shows the larger-sized pDNA NPs. While the smaller siRNA NPs enter cells via receptor mediated endocytosis (FIG. 2A), the larger pDNA NPs enter cells via phagocytosis (FIG. 2B). Since phagocytosis is limited to macrophages and other immune cells, NPs gene delivery may be specific for immunotherapy.

[0221] FIG. 32A illustrates how NPs can be used for immunotherapy targeting macrophagesor tissue-resident macrophages (microglial cells, and Kupffer cells etc.). In FIG. 32A: SLC7A1 and SLC7A2 are critical for transporting arginine to activate macrophage polarization (M1 and M2). FIG. 32B illustrates how regulating M1 and M2 polarization is critical for the treatment of cancer, autoimmune diseases, and age-related degenerative diseases including Alzheimer’s Disease. By sharing the same transporter with arginine, NPs gene delivery can be used for targeting macrophages and reprograming macrophage polarization via macrophage-specific gene delivery.

[0222] As a specialized population of macrophages-like cells in the CNS, microglia are considered as immune sentinels that can orchestrate a potent inflammatory response. Proliferation and activation of microglia in the brain, concentrated around amyloid plaques, is a prominent feature of AD. Human genetics data point to a key role for microglia in the pathogenesis of AD. Most risk genes for AD are highly expressed (and many are selectively expressed) by microglia in the brain. There is mounting evidence that microglia protect against the incidence of AD, as impaired microglial activities and altered microglial responses to p-amyloid are associated with increased AD risk. On the other hand, there is also abundant evidence that activated microglia can be harmful to neurons. Thus, it is critical to develop macrophage-targeted immunotherapy to induce M1 or M2 polarization of microglia at the specific stages of AD pathogenesis respectively. If fully developed, NPs may serve as novel microglia-targeted immunotherapy for AD treatment.

[0223] We performed NPs gene delivery via IV. NPs successfully target macrophages and tissue-resident macrophages in different tissues for gene delivery. However, the extent of gene delivery and expression varies between tissues. For example, the NPs gene delivery is 120-fold in liver, 1.7-fold in esophagus, and 1.2-fold in the brain. The different delivery rate may be determined by the number of macrophages in each tissue. On the other hand, it may depend on the metabolic state of cells, which regulate the expression levels of amino acid transporters including SLC7A1 and SLC7A2.

[0224] NPs Gene Delivery and PK in the brain of normal mice and Alzheimer’s Disease (AD) mouse model: as described above, we tested NPs delivery to CNS in both WT and AD mice at early as well as late stages of AD pathogenesis in the current period. In addition, we performed brain distribution, metabolism, and safety studies following in vivo nucleic acid delivery via IV. We accomplished these goals through a series of in vivo studies in young and older adult mice of both normal wildtype (WT) and APP / PS1 Alzheimer’s Disease (AD) mice. The detailed experiment, data, and conclusion are presented as follows.

[0225] NPs nucleic acid delivery in vivo: time course, metabolism, and safety: one of the important research goals is to perform metabolism and safety studies following NPs IV delivery in vivo. To accomplish that, we chemically labeled JBAK nanotubes with a 750nm wavelength fluorophore and an oligonucleotide (siRNA with scrambled nucleotide sequence) with a 650nm wavelength fluorophore respectively (FIG. 33). Labeling of the two components of NPs with fluorophores at different wavelengths enabled us to track them separately in the body after IV injection. We also used an oligonucleotide as cargo instead of plasmid DNA for this experiment. Both oligonucleotide and plasmid DNA belong to the nucleic acid category. However, it is more difficult to track a chemically modified plasmid DNA than an oligonucleotide because of the difference in their length. Formulation of chemically labeled siRNA and nanotubes resulted in dual fluorescently tagged NPs. This is shown as a single peak of the NPs of 157nm in size (FIG. 33).

[0226] FIG. 33 illustrates dual chemical labeling of fluorescently tagged Nanopieces (NPs) for time course and metabolism studies. In the left panel: JBAK nanotubes (NT) were chemically labeled with a 750nm wavelength fluorophore (750 nm FL) indicated by blue (darker grey) circles. Nucleic acid cargos (siRNA) were chemically labeled with a (650 nm FL) indicated by yellow (lighter grey) circles. In the middle panel: Formulation of siRNA with nanotubes resulted in the assembly of the dual fluorescently tagged NPs. In the right panel: NPs presented a uniform peak at 157 nm length.

[0227] After injection of dual-labeled NPs into IV for 24 hours, both siRNA signals (red or lighter grey) and JBAK nanotube signals (green or darker grey) were detected by Fluorescence Molecular Tomograph (FMT) (FIG. 34B). However, we failed to detect any fluorescence signals after IV injection of siRNA alone for 24 hrs. (FIG. 34A). This indicated that, without NPs delivery, siRNA cannot cross BBB to infiltrate the brain by itself. Interestingly, while the NT (green or darker grey) signals were detected throughout the brain after IV injection for 24hrs, the siRNA (red or lighter grey) signals were detected mainly in the middle part of the brain, overlapping with the NT green signals resulting in orange signals in the middle of the brain (FIG. 34B). This suggests that siRNA was delivered and sequestered in the CNS cells in the middle of the brain. On the other hand, JBAK green (or darker grey around periphery) signals were detected in the periphery of the brain independently without siRNA red (or lighter grey) signals (FIG. 34B). This suggested that, after NPs delivery of siRNA in the middle part of the brain, JBAKs were dissociated from siRNA and diffused into the periphery of the brain during the release process from the brain (FIG. 34D, FIG. 35A, and FIG. 35B). After NPs injection via IV for 72hrs, the siRNA positive areas were expanded in the middle of the brain (FIG. 34C), indicating that siRNA continued to beaccumulated in the brain during 72hrs after IV injection. JBAK green signals continued to be detected at the periphery of the brain, indicating the release of JBAKs from the brain after siRNA delivery. One week after NPs IV injection, neither JBAK NT (green) nor siRNA (red) was detected in the brain. This suggests that siRNA and JBAK were metabolized and cleared from the brain in one week after NPs delivery of nucleic acid cargo in the middle of the brain.

[0228] To determine the time course of the two components of NPs, JBAK NT and siRNA, during circulation, we quantified their content in blood and urine after NPs IV injection (FIG. 35A, FIG. 35B). The blood content of JBAK NT and siRNA peaked at 24hrs, indicating the presence of NPs in blood circulation after IV injection. Interestingly, the JBAK content in blood started to decline before siRNA (FIG. 35A, red or grey bars). The peaks of JBAK NT and siRNA content in urine followed those in the blood (FIG. 35B, blue or darker grey bars). They indicated that JBAK and siRNA were released out of the body through urine after they were metabolized and degraded. The mice were healthy and well throughout the one-week testing period. The blood work, and the liver and brain toxicity analysis did not reveal any significant toxicity associated with JBAK. Finally, genetic toxicity (ADME-Tox) analysis was performed by Eurofins labs. It indicated that the JBAK compound AAT is not mutagenic up to 10 pM in Ames assays. Since the therapeutic dosage of JBAK is less than 2.5nM, the JBAK concentration needs to increase 4000 times to have mutagenic effects.

[0229] Metabolism of dual labeled NPs in the mouse brain is illustrated. FIG. 34A shows siRNA injection alone via IV, brain fluorescent image was taken after 24 hrs. No siRNA signal (lighter grey or red, 650nm FL) was detected. FIG. 34B shows NPs injection via IV. Brain fluorescent image was taken after 24 hrs. Both siRNA signals (lighter grey, red) and Nanotube (NT) signals (darker grey, previously green in color images, 750nm FL) were detected. Overlapping siRNA and NT signals were orange. FIG. 34C shows NPs injection via IV for 72 hrs. Overlapping siRNA and NT signals expanded in the brain. FIG. 34D shows NPs injection via IV after a week. Neither siRNA nor NT signals were detected in the brain, indicating NPs degradation and clearance from the brain. N=4 in each group. Representative images were shown.

[0230] NPs targeted gene delivery in the brain is tissue-area specific, AD enhanced, and age independent: Fluorescent Molecular Tomography (FMT) analysis indicated that NPs targeted the mid-brain areas for gene delivery (FIG. 36B). On the other hand, blood vessels were distributed evenly on the surface of the cerebral hemispheres (FIG. 36C). Although the triangle tissue area in the mid-brain, including the superior and inferior colliculus (FIG. 36A), is devoid of blood vessels (FIG. 36C), it contained the strongest JBAK signals in the brain (FIG. 36B). Thissuggested that J BAK NPs target specific brain areas for delivery, rather than through passive diffusion-generated concentration gradients after crossing the BBB. The NPs targeted area corresponds to Cortex, Hippocampus & Thalamus Margin, and Midbrain in the Middle part of the mouse brain (FIG. 37A).

[0231] Transport, metabolism, and clearance of dual labeled NPs in mouse blood and urine are illustrated. FIG. 35A shows data for blood (lighter grey or red bars) and FIG. 35B shows data for urine (darker grey or blue bars) were collected after NPs were injected via IV for 24hrs, 72hrs, and 1 week. Control: no NPs injection. JBAK NT (dashed bars in the top panels) was detected via a 750nm wavelength fluorophore. siRNA (solid bars in the bottom panels) was detected via a 650nm wavelength fluorophore. JBAK NT clearance from the blood is followed by siRNA clearance at 72hrs, indicating the completion of NPs delivery into the tissues via blood transport (FIG. 35A). The clearance of JBAK and siRNA through urine occurred at 72hrs and 1 week respectively, indicating NPs degradation and excretion from the body at 1 week, n = 6. P values were indicated for experimental samples that have significant difference from the Control sample.

[0232] Fluorescently labeled JBAK indicates that the NPs targeted tissue areas are in the middle of the brain. FIG. 36A shows gross appearance and tissue areas of a mouse brain. FIG. 36B shows fluorescent molecular tomography of a mouse brain after IV injection of fluorescently labeled NPs for 72 hrs. Red signals (change in grey near center of image) indicate the NPs targeted tissue areas with the highest accumulation of JBAKs. FIG. 36C shows blood vessel distribution on the surface of the mouse brain (red arteries and blue veins). (Xiong et al., 2017).

[0233] FIG. 37A shows A. mouse brain parts and containing areas. Front Part: 1. Olfactory Bulb (OB). 2. Cortex (CT). Middle Part: 3. Hippocampus (HC). 4. Hippocampus & Thalamus Margin (H&T). 5. Thalamus (TM). 6. Hypothalamus (HTM). 7. Midbrain (MB). Back Part: 8. Cerebellum (CB). 9. Pon / Medulla / Spinal Cord (PMS). FIG. 37B shows eGFP mRNA levels in Front, Middle, and Back regions of the mouse brain. n=3. *p<0.05. **p<0.01. ns: p>0.05. FIG. 37C shows 18S RNA levels in Front, Middle, and Back regions of the mouse brain. n=3. *p<0.05. **p<0.01. ns: p>0.05.

[0234] To determine whether NPs gene delivery depends on age, disease state, and brain tissue areas, we performed a detail analysis to locate NPs gene delivery areas in the brain in both WT and AD mice in two different age groups. At two-month age in young adult mice, there is no p-amyloid plaques in the APP / PS1 AD mice while p-amyloid plaques are present in the 10-month older AD mice. In 10-month-old AD mice, the eGFP mRNA level is the highest in the middle partof the brain after IV injection of NPs / eGFP plasmid DNA for three days (FIG. 37B). This is consistent with the brain FMT imaging data showing that NPs target the mid-brain areas for brain delivery (FIGs. 34A-34D and FIGs. 36A-36C). The housekeeping 18S ribosome RNA levels were the same across the three brain areas, indicating that the targeted NPs delivery in the mid-brain areas did not affect the cellular ribosome RNA levels (FIG. 37C). This is consistent with the above safety data that NPs do not cause cellular toxicity in the tissue areas targeted by NPs gene delivery. The Middle part of the brain contains hippocampus (HC), Hippocampus & Thalamus Margin (H&T), Thalamus (TM), Hypothalamus (HTM) and Mid-brain (MB) (FIG. 37A).

[0235] To determine which brain areas are targeted by NPs for gene delivery, we cryosectioned the brain 72hrs after IV injection of NPs / eGFPpDNA. Fluorescence microscopy was performed to quantify the GFP protein expression in each tissue areas. In 2-month-old young mice, GFP was mainly detected in the middle part of the brain in both WT and AD mice (FIG. 38A). Low levels of GFP were also detected in the front part of the brain (FIG. 38A). This suggests that NPs mainly target the middle part of the brain for gene delivery. In 10-month older mice, NPs still target H&T tissue area in the middle part of the brain for delivery in WT mice, like the young mice (FIG. 38A and FIG. 38B). This suggests that the NPs targeted brain delivery is age independent. However, we observed a great increase of NPs gene delivery in the AD brain in 10-month-old mice, especially in the Middle part of the brain (FIG. 38B). It suggests that NPs gene delivery to the brain is enhanced by the AD phenotype.

[0236] Data is provided for quantification of eGFP expression in different parts (Front, Middle, and Back) of a mouse brain three days after IV injection of NPs / eGFPpDNA. FIG. 38A shows data for two-month-old young mice. FIG. 38B shows data for ten-month-old older mice. AD: APP / PS1 double transgenic mice. WT: wildtype littermate mice. See FIG. 37A for keys to each brain tissue area. Female mice were used in the study. Non-specific autofluorescence based on the control sample (no NPs / GFPpDNA delivery) was subtracted from the real fluorescence of the experimental samples (NPs / GFPpDNA delivery). n=4.

[0237] To determine which cells in CNS were targeted by NPs delivery, we performed side- by-side analyses of fluorescent microscopy and brain tissue histology of the NPs-targeted tissue areas in 10-month-old AD mice. In Hippocampus, GFP expression is associated with small-sized microglial cells (FIG. 39A). These microglial cells surrounded an AD lesion area comprising a group of neuronal cells with interstitial matrix (FIG. 39A). In the cortex tissue of the Middle part of the brain, GFP was expressed in a giant cell containing a fusion of multiple cells including a small microglial cell (FIG. 39B). Thus, NPs target multi-nucleated fusing cells for gene delivery in thebrain.

[0238] NPs targeted eGFP gene expression in mouse brain tissues after IV injection of NPs / eGFPpDNA for 72 hrs. In FIG. 39A and FIG. 39B, fluorescent microscopy of eGFP expression is shown on the left and H&E histology is shown on the right. FIG. 39A shows data for the hippocampus. Arrows point to GFP positive microglial cells surrounding an AD lesion area. FIG. 39B shows data for the cortex. A fused multi-nucleated cell containing a microglial cell is indicated by a red circle (or grey after red removal).

[0239] Immunohistochemistry analysis with an antibody against eGFP showed that, three days after a single IV administration of 2 pg NPs-eGFP plasmid DNA in a young AD mouse, the recombinant eGFP protein is distributed across the blood vessels between the thalamus and hippocampus (FIG. 40A). Numerous cell foci that express the eGFP gene are distributed throughout brain tissue regions including the thalamus and hippocampus (FIG. 40A), cortex (FIG. 40B), and cerebellum (FIG. 40C).

[0240] NPs targeted eGFP gene expression in mouse brain tissues after IV injection of NPs / eGFPpDNA for 72 hrs. Immunohistochemistry analyses with an antibody against eGFP recombinant protein in the brain of 2-month-old AD mice three days after a single IV injection of NPs-eGFP plasmid DNA are provided. FIG. 40A shows analysis of thalamus on the left and hippocampus on the right. Blood vessels stained positive for eGFP are seen at the boundary between thalamus and hippocampus. FIG. 40B shows analysis of the cortex. Numerous cell foci are seen expressing the eGFP gene product. FIG. 40C shows analysis of the cerebellum. Positive signals for eGFP gene product are seen around the blood vessels and in cell foci throughout cerebellum. Arrows: eGFP positive signals in and around blood vessels. Arrow heads: eGFP positive signals in cell foci in different regions of the brain.

[0241] We determined the mechanism by which NPs target a fusion cell containing macrophage-lineage cells. We observed such targeting of macrophage-lineage fusion cell not only in brain tissues as described above but also in tissues outside of the brain (see the last progress report). Recent studies have shown that nanotubes called the tunneling nanotubes (TNTs) mediate cell fusion with macrophages (FIG. 41). In tumors, TNTs mediate cancer cell fusion with macrophages resulting in advanced-stage tumor cells with a metastatic / invasive phenotype (FIG. 41). Thus, we postulate that NPs target macrophage fusion cells through nanotubes (NTs), which are morphologically similar to the TNTs. If so, NPs can target advanced stage cancer cells thereby providing a novel targeted immunotherapy for genetic treatment of advanced cancer. Because of the ability of NPs to infiltrate the BBB, the NPs gene therapy isparticularly suitable for brain cancers such as Glioblastoma (GBM), a devastating disease with a short and tragic survival period.

[0242] To test this hypothesis, we performed single-cell spatial transcriptome analysis of human GBM tissue sample by 10X Visium (FIG. 42A, FIG. 42B, FIG. 43). Super-imposing the cells expressing the NPs receptor (SLC7A1) (FIG. 42A) with the cancer cells expressing mTOR (FIG. 42B) indicated that the NPs targeted cells (yellow or lightest grey cells in FIG. 43) surround GBM cancer cells (green or darker grey cells in FIG. 43). Thus, NPs gene delivery may provide a targeted immunotherapy for GBM cancer cells.

[0243] NPs target macrophage fused tumor cells via nanotubes. FIG. 41 shows a proposed mechanism for tunneling nanotubes (TNTs) mediated partial cell fusion (left) and permanent cell fusion (right). Manjunath et al., UMS, 2020. Spatial transcriptomic analyses of tissue sections of human glioblastoma (GBM) are provided. FIG. 42A shows NPs receptor SLC7A1 mRNA distribution. Brown (lighter grey) areas represent SLC7A1 high expression cells, which are targeted by NPs. FIG. 42B shows mTOR mRNA distribution. Green (darker grey) areas represent mTOR high expression cells, which are tumor cells with high proliferative and metabolic activities. FIG. 43 shows super-imposing SLC7A1 and mTOR mRNA distribution areas. It shows that tumor cells (green or darker grey) are surrounded by NPs targeting cells (brown or lighter grey).

[0244] We not only discovered NPs targeted gene delivery in the brain by crossing the BBB but also made a technical breakthrough of formulating JABK nanotubes with plasmid DNA to enable non-viral cell transfection. In summary, we made several important discoveries as follows. First, we identified a cation amino acid transporter CAT-1 , encoded by the SLC7A1 gene, as the NPs receptor. This discovery suggests a mechanism for the NPs to cross endothelial cell barrier of the BBB via transcytosis, which is mediated by the association of the lysine residues of NPs with the lysine / arginine transporter CAT-1. It also explains the cell-specific NPs delivery mechanism via targeting the cells expressing CAT-1 on the cell surface. Second, we discovered that NPs target tissue-resident macrophages in different tissues including microglial cells in the brain and Kupffer cells in the liver. It suggests that NPs may be used for macrophage targeted immunotherapy for treating aging related diseases such as AD and cancer. Third, we also made an unexpected discovery that NPs target multi-nucleated giant cells containing macrophage fusion, possibly via a tunneling nanotube dependent mechanism. Such cells become prevalent during AD pathogenesis (FIGs. 38A-38B, FIGs. 39A-39B) and oncogenesis (FIGs. 41A-41 D). The unique properties of NPs to cross the BBB and target multi-nucleated macrophage fusion cells provide a novel treatment option for not only late-stage AD / ADRD but also advanced braincancer such as GBM.CHARACTERIZING THE DELIVERY OF ANTISENSE OLIGONUCLEOTIDE THERAPIES ACROSS THE BLOOD- BRAIN BARRIER USING DNA / LYSINE NANOPIECES:

[0245] Antisense oligonucleotides (ASOs) are a class of RNA-targeting nucleic acids with proven efficacy in treating a broad array of neurological diseases [1 , 2], However, they are limited in their ability to cross the blood-brain barrier (BBB), necessitating invasive and inefficient intrathecal injections for drug delivery. Here, we examine whether Nanopieces (NPs), a selfassembled nanocarrier composed of conjoined adenine / thymine bases attached to lysine, are capable of delivering Malatl -targeting ASOs across the BBB. In the pro-inflammatory miR-146a knockout mouse model, we observed time dependent knockdown by up to 90% in all brain regions besides the cerebellum, with peak effect sustained across 7 and 14 days. In the Alzheimer’s disease (AD) emulating APP / PS1 mouse model, we observed significant knockdown by 43.4% in the hippocampus of diseased mice. However, no effect was observed in wild-type mice. Our data suggest that Nanopieces are an efficient and protective carrier for ASOs across the BBB, but may have disease-specific effects. Further research will seek to elucidate what mechanisms cause this differential effect.

[0246] As introduced above, ASOs are single-stranded nucleic acids that prevent or modify translation of target RNAs. In 2016, Spinraza / nusinersen broke ground as the first neurological antisense agent to receive FDA approval, halting the progression of spinal muscular atrophy in infants [1], However, few ASO therapies targeting other neurodegenerative diseases followed. A principal factor halting progress is the blood-brain barrier (BBB). Long hailed as the greatest challenge to neurological drug development, the BBB excludes a majority of drugs, including ASOs. Therefore, therapies like nusinersen require intrathecal injections to enter the central nervous system. Administration to the spine is uncomfortable, challenging, and potentially dangerous. Furthermore, injection into a still, pressurized sac of cerebrospinal fluid does not distribute broadly [5], Successful therapies are mostly limited to lower motor neuron diseases. There is a strong clinical need for a drug vehicle that can cross the BBB after intravenous injection and degrade safely after delivery. If so, many doors would be opened for targeted therapies against presently incurable brain diseases. Janus base nanopieces (NPs) are a family of drug vehicles formed by two joined DNA 1 bases attached to a positively charged amino acid tail. Bases non-covalently assemble into long strands of nanotubes that wrap around nucleic acid cargo to disguise negative charge. Here, we used NPs to intravenously deliver ASOs across the BBB in mice and tested their efficiency by knocking down the ubiquitously expressed noncoding RNA Malatl via RNase H-mediated degradation.

[0247] The blood-brain barrier serves an important physiological function by gatekeeping what substances can and cannot enter the central nervous system (CNS). The tightly regulated movement of ions, molecules, and cells between blood and the brain maintains homeostasis and prevents toxins and pathogens from infiltrating our body’s most essential organ.

[0248] Conversely, the BBB poses a great challenge for the delivery of neurotherapeutics. Endothelial cells compose the walls of blood vessels, held together by tight junctions with pores just 4 nm in diameter, limiting diffusion [6], Paracellular transport is greatly limited for molecules with larger sizes, and tight junctions contain claudins that also confer charge selectivity. Certain low molecular weight, lipid-soluble molecules are capable of crossing the BBB through transmembrane diffusion. However, both blood and the extracellular fluid of the brain are aqueous-thus, molecules must not be too lipophilic, otherwise they are unable to reach the BBB or the cells behind the BBB [7], Finally, a broad array of efflux transporters use the hydrolysis of ATP to pump molecules from the brain back into the blood. Even if a molecule manages to cross the BBB, specificity to one of these efflux transporters will make its presence short-lived.

[0249] The intentional delivery of drugs across the blood-brain barrier remains one of the greatest unsolved bottlenecks in neurological drug development. In 2005, it was determined that 98% of small molecule drugs and ~100% of large molecule neurotherapeutics were excluded from the BBB [8], Furthermore, less than 1% of academic institutions and pharmaceutical companies had programs investigating BBB transport at the time [8], This stands in staggering contrast to the nearly 100 million Americans affected by neurological diseases and hundreds of billions of dollars invested in global neurological drug development [9],

[0250] Current clinical delivery strategies can be broadly divided into two categories: methods of administration bypassing the BBB and methods of BBB disruption. Methods of administration such as intracerebroventricular (ICV) injection and intrathecal (IT) injection involve physically penetrating into the cerebral ventricles or thecal sac to deliver therapeutics locally. These systems bypass the BBB entirely, but are invasive and susceptible to postoperative infection, subarachnoid hemorrhage, and malpositioning [10, 11], Furthermore, the flow of cerebrospinal fluid (CSF) is influenced by bulk displacement, injection currents, gravity, and baricity: positioning of the patient and alignment of the needle all confer variation in the flow of drug solution, resulting in inconsistent and patchy delivery [5], Once in the CSF, it remains a separate challenge whether drugs can be efficiently taken up by tissue in the brain or spinal cord. These factors make IT and ICV injection suboptimal choices for drug delivery.

[0251] Another category of methods used in conjunction with more traditional systemicdelivery methods involves transiently disrupting the BBB to allow drugs to pass through. For example, low-intensity focused ultrasound is being considered as a way to deliver therapeutics to the brain in otherwise unattainable dosages. Microbubbles are injected into the blood stream alongside the drug and a transducer causes vibrations that enlarge tight junctions, permitting greater diffusion

[0012] , However, this method is nonspecific and may introduce otherwise-excluded pathogens into the brain.

[0252] In preclinical research, modified viral vectors are being considered for their potential to introduce payloads across the BBB. Biotechnology companies like Voyager Therapeutics (Cambridge, MA) use directed evolution of adeno-associated virus (AAV) vectors to generate capsids with BBB-penetrating capabilities

[0013] , Another approach employed by companies like Roche and Denali Therapeutics involves attaching antibodies that promote uptake by nutrient receptors like the large neutral amino acid transporter 3 LAT-1

[0014] , Other notable transport systems that can be targeted as “Trojan horses” to aid drug delivery into the CNS include glucose transporter GLUT-1 , L-lactate transporter MCT-1 , and cationic amino acid transporter CAT-1 , which is encoded by SLC7A1 [6],

[0253] Antisense oligonucleotides (ASOs) have undergone nearly half a century of development since the first evidence of their efficacy in the 1970s. Their road to clinical success has been an uphill battle, punctuated with stalled translational efforts by industry, changes in the scientific understanding of their mechanism of action, and iterative changes in chemical design that have only recently culminated in a successful, functional form-one that can be customized and personalized to any disease, as long as a genetic target exists.

[0254] In 1978, Stephenson and Zamecnick first broke ground with a publication in the Proceedings of the National Academy of Sciences detailing the inhibition of Rous sarcoma virus replication using single-stranded DNA oligonucleotides

[0015] , They proposed that RNA specific knockdown was through Watson-Crick base pairing and that chemical modifications on the 3’ and 5’ termini conferred resistance to nucleases. Yet a modern understanding of ASOs suggests that these findings were most likely an artifact rather than a true antisense effect, since these modifications were insufficient in later industry efforts

[0016] ,

[0255] It is through three primary chemical modifications that ASOs achieve efficacy in many modern therapies. The phosphorothioate (PS) modification replaces a non-bridging oxygen atom with sulfur in the phosphate backbone of ASOs. This confers greater nuclease resistance, but ASOs experience lower binding affinity and off-target cytotoxic effects as a result. By attaching a methoxyethyl group to the 2’ end of bases, known as the 2’-MOE modification, ASOs display evengreater nuclease stability and enhanced binding capacity. However, bases with the 2’-MOE modification are unable to recruit ribonuclease H (RNase H), the 4 naturally-present enzymatic ’’scissors” that cleave complementary RNA. This limitation is circumvented by containing bases in ’’gapmer” formation. By flanking unmodified DNA bases with 2’-MOE modified RNA bases, RNase H activity is preserved while enhancing stability, efficacy, and specificity

[0017] ,

[0256] This basic structure was used by industry pioneer Isis Pharmaceuticals (later renamed lonis) in the development of formivirsen (Vitravene), mipomersen (Kynamro), and inotersen (Tegsedi), a portfolio of drugs targeting a range of diseases outside the central nervous system

[0018] , In 2016, an adjacent class of ASOs that block alternative splicing machinery rather than inducing RNase H was used to develop nusinersen (Spinraza), the first ever treatment for spinal muscular atrophy (SMA) and the first clinically successful ASO targeting a disease of the CNS [1], This basic structure was then subsequently modified to create milasen in 2018, an N-of-1 drug designed to treat a rare form of Batten’s disease in a six-year-old child [2], These developments present a clinically proven roadmap for other neurological precision medicine efforts in the future.

[0257] Evidence shows that when intravenously delivered, ASOs tend to accumulate in the kidney, liver, spleen, and lymph nodes, but can be detected in measureable levels in all organs besides the brain. For this reason, the aforementioned neurological therapies were subjected to intrathecal rather than intravenous delivery. As described below, the inability to cross the bloodbrain barrier remains one of the principal obstacles to the efficient usage of ASOs to treat brain diseases like Alzheimer’s, Huntington’s, and Parkinson’s disease.

[0258] Tissue distribution of total radioactivity at 24 hours after a single bolus IV injection of 5 mg / kg [3H]-labeled ASO to rats was measured (not shown). Figure obtained from Pharmacokinetics and Pharmacodynamics of Biotech Drugs by Bernd Meibohm, 2006 [3],

[0259] Experiments performed by Geary et al. analyzing the knockdown of PTEN mRNA using 6 Plasma, urine and tissue ASO concentrations were measured chimeric (gapmer) 2’-MOE modified ASOs indicate that a single subcutaneous injection of ASO results in peak knockdown at 3 days in the liver of mice (not shown). Maximal effects were retained for 5 days after injection. ASO half-life was measured to be 8 days long. Based on these results, it would be expected for intravenously delivered ASOs to have a similar drug profile, albeit with earlier onset since maximal drug concentration in the plasma is achieved immediately

[0019] ,

[0260] Liver ASO concentrations and knockdown response in PTEN mRNA over time after a single subcutaneous injection of 60 mg / kg ASO to mice were obtained, in an example, from Gearyet al., 2009 [4],

[0261] Recent research has also been published by Jafar-nejad et al. that comprehensively maps ASO distribution and activity in all major CNS regions across mice, rats, and nonhuman pri mates

[0020] , The ASO used in this study, ION-626112, is an RNase H-mediated, 2’-MOE and PS modified gapmer structure targeting Malatl. This is a ubiquitously conserved noncoding RNA, making it an excellent candidate for species-spanning translational experiments. Furthermore, Malatl is connected to the pathogenesis of a variety of diseases including brain metastasis of non-small cell lung cancer [21 , 22], Literature evidence suggests that Malatl may also enhance pro-inflammatory signaling pathways in macrophages

[0023] , Therefore, knocking down expression of this gene presents a viable proof-of-concept for a variety of more specific gene targets, such as mutated huntingtin (mHTT) in Huntington’s disease or microtubule-associated protein tau (MAPT) in Alzheimer’s disease.

[0262] The development of nanopieces: Nanopieces (NPs) are a class of non-covalently selfassembled Janus bases for nucleic acid delivery that were originally developed in 2015 by members of the Chen Lab

[0024] , Long strands of nanotubes envelop their cargo, hiding negative charge and polarity that would otherwise interfere with transfection into cells. They are 2000 times smaller in volume than lipid-based nanoparticles like I ipof ectamine, with a diameter of just 20-30 nm

[0025] , Additionally, they are composed entirely of non-covalent bonds, which allow them to safely degrade into biomimetic products with excellent biocompatibility and minimal cytotoxicity

[0025] , Finally, endosomal escape rates in NPs have been quantified to be much higher than 7 lipid nanoparticle counterparts due to the pH-buffering capacity of their lysine side chains

[0026] ,

[0263] Current evidence from the Chen Lab conducting experiments using eGFP-encoding plasmid DNA suggests that NPs target the receptor CAT-1 (encoded by SLC7A1), which is responsible for the uptake of lysine, an essential amino acid. The prevalence of this transporter in the blood-brain barrier [6], along with the proven capacity for NPs to deliver nucleic acid therapies to other disease models like chondrosarcoma

[0027] , suggest that NPs could be a potential nanocarrier for ASOs across the blood-brain barrier.

[0264] Examples goals of this disclosure are to (1) quantify the biodistribution of functional knock down in the brain regions and organs of diseased mice, (2) quantify the pharmacokinet ics / pharmacodynamics of this effect over the course of 14 days, and (3) validate delivery to the hippocampus in a translational mouse model for Alzheimer’s disease. We hypothesize that consistent Malatl knockdown profiles will be observed in all or gans, including the brain, of miR146a- / - mice similar to the hepatic dose-response curves observed in prior RNase H-mediated, 2’-M0E and PS modified gapmer ASO experiments (FIG. 43). Similarly, we hypothesize that Malatl knockdown will be identified in both wild-type C57BL / 6 and APP / PS1 (Alzheimer’s disease) mouse models and that Slc7a1 expression will be inversely proportional to target gene knockdown. More detailed Experiments are discussed in the Examples below.

[0265] The publication WO2019191151A1 is incorporated by reference herein in its entirety, all chemicals therein, all drawings therein, all text therein can be utilized and / or inter-changed with (any part of) this disclosure in claims. The publication WO2016081522A1 is incorporated by reference herein in its entirety, all chemicals therein, all drawings therein, all text therein can be utilized and / or inter-changed with (any part of) this disclosure in claims. The publication WO2015139051A2 is incorporated by reference herein in its entirety, all chemicals therein, all drawings therein, all text therein can be utilized and / or inter-changed with (any part of) this disclosure in claims. It is important to note that the discussion above are non-limiting. The methods disclosed herein can be carried out by use of instructions provided to a skilled person or to a subject, for example, on a label, electronic instructions, via telecommunication, or instructions provided by demonstration (e.g., video).

[0266] In a discussion, study or a reading of the details, features, embodiments, aspects, and / or examples of the technology disclosed herein, any of the features, embodiments, aspects, and / or examples can be inter-combined (or inter-discussed) with the example details listed below:

[0267] Detail 1 : A method for delivering a therapeutic agent across the blood-brain-barrier (BBB) or other tissue barrier of a subject in need thereof, the method comprising the steps of: (1) obtaining a small molecule comprising a Janus Base with Amino Acid (JBAA); (2) mixing the JBAA solution with the therapeutic agent and followed by processing the mixture with sonication, whereby a nanoparticle (NP) forms with the therapeutic agent inside or partially inside the NP; (3) administering the NP to the subject, whereby the NP subsequently crosses the BBB or other tissue barrier in the subject to deliver the therapeutic agent to a cell in the subject.

[0268] Detail 2: The method of detail 1 , wherein the J BAA forms a shape of a rosette nanotube surrounding the therapeutic agent and the NP comprises non-covalent bonds and / or a non- covalent assembly, and wherein the rosette nanotube has an inner diameter of about 1.1 nm to about 2.2 nm and an outer diameter of about 3.5 nm to about 4.5 nm.

[0269] Detail 3: The method of detail 1, wherein the therapeutic agent comprises a nucleic acid, a nucleotide, DNA, RNA (mRNA, rRNA, tRNA, siRNA, microRNA, IncRNA), and / or one or more oligonucleotides including native and modified forms, and wherein the nucleic acid oroligonucleotide has a length of about 10 to about 200 nucleotides.

[0270] Detail 4: The method of detail 1 , wherein the solution comprises a pH in a range from not less than 3.0 to not more than 4.2, and wherein the solution further comprises a buffer selected from the group consisting of acetate, citrate, succinate, and phosphate buffers.

[0271] Detail 5: The method of detail 1 , wherein the therapeutic agent comprises a small molecule with a molecular weight less than about 900 Da, a peptide with about 2 to about 50 amino acids, a protein with a molecular weight of about 1 kDa to about 500 kDa, a nucleic acid with about 10 to about 200 nucleotides, or a combination thereof.

[0272] Detail 6: The method of detail 1 , wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier, and wherein the therapeutic agent treats a disease or disorder associated with the target cell.

[0273] Detail 7: The method of detail 1 , wherein a zeta value of the nanoparticle is not less than 6 and not more than about 50, and wherein the zeta value is measured at a pH of about 7.4 in a solution comprising phosphate buffered saline.

[0274] Detail 8: The method of detail 1 , wherein an administration of the therapeutic agent to the subject without the J BAA will result in the therapeutic agent not contacting the cell in the subject in a therapeutically effective amount, and wherein the therapeutically effective amount is an amount sufficient to treat a disease or disorder associated with the cell.

[0275] Detail 9: The method of detail 1, wherein the JBAA nanoparticle has a diameter of about 1 nm to about 1000 nm, and wherein the diameter is an average hydrodynamic diameter measured by dynamic light scattering.

[0276] Detail 10: The method of detail 1, wherein the other tissue barrier comprises a bloodretina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or the glomerular filtration barrier in the kidney, and wherein the NP crosses the other tissue barrier to deliver the therapeutic agent to a cell protected by the other tissue barrier.

[0277] Detail 11 : A JBAA nanoparticle for delivering a therapeutic agent across a blood-brain- barrier or other tissue barrier, the JBAA nanoparticle comprising: a Janus Base with Amino Acid (JBAA) molecule; and a therapeutic agent, wherein the therapeutic agent is inside or partially inside the JBAA nanoparticle, and wherein the JBAA nanoparticle is capable of crossing theblood-brain-barrier or other tissue barrier to deliver the therapeutic agent to a target cell.

[0278] Detail 12: The JBAA nanoparticle of detail 11 , wherein the JBAA molecule is a small molecule comprising a Janus Base with Amino Acid (JBAA), and wherein the JBAA molecule has a molecular weight of about 200 Da to about 2000 Da.

[0279] Detail 13: The JBAA nanoparticle of detail 11 , wherein the therapeutic agent is selected from the group consisting of a small molecule drug with a molecular weight less than about 900 Da, a peptide with about 2 to about 50 amino acids, a protein with a molecular weight of about 1 kDa to about 500 kDa, a nucleic acid with about 10 to about 200 nucleotides, and combinations thereof.

[0280] Detail 14: The JBAA nanoparticle of detail 11 , wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier, and wherein the therapeutic agent treats a disease or disorder associated with the target cell.

[0281] Detail 15: The JBAA nanoparticle of detail 11, having a diameter of about 1 nm to about 1000 nm, and wherein the diameter is an average hydrodynamic diameter measured by dynamic light scattering.

[0282] Detail 16: The JBAA nanoparticle of detail 11 , formulated in a pharmaceutically acceptable carrier for administration to a subject, wherein the pharmaceutically acceptable carrier comprises water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, mannitol solution, or a combination thereof.

[0283] Detail 17: The JBAA nanoparticle of detail 16, wherein the pharmaceutically acceptable carrier is suitable for at least one of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intraventricular, intracranial, intranasal, or intratracheal administration, and wherein the administration delivers the JBAA nanoparticle to the blood-brain-barrier or other tissue barrier.

[0284] Detail 18: The nanoparticle of detail 11 , wherein the other tissue barrier comprises a blood-retina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or the glomerular filtration barrier in the kidney, and wherein the JBAA nanoparticle crosses the other tissue barrier to deliver the therapeutic agent to a cell protected by the other tissue barrier.

[0285] Detail 19: The JBAA nanoparticle of detail 11, wherein the subject is a mammal, and wherein the mammal is selected from the group consisting of a human, a non-human primate, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, a mouse, a rat, a guinea pig, and a hamster.

[0286] Detail 20: The JBAA nanoparticle of detail 11 , wherein the therapeutic agent is delivered to the target cell to provide a therapeutic effect, and wherein the therapeutic effect comprises treating a disease or disorder associated with the target cell.

[0287] Detail 21: The nanoparticle of detail 20, wherein the disease or disorder comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, epilepsy, brain tumor, meningitis, encephalitis, cerebral palsy, Rett syndrome, Fragile X syndrome, Down syndrome, autism spectrum disorder, schizophrenia, depression, anxiety, bipolar disorder, addiction, attention deficit hyperactivity disorder, migraine, chronic pain, neuropathic pain, or a combination thereof.

[0288] Detail 22: The method of detail 1 , wherein the method is used to treat Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, epilepsy, brain tumor, meningitis, encephalitis, cerebral palsy, Rett syndrome, Fragile X syndrome, Down syndrome, autism spectrum disorder, schizophrenia, depression, anxiety, bipolar disorder, addiction, attention deficit hyperactivity disorder, migraine, chronic pain, neuropathic pain, or a combination thereof.

[0289] Detail 23: A method for delivering a therapeutic agent across a biological barrier of a subject, the method comprising: obtaining a molecule comprising a Janus Base with Amino Acid (JBAA) moieties, wherein the JBAA molecule is a small molecule comprising a Janus Base moiety and an Amino Acid moiety; mixing the JBAA molecule with the therapeutic agent in an aqueous solution to form a nanoparticle, wherein the therapeutic agent is fully or partially encapsulated within the nanoparticle; administering the nanoparticle to the subject via a route selected from the group consisting of oral, intravenous, intramuscular, subcutaneous, transdermal, and inhalation, whereby the nanoparticle crosses the biological barrier to deliver the therapeutic agent to a target cell in the subject; and delivering the therapeutic agent to the target cell in the subject, wherein the target cell is selected from the group consisting of a brain cell, a spinal cord cell, and a tissue cell.

[0290] Detail 24: The method of detail 23, wherein the biological barrier is a blood-brainbarrier (BBB) or a tissue barrier, and wherein the tissue barrier comprises a blood-retina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or the glomerular filtration barrier in the kidney.

[0291] Detail 25: The method of detail 23, wherein the JBAA molecule has a molecular weight ranging from about 100 Daltons to about 1000 Daltons.

[0292] Detail 26: The method of detail 23, wherein the therapeutic agent is partially encapsulated within the nanoparticle, with a portion of the therapeutic agent exposed on a surface of the nanoparticle.

[0293] Detail 27: The method of detail 23, wherein the subject is a mammal in need of delivery of the therapeutic agent across the biological barrier for treatment of a condition selected from the group consisting of a neurological disorder, a brain tumor, and a tissue disorder.

[0294] Detail 28: The method of detail 23, further comprising delivering the therapeutic agent to the target cell in the subject, wherein the therapeutic agent is released from the nanoparticle upon crossing the biological barrier and entering the target cell.

[0295] Detail 29: A method for targeted delivery of a therapeutic agent across a biological barrier using a nanoparticle, the method comprising: preparing a Janus Base with Amino Acid (JBAA) molecule, wherein the JBAA molecule comprises a Janus Base moiety and an Amino Acid moiety; forming the nanoparticle by mixing the JBAA molecule with the therapeutic agent in an aqueous solution, wherein the therapeutic agent is fully or partially encapsulated within the nanoparticle; administering the nanoparticle to a subject via a route selected from the group consisting of oral, intravenous, intramuscular, subcutaneous, transdermal, and inhalation, whereby the nanoparticle crosses the biological barrier and delivers the therapeutic agent to a target cell in the subject; and delivering the therapeutic agent to the target cell in the subject, wherein the target cell is selected from the group consisting of a brain cell, a spinal cord cell, and a tissue cell.

[0296] Detail 30: The method of detail 29, wherein the Janus Base moiety and the Amino Acid moiety are covalently linked in the JBAA molecule.

[0297] Detail 31 : The method of detail 29, wherein preparing the JBAA molecule comprises obtaining the JBAA molecule by chemical synthesis, purification, or a combination thereof.

[0298] Detail 32: The method of detail 29, wherein forming the nanoparticle comprises mixing the JBAA molecule and the therapeutic agent in the aqueous solution under conditions thatpromote self-assembly of the JBAA molecule and encapsulation of the therapeutic agent.

[0299] Detail 33: The method of detail 29, wherein administering the nanoparticle comprises administering the nanoparticle in a pharmaceutically acceptable carrier, excipient, or diluent

[0300] Detail 34: The method of detail 29, wherein the nanoparticle crosses the biological barrier by transcytosis, endocytosis, or a combination thereof, and wherein the therapeutic agent is released from the nanoparticle upon entering the target cell.

[0301] Detail 35: The method of detail 29, wherein the biological barrier is selected from the group consisting of a blood-brain barrier (BBB), a skin barrier, a mucous membrane barrier, and an organ barrier.

[0302] Detail 36: The method of detail 29, wherein the subject is a mammal in need of delivery of the therapeutic agent across the biological barrier for treatment of a condition selected from the group consisting of a neurological disorder, a brain tumor, and a tissue disorder.

[0303] Detail 37: The method of detail 29, wherein the therapeutic agent is partially encapsulated within the nanoparticle, with a portion of the therapeutic agent exposed on a surface of the nanoparticle to facilitate interaction with the target cell.

[0304] Detail 38: The method of detail 29, wherein the JBAA molecule has a molecular weight ranging from about 100 Daltons to about 1000 Daltons and is capable of self-assembly in the aqueous solution.

[0305] Detail 39: A nanoparticle for targeted delivery of a therapeutic agent across a biological barrier, the nanoparticle comprising: a Janus Base with Amino Acid (JBAA) molecule, wherein the JBAA molecule comprises a Janus Base moiety and an Amino Acid moiety; and the therapeutic agent fully or partially encapsulated within the nanoparticle, wherein the nanoparticle is formed by mixing the JBAA molecule with the therapeutic agent in an aqueous solution under conditions that promote self-assembly of the JBAA molecule and encapsulation of the therapeutic agent, and wherein the nanoparticle is capable of crossing the biological barrier and delivering the therapeutic agent to a target cell when administered to a subject via a route selected from the group consisting of oral, intravenous, intramuscular, subcutaneous, transdermal, and inhalation.

[0306] Detail 40: The nanoparticle of detail 39, wherein the Janus Base moiety and the Amino Acid moiety are covalently linked in the JBAA molecule, and wherein the JBAA molecule has a molecular weight ranging from about 100 Daltons to about 1000 Daltons.

[0307] Detail 41 : The nanoparticle of detail 39, wherein the biological barrier is selected fromthe group consisting of a blood-brain barrier (BBB), a skin barrier, a mucous membrane barrier, and an organ barrier, and wherein the target cell is selected from the group consisting of a neuron, a glial cell, an epithelial cell, an endothelial cell, a muscle cell, a skin cell, a liver cell, a kidney cell, a lung cell, a heart cell, a pancreatic cell, and a stem cell.

[0308] Detail 42: The nanoparticle of detail 39, wherein the therapeutic agent is partially encapsulated within the nanoparticle, with a portion of the therapeutic agent exposed on a surface of the nanoparticle to facilitate interaction with the target cell, and wherein the nanoparticle has a size ranging from about 10 nanometers to about 200 nanometers.

[0309] Detail 43: A method for delivering a therapeutic agent across a blood-brain-barrier (BBB) or other tissue barrier of a subject in need thereof, the method comprising: obtaining a small molecule comprising a Janus Base with Amino Acid (JBAA); mixing the JBAA with the therapeutic agent in a solution, whereby a nanoparticle (NP) forms with the therapeutic agent encapsulated or partially encapsulated within the NP; administering the NP to the subject, whereby the NP subsequently crosses the BBB or other tissue barrier in the subject to deliver the therapeutic agent to a cell in the subject; wherein the solution comprises a pH in a range from about 3.0 to about 4.2; and wherein a zeta value of the nanoparticle is not less than about 6.

[0310] Detail 44: The method of detail 43, wherein the small molecule further comprises at least one of a lipid selected from the group consisting of a phospholipid, a glycolipid, a sphingolipid, and combinations thereof; a surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and combinations thereof; or a polymer selected from the group consisting of a natural polymer, a synthetic polymer, and combinations thereof.

[0311] Detail 45: The method of detail 43, wherein the therapeutic agent is selected from the group consisting of a small molecule drug with a molecular weight less than about 1000 Da, a peptide comprising from about 2 to about 50 amino acids, a protein with a molecular weight greater than about 5000 Da, a nucleic acid selected from the group consisting of DNA, RNA, siRNA, miRNA, antisense oligonucleotides, aptamers, ribozymes, and combinations thereof.

[0312] Detail 46: The method of detail 43, wherein the cell is selected from the group consisting of a neuron in the central nervous system (CNS), a glial cell in the CNS selected from the group consisting of an astrocyte, an oligodendrocyte, a microglia, and combinations thereof; an endothelial cell of the BBB, and combinations thereof.

[0313] Detail 47: The method of detail 43, wherein the subject is a mammal selected from thegroup consisting of a rodent, a lagomorph, a bovine, an ovine, a porcine, a feline, a canine, an equine, a primate, and a human.

[0314] Detail 48: The method of detail 47, wherein the mammal is a human patient suffering from a CNS disorder selected from the group consisting of a neurodegenerative disease, a neuroinflammatory disease, a cerebrovascular disease, a brain tumor, a brain injury, a psychiatric disorder, a lysosomal storage disorder, and combinations thereof.

[0315] Detail 49: The method of detail 43, wherein the NP has a diameter in the range of about 10 nm to about 200 nm as measured by a method selected from the group consisting of dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and combinations thereof.

[0316] Detail 50: The method of detail 43, wherein the NP has a polydispersity index of less than about 0.4 as measured by a method selected from the group consisting of DLS, TEM, SEM, AFM, and combinations thereof.

[0317] Detail 51 : The method of detail 43, wherein the NP has an encapsulation efficiency of the therapeutic agent of at least about 50% as measured by high-performance liquid chromatography (HPLC) or UV-Vis spectroscopy.

[0318] Detail 52: The method of detail 43, wherein the NP is administered via a route selected from the group consisting of intravenous injection, intranasal instillation, intrathecal injection, and combinations thereof at a dose ranging from about 0.1 mg / kg to about 100 mg / kg of body weight.

[0319] Detail 53: The method of detail 43, wherein the NP crosses the BBB or other tissue barrier by a mechanism selected from the group consisting of receptor-mediated transcytosis via binding to a receptor selected from the group consisting of transferrin receptor, insulin receptor, low-density lipoprotein receptor, and combinations thereof; adsorptive-mediated transcytosis via electrostatic interactions with the negatively charged surface of the endothelial cells; and passive diffusion through the lipid bilayer of the endothelial cells.

[0320] Detail 54: The method of detail 43, wherein the therapeutic agent is delivered to the cell in a sustained manner for a period of at least about 24 hours as measured by in vitro drug release assay or in vivo pharmacokinetic study.

[0321] Detail 55: The method of detail 43, wherein the therapeutic agent is delivered to the cell in a targeted manner with a specificity of at least about 80% as measured by in vitro cell uptake assay or in vivo biodistribution study.

[0322] Detail 56: The method of detail 43, wherein the mixing step is performed by a method selected from the group consisting of sonication using a probe sonicator or a bath sonicator at a frequency ranging from about 20 kHz to about 100 kHz for a duration ranging from about 1 minute to about 60 minutes; vortexing using a vortex mixer at a speed ranging from about 500 rpm to about 3000 rpm for a duration ranging from about 1 minute to about 60 minutes; homogenization using a homogenizer at a pressure ranging from about 500 bar to about 2000 bar for a duration ranging from about 1 minute to about 60 minutes; and combinations thereof.

[0323] Detail 57: The method of detail 43, wherein the NP is lyophilized prior to the administering step using a lyophilizer at a temperature ranging from about -50°C to about -80°C and a pressure ranging from about 0.01 mbar to about 0.1 mbarfor a duration ranging from about 12 hours to about 72 hours.

[0324] Detail 58: The method of detail 43, wherein the NP is reconstituted in an aqueous solution prior to the administering step, wherein the aqueous solution is selected from the group consisting of water for injection (WFI), saline, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and combinations thereof.

[0325] Detail 59: The method of detail 43, wherein the method further comprises monitoring a pharmacokinetic parameter of the therapeutic agent in the subject using a method selected from the group consisting of liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and combinations thereof.

[0326] Detail 60: The method of detail 59, wherein the pharmacokinetic parameter is selected from the group consisting of maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the curve (AUC), half-life (t1 / 2), volume of distribution (Vd), clearance (CL), and combinations thereof.

[0327] Detail 61: The method of detail 43, wherein the method further comprises monitoring a pharmacodynamic parameter of the therapeutic agent in the subject using a method selected from the group consisting of behavioral test, cognitive test, neuroimaging, electrophysiology, biochemical assay, histological examination, and combinations thereof.

[0328] Detail 62: The method of detail 61 , wherein the pharmacodynamic parameter is selected from the group consisting of a biomarker level in a biological sample, a clinical symptom score, a disease progression rate, and combinations thereof.

[0329] Detail 63: The method of detail 43, wherein the amino acid in the JBAA is selectedfrom the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and combinations thereof.

[0330] Detail 64: A method for delivering a therapeutic agent across the blood-brain-barrier (BBB) or other tissue barrier of a subject in need thereof, the method comprising the steps of: (1) obtaining a small molecule comprising a Janus Base with Amino Acid (JBAA), wherein the JBAA has a molecular weight of about 200 Da to about 2000 Da and comprises a hydrophobic face and a hydrophilic face; (2) mixing the JBAA solution with the therapeutic agent and processing the mixture with sonication, whereby a nanoparticle (NP) forms with the therapeutic agent inside or partially inside the NP, wherein the solution has a pH in a range from about 3.0 to about 4.2 and comprises a buffer selected from the group consisting of acetate, citrate, succinate, and phosphate buffers; (3) administering the NP to the subject, whereby the NP subsequently crosses the BBB or other tissue barrier in the subject to deliver the therapeutic agent to a cell in the subject, wherein the NP has a diameter of about 1 nm to about 1000 nm and a zeta potential of about +6 mV to about +50 mV at a pH of about 7.4.

[0331] Detail 65: The method of detail 64, wherein the JBAA forms a shape of a rosette nanotube surrounding the therapeutic agent and the NP comprises non-covalent bonds and / or a non-covalent assembly, and wherein the rosette nanotube has an inner diameter of about 1.1 nm to about 2.2 nm, an outer diameter of about 3.5 nm to about 4.5 nm, and a length of about 10 nm to about 1000 nm.

[0332] Detail 66: The method of detail 64, wherein the therapeutic agent comprises a nucleic acid, a nucleotide, DNA, RNA (mRNA, rRNA, tRNA, siRNA, microRNA, IncRNA), and / or one or more oligonucleotides including native and modified forms, and wherein the nucleic acid or oligonucleotide has a length of about 10 to about 200 nucleotides and a molecular weight of about 3 kDa to about 100 kDa.

[0333] Detail 67: The method of detail 64, wherein the solution further comprises a cryoprotectant selected from the group consisting of trehalose, sucrose, glucose, mannose, lactose, mannitol, glycerol, dimethyl sulfoxide (DMSO), and polyethylene glycol (PEG), and wherein the cryoprotectant is present at a concentration of about 1 % to about 20% (w / v).

[0334] Detail 68: The method of detail 64, wherein the therapeutic agent comprises a small molecule with a molecular weight less than about 900 Da, a peptide with about 2 to about 50 amino acids, a protein with a molecular weight of about 1 kDa to about 500 kDa, a nucleic acidwith about 10 to about 200 nucleotides, or a combination thereof, and wherein the therapeutic agent is present at a concentration of about 0.1 mg / mL to about 100 mg / mL in the solution.

[0335] Detail 69: The method of detail 64, wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier, and wherein the therapeutic agent treats a disease or disorder associated with the target cell selected from the group consisting of a neurodegenerative disease, a neurological disorder, a neuropsychiatric disorder, a neurodevelopmental disorder, a brain injury, a spinal cord injury, a brain tumor, an infection of the central nervous system, and a combination thereof.

[0336] Detail 70: The method of detail 64, wherein the zeta potential is measured by electrophoretic light scattering using a Zetasizer Nano ZS instrument (Malvern Instruments) with a folded capillary cell (DTS1070) at 25°C, and wherein the diameter is an average hydrodynamic diameter measured by dynamic light scattering using a Zetasizer Nano ZS instrument (Malvern Instruments) with a disposable cuvette (DTS0012) at 25°C and a scattering angle of 173°.

[0337] Detail 71 : The method of detail 64, wherein an administration of the therapeutic agent to the subject without the JBAA will result in the therapeutic agent not contacting the cell in the subject in a therapeutically effective amount, and wherein the therapeutically effective amount is an amount sufficient to treat a disease or disorder associated with the cell and ranges from about 0.001 mg / kg to about 100 mg / kg body weight of the subject.

[0338] Detail 72: The method of detail 64, wherein the NP is lyophilized and reconstituted in an aqueous solution prior to administration, and wherein the lyophilized NP has a shelf life of at least 6 months at room temperature or at least 1 year at 4°C.

[0339] Detail 73: The method of detail 64, wherein the other tissue barrier comprises a bloodretina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or the glomerular filtration barrier in the kidney, and wherein the NP crosses the other tissue barrier to deliver the therapeutic agent to a cell protected by the other tissue barrier selected from the group consisting of a retinal cell, a placental cell, a testicular cell, a thymic cell, a liver cell, a kidney cell, and a combination thereof.

[0340] Detail 74: A JBAA nanoparticle for delivering a therapeutic agent across a blood-brain- barrier or other tissue barrier, the JBAA nanoparticle comprising: a Janus Base with Amino Acid (JBAA) molecule, wherein the JBAA has a molecular weight of about 200 Da to about 2000 Da and comprises a hydrophobic face and a hydrophilic face; and a therapeutic agent, wherein thetherapeutic agent is selected from the group consisting of a small molecule drug with a molecular weight less than about 900 Da, a peptide with about 2 to about 50 amino acids, a protein with a molecular weight of about 1 kDa to about 500 kDa, a nucleic acid with about 10 to about 200 nucleotides, and combinations thereof, wherein the therapeutic agent is inside or partially inside the J BAA nanoparticle, and wherein the J BAA nanoparticle is capable of crossing the blood-brain- barrier or other tissue barrier to deliver the therapeutic agent to a target cell, and wherein the J BAA nanoparticle has a diameter of about 1 nm to about 1000 nm and a zeta potential of about +6 mV to about +50 mV at a pH of about 7.4.

[0341] Detail 75: The JBAA nanoparticle of detail 74, wherein the JBAA forms a shape of a rosette nanotube surrounding the therapeutic agent and the JBAA nanoparticle comprises non- covalent bonds and / or a non-covalent assembly, and wherein the rosette nanotube has an inner diameter of about 1.1 nm to about 2.2 nm, an outer diameter of about 3.5 nm to about 4.5 nm, and a length of about 10 nm to about 1000 nm.

[0342] Detail 76: The JBAA nanoparticle of detail 74, wherein the therapeutic agent comprises a nucleic acid, a nucleotide, DNA, RNA (mRNA, rRNA, tRNA, siRNA, microRNA, IncRNA), and / or one or more oligonucleotides including native and modified forms, and wherein the nucleic acid or oligonucleotide has a length of about 10 to about 200 nucleotides and a molecular weight of about 3 kDa to about 100 kDa.

[0343] Detail 77: The JBAA nanoparticle of detail 74, wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier, and wherein the therapeutic agent treats a disease or disorder associated with the target cell selected from the group consisting of a neurodegenerative disease, a neurological disorder, a neuropsychiatric disorder, a neurodevelopmental disorder, a brain injury, a spinal cord injury, a brain tumor, an infection of the central nervous system, and a combination thereof.

[0344] Detail 78: The JBAA nanoparticle of detail 74, wherein the zeta potential is measured by electrophoretic light scattering using a Zetasizer Nano ZS instrument (Malvern Instruments) with a folded capillary cell (DTS1070) at 25°C, and wherein the diameter is an average hydrodynamic diameter measured by dynamic light scattering using a Zetasizer Nano ZS instrument (Malvern Instruments) with a disposable cuvette (DTS0012) at 25°C and a scattering angle of 173°.

[0345] Detail 79: The JBAA nanoparticle of detail 74, formulated in a pharmaceutically acceptable carrier for administration to a subject, wherein the pharmaceutically acceptable carrier comprises water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, mannitol solution, or a combination thereof, and wherein the pharmaceutically acceptable carrier further comprises a cryoprotectant selected from the group consisting of trehalose, sucrose, glucose, mannose, lactose, mannitol, glycerol, dimethyl sulfoxide (DMSO), and polyethylene glycol (PEG) at a concentration of about 1% to about 20% (w / v).

[0346] Detail 80: The JBAA nanoparticle of detail 79, wherein the pharmaceutically acceptable carrier is suitable for at least one of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intraventricular, intracranial, intranasal, or intratracheal administration, and wherein the administration delivers the JBAA nanoparticle to the blood-brain-barrier or other tissue barrier at a dose of about 0.001 mg / kg to about 100 mg / kg body weight of the subject.

[0347] Detail 81 : The nanoparticle of detail 74, wherein the other tissue barrier comprises a blood-retina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or the glomerular filtration barrier in the kidney, and wherein the JBAA nanoparticle crosses the other tissue barrier to deliver the therapeutic agent to a cell protected by the other tissue barrier selected from the group consisting of a retinal cell, a placental cell, a testicular cell, a thymic cell, a liver cell, a kidney cell, and a combination thereof.

[0348] Detail 82: The JBAA nanoparticle of detail 74, wherein the subject is a mammal, and wherein the mammal is selected from the group consisting of a human, a non-human primate, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, a mouse, a rat, a guinea pig, and a hamster, and wherein the subject has or is at risk of developing a disease or disorder associated with the target cell.

[0349] Detail 83: The JBAA nanoparticle of detail 74, wherein the therapeutic agent is delivered to the target cell to provide a therapeutic effect, and wherein the therapeutic effect comprises treating a disease or disorder associated with the target cell selected from the group consisting of a neurodegenerative disease, a neurological disorder, a neuropsychiatric disorder, a neurodevelopmental disorder, a brain injury, a spinal cord injury, a brain tumor, an infection of the central nervous system, and a combination thereof.

[0350] Detail 84: The nanoparticle of detail 83, wherein the disease or disorder comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, epilepsy, brain tumor,meningitis, encephalitis, cerebral palsy, Rett syndrome, Fragile X syndrome, Down syndrome, autism spectrum disorder, schizophrenia, depression, anxiety, bipolar disorder, addiction, attention deficit hyperactivity disorder, migraine, chronic pain, neuropathic pain, or a combination thereof, and wherein the therapeutic agent is selected from the group consisting of a small molecule drug, a peptide, a protein, a nucleic acid, an antibody, a gene therapy vector, a cell therapy composition, and a combination thereof.

[0351] Detail 85: The method of detail 64, wherein the method is used to treat Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, epilepsy, brain tumor, meningitis, encephalitis, cerebral palsy, Rett syndrome, Fragile X syndrome, Down syndrome, autism spectrum disorder, schizophrenia, depression, anxiety, bipolar disorder, addiction, attention deficit hyperactivity disorder, migraine, chronic pain, neuropathic pain, or a combination thereof, and wherein the therapeutic agent is selected from the group consisting of a small molecule drug, a peptide, a protein, a nucleic acid, an antibody, a gene therapy vector, a cell therapy composition, and a combination thereof, and wherein the method further comprises monitoring the subject for an improvement in one or more symptoms associated with the disease or disorder.

[0352] The technology herein solves a long-felt but unmet need in the face of years of failure by others. For example, it is known in the field of making and in developing pharmaceutical formulations (e.g., both pre-clinical and clinical formulations) that crossing tissue barriers such as the BBB or even digestive barriers is extremely difficult, requires lengthy dissolution tests at varying pH values, requires lengthy animal testing with organ sampling, and then is highly unpredictable in the human models and / or in use with nucleic acids; Efforts have gone on for years, raising the costs of a single drug development to billions (USD). Thus, the technology herein solves decades of attempts at delivering therapeutic agents, and in particular with new uses for nucleic acid deliveries (e.g., mRNA use over the past 5 years) the technology can be immediately lifesaving.

[0353] The methods herein can be applied prophylactically to a normal healthy subject. A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a genetic disorder) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. For example, a subject can be one whoexhibits one or more risk factors for a condition, or one or more complications related to a condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.EXAMPLES

[0354] The invention now being generally described above, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.EXAMPLE 1. ASO PREPARATION

[0355] Antisense oligonucleotides targeting Malatl were obtained from Integrated DNA Technologies based on the sequence ION-626112, developed by lonis Pharmaceuticals, Inc. (Carlsbad, CA). 2’-methoxyethyl ribose (2’-MOE) modifications flank the DNA sequence in gapmer formation, strung together by either unmodified phosphodiester (PO) or modified phosphorothioate (PS) linkages (FIG. 44). The nucleotide sequence depicted in FIG. 44 is SEQ ID NO: 1. The pharmacokinetics and pharmacodynamics of ION-626112 in the brain have been previously well-characterized in mice, rats, and non-human primates following local injection

[0028] , Lyophilized ASO was dissolved in deionized water and aliquoted at a dose of 3.24 pg per mouse for intravenous injections, corresponding to 0.1 mg / kg body weight.

[0356] FIG. 44 shows SEQ ID NO: 1, a sequence of antisense oligonucleotide used to knock down Malatl. 2’-MOE modifications highlighted in orange (very light grey), PO linkages highlighted in red (or grey), PS linkages highlighted in teal (or darker grey), DNA left in black.

[0357] Nanopieces formulation: Conjoined adenine-thymine (AAT) Janus bases attached to lysine (J BAK) were synthesized by a third-party vendor (Piramal Pharma Solutions, Ltd., Lexington, KY). Quality checks for identity and purity were confirmed via liquid chromatographymass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance spectroscopy (NMR).

[0358] Stock powder of AAT J BAK was dissolved with ASOs in deionized water, then pH adjusted to acidic conditions in order to promote self-assembly. This was sterilized by passage through a 0.2 pm filter, then sonicated and vortexed to form NPASO(or NPASO) assemblies (FIG. 45). PEG 400 was added to minimize erythrocyte sequestration in vivo, glucose was addedto ensure that the solution was isotonic to blood, since phosphate-buffered saline disrupts selfassembly. Total solution volume was tuned to a range between 140 and 246 pL. Quality was assessed by measuring Zeta potential followed by TEM imaging.

[0359] FIG. 45 shows an example of self-assembly of Nanopieces. AAT JBAK bases form rosettes through hydrogen bonding, which stack into Rosette nanotubes (NTs) and encircle ASOs to form the NPASOassembly. Nanopieces assemblies (not shown) and formulations with siRNA / ASO encapsulation and with TEM images were studied.EXAMPLE 2. INTRAVENOUS INJECTION IN MIR-146A MICE AND IN APP / PS1 MICE

[0360] The use of animals was approved by the Lifespan IACUC animal studies committee. All animal studies described herein were performed in accordance with institutional guidelines. Homozygous miR-146a knock-out mice (MMRRC stock #016239) were obtained from the Jackson Laboratory and bred in-house to generate experimental cohorts. This microRNA is critical to the anti-inflammatory function of regulatory T (Treg) cells and knockout of the Mir146 gene causes autoimmune and inflammatory phenotypes

[0029] , Reductions in miR 146a is observed in several neurological and neurodegenerative disorders, and studies on mice have shown that miR- 146a- / - is associated with a reduction in exploratory locomotion and increase in anxiety-like behavior, along with an increase in inflammatory cytokines and induction of oxidative stress in brain tissue

[0030] ,

[0361] Twelve mixed sex, miR-146a- / - mice in total were assigned to receive NPASOtreatment and sacrificed at different timepoints (see FIG. 46A, FIG. 46B). Three, four, and five mice, respectively, received injections of NPASOwith a sacrifice timepoint of 3, 7, and 14 days. All mice were aged 3.5 to 4 months. At sacrifice, brain tissue was dissected, and organs were harvested from the mice.

[0362] Intravenous injection in APP / PS1 mice: APPswe / PSEN1dE9 transgenic mice (MMRRC stock #34832), from here on referred to as APP / PS1 mice, were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in-house to generate experimental cohorts. The APPSwe transgene expresses a mouse amy loid beta (A4) precursor protein gene modified to emulate human residues and to contain the mutation K595N / M596L, linked to familial Alzheimer’s disease

[0031] , The PSEN1de9 trans gene contains mutant human presenilin 1, which is likewise linked to early-onset Alzheimer’s disease

[0031] , Breeding strategy involved crossing hemizygous APP / PS1 mice with C57BL / 6 mice.

[0363] Sixteen mixed sex, wild type C57BL / 6 mice and seven mixed sex APP / PS1 mice wereeither left untreated or injected with NPASOthrough the tail vein (see FIG. 46B, FIG. 46A). All mice were aged between 1 year, 3 months to 1 year, 4 months. At day 7, all mice were sacrificed and brain tissue was dissected.EXAMPLE 3. EXPERIMENTAL DISSECTIONS, ANALYSES, ALZHEIMER’S DISEASE MODEL, AND DISCUSSION

[0364] Following sacrifice, mice bodies were immediately placed on ice. Two sets of dissection protocol were used for miR-146a- / - and APP / PS1+ mice. For the miR-146a groups, the biodistribution of knockdown was quantified by separating the brain into seven anatomical regions and dissecting out nine other body parts, including the heart, lung, stomach, kidney, liver, spleen, large intestine, small intestine, and transversus abdominus muscle. For the APP / PS1groups, atranslational focus towards alleviating neuroinflammation in Alzheimer’s disease meant that the hippocampus — the main brain region driving the pathophysiology of AD — was specifically examined for MALAT-1 knockdown.

[0365] Using sterile surgical scissors, a small superficial incision at base of the mouse skull was performed and skin fascia was separated from the skull up to the ear. Sterile saline was used to wash away any residual fur. An incision was performed along the circumference of the skull, leaving bone near the olfactory lobe such that the superior hemisphere of the skull could be hinged outwards. Finally, the brain was fully detached and lifted, taking care not to damage the olfactory lobe and cervical spinal cord.

[0366] For mice in the APP / PS1 groups, the mouse brain was then bisected along the mid- sagittal plane using a scalpel. The left hippocampus was isolated by lifting the cerebral hemisphere, identifying a continuous pale crescent under 10X magnification (FIG. 47A). The front (F), middle (M), and back (B) brain were isolated by performing three equispaced cuts along the coronal axis of the right hemisphere (see FIG. 47B).

[0367] Brain dissection of the APP / PS1 mice is detailed. FIG. 47A shows a saggittal cut bisects the hemispheres of the brain, from which the hippocampus was isolated. FIG. 47B shows and example of how the right hemisphere was divided via three equispaced cuts along the coronal axis.

[0368] For mice in the miR-146a groups, the mouse brain was likewise bisected. The olfactory lobe (OL), cerebellum (CB), and spinal cord (CB) were isolated from both sides. The superior colliculus and inferior collicus (SIC) were extracted together and consolidated as one group. The cerebral hemisphere was split into groups CH1 , CH2, CH3, corresponding to three equispaced coronal cuts (FIG. 48). FIG. 48 illustrates how brain dissection of miR-146a knockout mice wasperformed based on major anatomical regions.

[0369] Organ dissection was performed by exposing the abdomen using surgical scissors. In total, the heart, lung, stomach, kidney, liver, spleen, large intestine, small intestine, and transversus abdominus (TA) muscles were extracted. All brain tissue and organ samples were immediately placed in 2 ml_ extraction tubes with 300 pL of QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) and then homogenized under the Fisher Scientific Power Gen 125 (Thermo Fisher Scientific, Inc., Waltham, MA) at power setting 6 while remaining on ice. Finally, 400 pL of lysis reagent was again added to each extraction tube and samples were kept in-80°C storage before RNA extraction.

[0370] RNA extraction and RT-PCR: Total RNA was purified using the miRNeasy Mini Kit (cat. no 217004, QIAGEN) with protocol modifications to enhance yield and purity. Homogenate was incubated at room temperature (15-25°C) for 5 minutes. 140 pL chloroform was added to each tube and capped securely, then vortexed for 30 seconds. After another 2-3 minutes of incubation at room temperature, each tube was centrifuged for 15 minutes at 12,000 x g at 4°C. 300 pL of the upper aqueous layer was transferred to a separate tube of 450 pL of 100% ethanol and transferred to an RNeasy Mini column in a 2 mL collection tube. The columns were centrifuged for 1 minute at 10,000 x g at room temperature, discarding the flow-through. This process was repeated after the addition of 700 pL of Buffer RWT. This was repeated again for 500 pL of Buffer RWE; and for a final time for 2 minutes after the addition of 500 pL of Buffer RWE. The Mini columns were then centrifuged at full speed for 1 min in a new 2 mLcollection, then transferred to new 1.5 mL microcentrifuge tubes. 40 pL of RNase-free was added to the membranes and the tubes were incubated for 10 minutes on ice. Finally, all samples were centrifuged for 1 minute at 10,000 x g to isolate total RNA in the collection tubes.

[0371] RNA concentration was determined and quality control checks for RNA purity were performed using a NanoDrop Spectrophotometer (Thermo Fisher Scientific). A measurement using the spectrophotometer reliably provides information on the absolute and relative concentrations of RNA and DNA indicated by absorbance values. Threshold values for pure RNA were set at greater than 2.0 for the ratio of absorbance at 260 nm and 280 nm (260 / 280) and between 2.0 to 2.2 for the ratio of absorbance at 260 nm and 230 nm (260 / 230)

[0032] ,

[0372] Reverse-transcriptase PCR was performed using the miScript II RT Kit (QIAGEN) in accordance with standard manufacturer protocol. 4 pL of 5x miScript HiFlex Buffer, 2 pL of 10x miScript Nucleics Mix, 2 pL miScript Reverse Transcriptase Mix, and 12 pL of template RNA diluted in RNase-free water was added to each RT-PCR tube, such that 300 ng of RNAwascontained in each PCR tube. The strips then underwent 60 minutes of incubation at 37°C, followed by 5 minutes at 95°C to inactivate the mix using the Applied Systems ProFlex PCR System (Thermo Fisher Scientific). After completion, all samples underwent 2X dilution to 7.5 ng / pL cDNA in RNAse-free water.

[0373] qPCR analysis: Primers for qPCR were obtained through the Mass General PrimerBank database, a publicly available resource covering most human and mouse genes

[0033] , Additional candidate qPCR sequences were found through literature searches. Each sequence was verified to have less than 200 bp amplicon size, less than 30 bp length, and specificity to the target RNA based on the Basic Local Alignment Search Tool (BLAST) tool by the National Center for Biotechnology (NCBI)

[0034] ,

[0374] Candidate primers were acquired from Eurofins Genomics, LLC (Louisville, KY) in lyophilized form and diluted in 1X TE Buffer in accordance with the manufacturer’s specifications. Forward and reverse primers were then combined in a 1 : 1 ratio and diluted to 250 nM with RNase- free water. Candidate primers were then tested on samples to ensure that all quality control metrics were met, including consistency in melt curve and melt peak as well as Ct values less than 35.

[0375] qPCR run conditions: all qPCR experiments were performed on the CFX Opus 96 Real-time PCR System and CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA). Reactions started with an initial step of 15 minutes at 95°C to activate the Taq poly merase. This was followed by 40 cycles of (1) denaturation for 15 seconds at 94°C, (2) annealing for 30 seconds at 55°C, and (3) extension for 30 seconds at 70°C followed by data capture. After every cycle is completed, the machine incubates at 72°C for 5 minutes before beginning melt curve steps: (1) 95°C for 30 seconds, followed by escalating temperature from 65°C to 95°C in 5 second steps.

[0376] Run conditions are described in detail above and exact examples of sequences are documented in FIG. 49. FIG. 49 shows example primer sequences used for qPCR analysis, namely, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. All primers designed with Mus mus cuius genome as reference; SLC7A1 is species-spanning.

[0377] Samples were loaded in 96-well plates with a final reaction volume of 20 pL per well. This was composed of 2 pL cDNA (15 ng), 6 pL RNase-free water, 2 pL primer mix, and 10 pL ofSYBR Green Master Mix (Applied Biosystems). All primers were run in triplicate.

[0378] 18s and Gapdh were used as reference genes (also known as housekeeping genes) for reactions. 18s encodes the rRNA that composes the eukaryotic small ribosomal subunit. It is generally considered one of the most stable housekeeping genes for a variety of stresses, including viral infection and hypoxia [35, 36], Gapdh encodes glyceraldehyde-3-phopsphate dehydrogenase, a glycolytic enzyme. Although it is now widely considered an unreliable housekeeping gene, we did not detect any greater variation in its expression relative to 18S, and thus chose to include it for later experiments as a second housekeeping gene to reduce noise from the reference. The Livak Method was used to calculate fold change

[0037] ,

[0379] Results; Pro-inflammatory miR-146a- / - mouse model: MiR-146a- / - mice injected with Nanopieces broadly experienced Malatl knockdown in the brain when compared to baseline levels in C57BL / 6 mice across all three timepoints at 3, 7 and 14 days (see FIGs. 50A-50G). The knockdown profiles in the olfactory lobe (OL), cerberal cortex (CH1, CH2, CH3), superior and inferior colliculus (SIC), and spinal cord (SC) are gradually decreasing, indicating that peak knockdown is observed on or after 14 days (FIGs. 50A-50E, FIG. 50G). Malatl knockdown begins to reach close to maximum levels at 7 days before leveling off for the anterior brain regions OL, CH1, CH2, and CH3 (FIGs. 50A-50D), while Malatl levels are reduced linearly in the posterior SIC and SC regions (FIG. 50E, FIG. 50G). This suggests that NPASOs induce knockdown more rapidly in the frontal regions of the brain. Notably, no statistically significant change in Malatl knockdown was observed in the cerebellum, the brain region responsible for coordinating movement and balance (FIG. 50F).

[0380] Malatl knockdown in the brain of miR-146a- / - mice over the timespan 3 days, 1 week, and 2 weeks (mean ± SEM; 3, 4, 5 mice per group, respectively) is shown in FIGs. 50A-50F. Fold change is normalized to 18S and compared to wild-type C57BL / 6mice (n=3). *p<0.05, **p<0.001 , Student’s one-tailed t-test.

[0381] FIG. 50A shows miR-146a- / - mice data for olfactory lobe. FIG. 50B shows miR-146a- / - mice data for cerebral hemisphere 1. FIG. 50C shows miR-146a- / - mice data for cerebral hemisphere 2. FIG. 50D shows miR-146a- / - mice data for cerebral hemisphere 3. FIG. 50E shows miR-146a- / - mice data for the superior and inferior colliculus. FIG. 50F shows miR-146a- / - mice data for the cerebellum. FIG. 50G shows miR-146a- / - mice data for the spinal cord.

[0382] In FIGs. 51A-51G, Malatl knockdown in the brain of miR-146a- / - mice two weeks after injection is compared to wild-type C57BL / 6 mice (mean±SEM, 5 and 3 mice per group,respectively). *p<0.05,**p<0.01,***p<0.001 , Student’s one-tailed t-test.

[0383] FIG. 51 A shows comparative data for olfactory lobe. FIG. 51 B shows comparative data for cerebral hemisphere 1. FIG. 51 C shows comparative data for cerebral hemisphere 2. FIG. 51 D shows comparative data for cerebral hemisphere 3. FIG. 51 E shows comparative data for the superior and inferior colliculus. FIG. 51 F shows comparative data for the cerebellum. FIG. 51 G shows comparative data for the spinal cord.

[0384] Expression levels in the liver, heart, lung, stomach, spleen, small intestine, and large intestine achieved similar knockdown profiles to the anterior brain regions, with evidence of rapid depression in Malatl levels at 7 days that level off through 14 days (FIGs. 52A-52C, FIGs. 52E- 52H). Malatl knockdown in the kidney was most immediate, reaching 22.9% of the untreated C57BL / 6 control at 3 days and plateauing to 10.2% and 10.1% at days 7 and 14, respectively (FIG. 52D).

[0385] A 0.119- and 0.115- fold change, corresponding to a 88.1% and 88.5% decrease in expression levels, respectively, was observed at these timepoints for the liver (FIG. 52E), the organ that is generally regarded as the final site of accumulation for both metal and lipid nanoparticles[38,39]. The standard deviation in liver expression levels was 5.3% and 5.4%, respectively. However, no timepoints were deemed statistically significant according to a one- tailed Student’s t-test. This was due to very high variance in Malatl levels of the uninjected control group of C57BL / 6 mice (FIG. 53E). Similarly, the Malatl knockdown levels in the kidney and small intestine was not deemed statistically significant at any timepoints, including 2 weeks, due to variance in the control group (FIG. 53D, FIG. 53H).

[0386] The transversus abdominus (TA) muscle was the only experimental group to yield a statistically significant, 12.8-fold increase in the expression of Malatl at three days (FIG. 521). However, this was reduced to a 0.44-fold decrease in Malatl expression by day 14.

[0387] FIGs. 52A-52I show Malatl knockdown in the non-brain regions of miR-146a- / - mice over the timespans 3 days, 1 week, and 2 weeks (mean ± SEM; 3, 4, 5 mice per group, respectively). Fold change normalized to 18S and compared to wild-type C57BL / 6 mice (n=3). *p<0.01,***p<0.001 , Student’s one-tailed t-test.

[0388] FIGs. 53A-53I show Malatl knockdown in the non-brain regions of miR-146a- / - mice two weeks after injection compared to wild-type C57BL / 6 mice (mean±SEM, 5 and 3 mice per group, respectively). *p<0.05, **p<0.01 , ***p<0.001 , Student’s one-tailed t-test.

[0389] Alzheimer’s disease APP / PS1 mouse model: Baseline levels of Malatl in thehippocampus of wildtype and APP / PS1 mice was approximately the same, with no significant difference between the two groups (not shown). APP / PS1 mice experienced a statistically significant 0.566-fold decrease in Malatl expression in the hippocampus of those treated with NPASO. However, no significant knockdown was exhibited in wild-type mice treated with the same NPASO. The differential response between the two groups could be due to a variety of factors implicated in the pathogenesis of Alzheimer’s disease, or perhaps a differential overexpression of cationic amino acid transporter SLC7A1 in diseased mice.

[0390] To test this hypothesis, Slc7a1 levels were measured from the same samples. Here, there was a non-significant increase in the expression of Slc7a1 between untreated wild-type and APP / PS1 mice. Additionally, the introduction of NPASO did not significantly change RNA levels.

[0391] Discussion: empirically, we show that NPASOs are capable of efficiently knocking down their target mRNA, Malatl, at significant levels in most brain regions of diseased mice. However, efficacy appears to be limited in wild-type mice, and Slc7a1 mRNA expression is not modulated by treatment.

[0392] In the pro-inflammatory miR-146a- / - model, peak knockdown was observed earlier in anterior brain regions (FIGs. 50A-50G). This could be due to a variety of factors: the BBB is heterogeneous, with differences in permeability according to region

[0040] , Differences in vascular density may also affect the order in which tissues experience knockdown

[0040] , All brain regions experienced between 70% to 90% knockdown at 2 weeks post-administration, with the notable exception of the cerebellum, for which no significant knockdown was observed at any time point (FIGs. 50A-50G, FIGs. 51 A-51G). Although this may seem abnormal, this result is consistent with prior experiments performed by Jafar-nejad et al. using ION-626112 delivered as an intrathecal bolus in rats

[0020] , Based on whole tissue qPCR data, they noticed that cerebellar Malatl knockdown levels was less robust than other brain regions. Immunohisto chemical (IHC) staining revealed that although ASOs affect the Purkinje cells and molecular layer of the cerebellum, the dense granular layer shows little reduction in Malatl. Here, our data suggests that NPs did not grant ASOs the ability to penetrate the granular layer to any greater extent.

[0393] An alternative hypothesis for the localized absence of knockdown in the cerebellum could be due to a lower expression of CAT-1 in this brain region. According to the mouse brain RNA-seq dataset from the Human Protein Atlas project, the cerebellum has the lowest SLC7A1 expression of any measured brain regions, with nearly three times less expression than that of the cerebral cortex

[0041] , Future work should corroborate qPCR data with IHC staining of CAT-1 protein and Malatl RNA to analyze whether this is truly the case.

[0394] In the non-brain organs harvested from miR-146a- / - mice, the greatest magnitude of knockdown at 7 days was observed in the kidney, liver, spleen, and small intestine, ranging from an 87.8% to 89.8% reduction, although this was only statistically significant in the spleen due to high variance in the control group as mentioned earlier (see FIGs. 52A-52I). This aligns with the existing literature evidence that ASOs tend to accumulate in the kidney, liver, and spleen [3], Since NPASOs do not have tissue-specific targets, this consistency in biodistribution was expected. Furthermore, since the magnitude of Malatl knockdown in the brain is within the same range as that of the kidney, liver, and spleen, it is less likely that these organs will be saturated to pathological levels before ASOs reach therapeutic levels in the brain. This is an optimistic sign that potential NPASOtherapies may not require extremely high dosages to have a neurological effect.

[0395] Interestingly, the time curve of Malatl knockdown was different than expected. Although we predicted a more immediate or similarly rapid treatment effect as the experiments shown, ASO knockdown did not reach peak levels until much later, sometime between 3 and 7 days rather than at day 3 (FIGs. 50A-50G and FIGs. 52A-52I). Furthermore, peak effect was sustained over the course of 7 days rather than across 5 days as measured in the subcutaneously delivered, hepatic knockdown experiments performed by Geary et al. [4], Therefore, it is likely that NPs promote slower uptake and clearance of ASOs in vivo.

[0396] Malatl levels were significantly reduced at 7 days post-administration in the hippocam pus of the APP / PS1 mouse model, further supporting that NPASOs are capable of crossing the BBB (not shown). Unexpectedly, there was no change in Malatl levels for wild-type mice. This result suggests that some aspect common to both of the diseased mouse models promotes delivery across the BBB. In this case, it is likely linked to inflammation, a phenotype observed in both miR-146a- / - and APP / PS1 mice. Further work has begun on elucidating the relationship between immunomodulation and Nanopiece delivery.

[0397] Slc7a1 mRNA expression was unchanged across wild-type and APP / PS1 mice, for both treated and untreated groups. The lack of a clear relationship between Malatl knockdown and Slc7a1 expression could indicate any number of possible explanations, including that Slc7a1 is expressed in such excess that it is not rate-limiting in NP delivery. Unlike Malatl which is noncoding, Slc7a1 must also undergo translation before reaching its functional form, so in the absence of protein-level data our interpretation of this is restricted.

[0398] Other limitations in interpretability include the use of a non-ideal baseline control for the miR-146a knockout portion of the experiment. Two independent things are different betweenthe two groups in FIGs. 50A-50G and FIGs. 51A-51G: treatment and genotype. But (1) since MALAT1 is associated with inflammation, which is upregulated in miR-146a- / - mice, the difference between baseline miR-146a- / - and NPASO-treated mice should theoretically be greater than the existing difference

[0042] , Regardless, (2) the treatment effect curve in FIGs. 50A-50G are preserved regardless of reference group-by the Livak method of qPCR analysis, changing the reference group only translates data vertically, since the experimental delta Ct values will not be different. This may also be the reason why knockdown at 3 days sometimes starts with upregulation, as seen in the TA muscle (FIG. 52I). We intend to eventually acquire untreated, miR-146a- / - mice such that they are the reference control group. Finally, skewed sex ratios in the experimental and control groups may bias results if sex differences are present (FIG. 46A, FIG. 46B). Although outlier tests confirmed that no sex differences in Malatl or Slc7a1 expression were identified, further work will seek to remediate this imbalance by including sex-matched controls.

[0399] It is noted that Antisense oligonucleotides hold great therapeutic potential in the development of precision neurological drugs. Here, we show that Nanopieces act as a nanocarrier promoting the de livery of ASOs across the blood-brain barrier in diseased mouse models. In time dependence studies on miR-146a- / - mice, we show that Nanopieces slow down uptake and clearance, with a delayed but sustained peak effect through 7 and 14 days in most brain regions and organs, resulting in up to 90% knockdown in the test gene Malatl. In the APP / PS1 mouse model of Alzheimer’s disease, we note that significant levels of knockdown in the hippocampus is also observed at 7 days. However, it appears that Nanopieces are ineffective at inducing knockdown in wild-type mice. Further studies will seek to analyze why this is the case, investigating whether factors like inflammation and immunomodulation are involved in the NP mechanism of action.EXAMPLE 4. CYTOTOXICITY STUDY OF NP DELIVERY

[0400] Experimental design of the cytotoxicity study of NP’s systemic delivery of nucleic acid via IV injection of mouse tail vein is shown in Table 1 below.

[0401] In the study, the total number of mice: 140.

[0402] Species: mouse, e.g. C57BL / 6, n-=5, male, age 6-8 weeks (22-30g).

[0403] Time points: 24 hours and 7 days. Administration: tail vein IV injection, single injection. Dose: 5 pg, 15 pg, and 30 pg in 150 pL volume, control JBAK1 (RNT) or JBAK2 (TBL) unloaded, buffer alone, beacon aloneTable 1. Experimental Design of the Cytotoxicity Study

[0404] Results: No test articles (JPAK Nanopieces and Nanotubes) related clinical observations were made for changes in: weight, skin, fur, eyes and mucous membranes.

[0405] Further pre-clinical animal studies (e.g., behavior observations and dissections) were made in detail and no changes were observed in respiratory system, circulatory system, autonomic central nervous system, somatomotor activity, locomotor activity, and / or behavior patterns.

[0406] As such, based on pre-clinical animal models, the NP delivery herein indicates a streamlined formulation that can be utilized to deliver a large variety of therapeutic agents across various tissue barriers in a subject without causing cytotoxicity, thereby representing a solution to a long-felt but unmet need in the face of failure (over many decades) in attempts to develop pharmaceutical formulations that can cross sensitive barriers. It is further contemplated that thetechnology disclosed herein can include one or more targeting moieties that will be carried as tissue barriers are crossed and then will target specific cells (e.g., such as cancer cells).

[0407] In this example, the NP delivery indicates a streamlined formulation that can be utilized to deliver a large variety of therapeutic agents across various tissue barriers in a subject without causing cytotoxicity, thereby representing a solution to a long-felt but unmet need in the face of failure (over many decades) in attempts to develop pharmaceutical formulations that can cross sensitive barriers. It is further contemplated that the technology disclosed herein can include one or more targeting moieties that will be carried as tissue barriers are crossed and then will target specific cells (e.g., such as cancer cells).

[0408] Complete methods of treating patients are developed. Various clinical trials are envisioned whereby the clinical properties of the therapeutic agent(s) can be studied without interference (e.g., toxicity, low bioavailability) from the delivery formulations.

[0409] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0410] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.REFERENCES:[1] U.S Food and Drug Administration. Fda approves first drug for spinal muscular atrophy. 2016.[2] Jinkuk Kim, Chunguang Hu, Christelle Moufawad El Achkar, Lauren E Black, Julie Douville, AustinLarson, Mary K Pendergast, Sara F Goldkind, Eunjung A Lee, Ashley Kuniholm, et al. Patient- customized oligonucleotide therapy for a rare genetic disease. New England Journal of Medicine, 381(17):1644-1652, 2019.[3] Bernd Meibohm and Hartmut Derendorf. Pharmacokinetics and pharmacodynamics of biotech drugs. Pharmaceutical biotechnology: Drug discovery and clinical applications, pages 145-172, 2004.[4] Richard S Geary, Ed Wancewicz, John Matson, Megan Pearce, Andrew Siwkowski, Eric Swayze, and Frank Bennett. Effect of dose and plasma concentration on liver uptake and pharmacologic activity of a 2-methoxyethyl modified chimeric antisense oligonucleotide targeting pten. Biochemical pharmacology, 78(3):284-291 , 2009.[5] GHocking and JAWWildsmith. Intrathecal drug spread. British journal of anaesthesia, 93(4):568-578, 2004.[6] Richard Daneman and Alexandre Prat. The blood-brain barrier. Cold Spring Harbor perspectives in biology, 7(1 ):a020412, 2015.[7] William A Banks. Characteristics of compounds that cross the blood-brain barrier. BMC neurology,9(1):1-5, 2009.[8] William M Pardridge. The blood-brain barrier: bottleneck in brain drug development. NeuroRx, 2:3-14, 2005.[9] GBD 2017 US Neurological Disorders Collaborators. Burden of Neurological Disorders Across theUS From 1990-2017: A Global Burden of Disease Study. JAMA Neurology, 78(2): 165— 176, 02 2021.

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Claims

CLAIMS l / l / e claim:

1. A method for delivering a therapeutic agent across a blood-brain-barrier (BBB) or other tissue barrier of a subject in need thereof, the method comprising steps of:(1) obtaining a small molecule comprising a Janus Base with Amino Acid (JBAA);(2) mixing the JBAA in a solution with the therapeutic agent, whereby a mixture is formed, and processing the mixture with sonication, whereby a nanoparticle (NP) forms with the therapeutic agent inside or partially inside the NP; and(3) administering the NP to the subject, whereby the NP subsequently crosses the BBB or other tissue barrier in the subject to deliver the therapeutic agent to a cell in the subject.

2. The method of claim 1 , wherein the JBAA forms a shape of a rosette nanotube coassembling with and surrounding the therapeutic agent with non-covalent bonds and / or a non- covalent assembly.

3. The method of claim 1 , wherein the therapeutic agent comprises a nucleic acid, a nucleotide, DNA, RNA (mRNA, rRNA, tRNA, siRNA, microRNA, IncRNA), and / or one or more oligonucleotides including native and modified forms.

4. The method of claim 1 , wherein the solution comprises a pH in a range from not less than 3.0 to not more than 4.2.

5. The method of claim 1 , wherein the therapeutic agent comprises a small molecule, a peptide, a protein, a nucleic acid, or a combination thereof.

6. The method of claim 1 , wherein a target cell for the therapeutic agent is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier.

7. The method of claim 1 , wherein a zeta value of the nanoparticle is not less than 2.

8. The method of claim 1 , wherein an administration of the therapeutic agent to the subject without the JBAA will result in the therapeutic agent not crossing the BBB in the subject in atherapeutically effective amount.

9. The method of claim 1 , wherein the NP and / or a JBAA nanoparticle has a diameter of about 1 nm to about 1000 nm.

10. The method of claim 1 , wherein the other tissue barrier comprises a blood-retina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, blood- synovial barrier, blood-tumor barrier, and / or a glomerular filtration barrier in a kidney.

11. A JBAA nanoparticle for delivering a therapeutic agent across a blood-brain-barrier or other tissue barrier, the JBAA nanoparticle comprising: a Janus Base with Amino Acid (JBAA) molecule, a therapeutic agent, and wherein the therapeutic agent is inside or partially inside the JBAA nanoparticle, wherein the JBAA nanoparticle is capable of crossing the blood-brain-barrier or other tissue barrier to deliver the therapeutic agent to a target cell.

12. The JBAA nanoparticle of claim 11 , wherein a JBAA molecule is a small molecule comprising a Janus Base with Amino Acid (JBAA).

13. The JBAA nanoparticle of claim 11 , wherein the therapeutic agent is selected from a group consisting of a small molecule drug, a peptide, a protein, a nucleic acid, and combinations thereof.

14. The JBAA nanoparticle of claim 11 , wherein the target cell is a brain cell, a spinal cord cell, a neuron, a glial cell, an astrocyte, an oligodendrocyte, a microglial cell, an ependymal cell, a neural stem cell, a cerebral endothelial cell, a pericyte, or a cell of another tissue protected by a tissue barrier.

15. The JBAA nanoparticle of claim 11 , having a diameter of about 1 nm to about 1000 nm.

16. The JBAA nanoparticle of claim 11 , formulated in a pharmaceutically acceptable carrier for administration to a subject.

17. The JBAA nanoparticle of claim 16, wherein the pharmaceutically acceptable carrier is suitable for at least one of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intraventricular, intracranial, intranasal, or intratracheal administration.

18. The JBAA nanoparticle of claim 11 , wherein the other tissue barrier comprises a bloodretina barrier, blood-placenta barrier, blood-testis barrier, blood-thymus barrier, blood-bile barrier, and / or a glomerular filtration barrier in a kidney.

19. The JBAA nanoparticle of claim 11 within a subject, wherein the subject is a mammal.

20. The JBAA nanoparticle of claim 11 , wherein the therapeutic agent is delivered to the target cell to provide a therapeutic effect.

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