Systems and methods to facilitate delivery of stem cells and exosomes to brain

Abstract

Methods and systems for improving or enabling delivery of allogenic exosomes or stem cells across the Blood-Brain Barrier (BBB) are disclosed. A dose of integrin blocking agents is administered to the patient. Plasmapheresis is applied to the patient to remove free integrin blocking agent and autologous exosomes or stem cells from the patient's blood. A targeted region of the BBB is stimulated, for example through focused ultrasound or shockwave, and allogenic exosomes or stem cells are applied to the targeted region of the BBB.

Description

Attorney Docket No. 103366.0024PCTSYSTEMS AND METHODS TO FACILITATE DELIVERY OF STEM CELLS AND EXOSOMES TO BRAIN

[0001] This application claims the benefit of priority to United States Provisional Patent No. 63 / 791,041 filed April 18. 2025, which is incorporated by reference in its entirety herein.Field of the Invention

[0002] The field of the invention is treatment of neurological conditions.Background

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] It is increasingly desirable to enable or improve delivery of therapeutic agents across the Blood-Brain Barrier (BBB). The BBB prevents foreign elements from entering the brain, and often prevents objects from entering the brain that may have therapeutic effect. For example, exosomes and stem cells generally will not natively cross the BBB to enter the brain. While it is known to mechanically bypass the BBB with a needle or laceration, this is invasive and can cause unnecessary or unacceptable damage to a patient.

[0006] Less invasive methods for crossing the BBB are known. For example, “Targeted Delivery of Neural Stem Cells to the Brain Using MRI-Guided Focused Ultrasound to Disrupt the Blood-Brain Barrier,” PLoS ONE 6(11): e27877 by Burgess, et al., reports use of focused ultrasound with microbubbles to open targeted regions of the BBB in animal models. “Cellular Mechanisms Of The Blood-Brain Barrier Opening Induced By Ultrasound In Presence OfAttorney Docket No. 103366.0024PCTMicrobubbles,” Ultrasound in Med. & Biol., Vol. 30, No. 7, pp. 979- 989, 2004 by Sheikov, et al., reports use of ultrasound and microbubbles in animal models to open the BBB, noting the BBB can be damaged at 3W or greater sonication. “Noninvasive Localized Delivery Of Herceptin To The Mouse Brain By Mri-Guided Focused Ultrasound-Induced Blood-Brain Barrier Disruption,” PNAS, Vol. 103, No. 31, 11719-23 by Kinoshita, et al., reports use of ultrasound with microbubbles in animal models to disrupt BBB, noting microbubbles are required for consistent BBB opening. US Patent Publ. 2020 / 0108241 to Jordan, et al., also teaches use of ultrasound to improve delivery of exosomes across the BBB.

[0007] All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0008] Further setbacks or hinderances of methods in the art have been identified. Focused ultrasound and shock wave when targeting the area of interest may temporarily improve penetration of stem cells or exosomes to the brain that had been given intravenously from an outside source (allogeneic), however, these approaches also enhance penetration of white cells and exosomes already present in the bloodstream (autologous). The inadvertent delivery of exosomes and white cells would have the deleterious effect of delivering senescent signals to the brain which neutralize some or all benefits from the delivery of youthful exosomes and stem cells.

[0009] Thus, there remains a need for systems and methods to improve delivery of exosomes and stem cells across the BBB without damaging the BBB.Summary of The Invention

[0010] Systems and methods for delivering a stem cell or an exosome, typically allogenic, across a blood-brain barrier (BBB) of a patient are contemplated. A plasmapheresis process is applied to the patient, thereby removing one or more autologous matter (e.g., exosomes) from a blood volume of the patient. A region of the BBB is stimulated to facilitate delivery of the stem cell or the exosome across the BBB at the region of interest. The stem cell or the exosome is thenAttorney Docket No. 103366.0024PCTadministered to the patient peripherally by intravenous, subcutaneous, nasal or oral inhalation, or intrathecal route. In some cases an integrin blocking agent (one or more agents, of one or more type, e.g., natalizumab, etc.) is administered to the patient before the plasmapheresis process is applied.

[0011] It should be appreciated the inventive subject matter contemplates (1) performing plasmapheresis to improve delivery of allogenic exosomes or stem cells across the BBB using targeted ultrasound or shockwave treatment without integrin blockers, (2) administering a dose of integrin blockers to improve delivery of allogenic exosomes or stem cells across the BBB using targeted ultrasound or shockwave treatment without performing plasmapheresis, or (3) performing plasmapheresis in conjunction with administering a dose of integrin blockers to improve delivery of allogenic exosomes or stem cells across the BBB using targeted ultrasound or shockwave treatment.

[0012] Typically the time between applying the plasmapheresis process and introducing the stem cell or the exosome is no more than 8 hours. In preferred embodiments, the majority of the subject’s circulating exosomes are removed from the blood volume during the plasmapheresis process; though it is contemplated removal of 40% to 95% or up to 100% of exosomes (e.g., autologous) yields suitable results. The plasmapheresis process replaces a plasma volume of the blood volume with either a donor plasma volume, an albumin volume, or a combined saline and albumin volume (e.g., 70% albumin, 30% saline).

[0013] In some cases both the stem cell and the exosome (preferably both allogenic) are administered to the patient or otherwise introduced to the region of the BBB. For example, the stem cell or the exosome can be introduced to the patient intravenously, intramuscularly, intrathecally, subcutaneously, orally, or inhaled. It is contemplated the stem cell and exosome are subsequently exposed to a brain tissue of the patient.

[0014] Stimulating the region of the BBB typically requires directing focused energy at the region. Directing focused energy includes any one or more of directing a focused ultrasound, a transcranial magnetic stimulation, a shockwave, a light energy, or other electromagnetic radiation.Attorney Docket No. 103366.0024PCT

[0015] Further methods of delivering a stem cell or an exosome across a blood-brain barrier (BBB) of a patient are contemplated. A dose of integrin blocking agents is administered to the patient followed by applying a plasmapheresis process to the patient. It is contemplated that the integrin blocking agents block ligands of autologous white blood cells, yielding a majority of the patient’s white blood cells integrin blocked (or within 40% to 100%) and an amount of free integrin blocking agents circulating in the patient’ s blood. Plasmapheresis is then applied to remove the free integrin blocking agents or exosomes (e.g., autologous) in blood circulation, or both. Where plasmapheresis is intended to remove both free integrin blocking agents and exosomes, the plasmapheresis process occurs after administering the dose of integrin blocking agents (e.g., at least 5, 10, or 30 mins after, or between 1 to 24 hours after). A target region of the BBB is stimulated to facilitate delivery of the allogenic stem cell or exosome across the BBB at the region, and the allogenic stem cell or exosome are administered to the patient or otherwise introduced to the region of the BBB.

[0016] In some embodiments the plasmapheresis process replaces a plasma volume of the blood volume with one of a donor plasma volume, an albumin volume, or a combined saline and albumin volume (e.g., 70% albumin, 30% saline). Preferably at least a majority or 40% to 95% up to 100% of autologous exosomes are removed from the blood volume during the plasmapheresis process.

[0017] In some embodiments the exosome or stem cell is modified to improve adhesion of the exosome with the BBB or express an antibody receptor. The region of the BBB is preferably stimulated by one or more of directing a focused ultrasound, a transcranial magnetic stimulation, a shockwave, a light energy, or other EM radiation at the region.

[0018] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.Brief Description of the Drawings

[0019] Figure 1 depicts a flow chart of a method of the inventive subject matter.

[0020] Figure 2 depicts a flow chart of another method of the inventive subject matter.Attorney Docket No. 103366.0024PCT

[0021] Figure 1 depicts a flow chart of yet another method of the inventive subject matter.Detailed Description

[0022] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0023] Figure 1 depicts flow chart 100 of method 110 having ordered steps 120, 130, and 140.

[0024] Figure 2 depicts flow chart 200 of method 210 having ordered steps 220, 230, 240, and 250.

[0025] Figure 3 depicts flow chart 300 of method 310 having ordered steps 320, 330, and 340.

[0026] Plasmapheresis has the property of removing exosomes from the blood. Performing a plasmapheresis of one plasma volume and replacing this with albumin with or without saline, will have the effect of removing most of the autologous exosomes. The new art is to apply a plasmapheresis of one plasma volume before delivery of stem cells or exosomes. The stem cell or exosome brain area target is treated with ultrasound which has the effect of temporarily increasing the expression of VCAM and ICAM which are endothelial adhesion receptors for the ligands that allow stem cells and exosomes to attach to vessels in the area of interest. The art is to precede exosome or stem cell intravenous delivery with a plasmapheresis session within eight hours and to act prior to repopulation of the blood with autologous exosomes.

[0027] White cells including monocytes and lymphocytes have integrins that may attach to VCAM and ICAM which are stimulated by ultrasound treatment. White cells are not depleted by plasmapheresis so that they remain a competing element for allogeneic stem cells and exosomes. Integrin blocking agents including but not limited to natalizumab will block the ligands expressed by those white cells which are circulating at the time of administration. If plasmapheresis follows the integrin blocking administration, most of the latter drug will be removed so that subsequent stem cell or exosome administration may not be exposed substantially to drug blockade. In addition, stem cells and exosomes have other ligands whichAttorney Docket No. 103366.0024PCTinteract with endothelial receptors other than VCAM and ICAM. These other receptors may also be stimulated by ultrasound allowing for facilitated delivery with potentially less competition from autologous white cells and autologous exosomes.

[0028] In some embodiments, the exosome is derived from full (or partial) term placental tissue, umbilical tissue, mesenchymal stem cell, is derived synthetically, or some combination thereof. For example, exosomes could be cultured in a supernatant solution, with the solution containing the exosomes administered to the patient. Viewed from another perspective, the administration of exosomes to the patient includes the administration of exosomes or media (e.g., fluids, solutions, plasmas, infusions, powders, inhalants, dehydrates, etc.) comprising, containing, or conveying the exosomes, or combinations thereof. In some embodiments it is favorable to deliver more than one exosome across the BBB of the patient, in some cases delivering multiple exosomes of multiple types or derivations. The exosomes can also contain therapeutic agents (e.g., medication, protein, antibody, etc), whether native, engineered, or synthetic. .

[0029] The exosome can also be derived from cell lines containing receptors for immunoglobulins, for example a white blood cell, a lymphocyte, a monocyte, a T cell, a B cell, a dendritic cell, a neutrophil, a basophil, an eosinophil, a stem cell, or an immune cell progenitor. The exosome can otherwise be cultured or derived from an immune cell line, derived from a cell line that naturally includes an immune receptor (e.g., Ig receptor), derived from a cell line engineered to include an immune receptor, or the exosome is otherwise derived in a way to include an immune receptor. Generally the exosome comprises an antibody receptor. In some embodiments the exosome is modified to improve adhesion of the exosome with the BBB or to express an antibody receptor.

[0030] Stem cells can include embryonic stem cells, induced pluripotent stem cells, tissue stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, or epithelial stem cells. The stem cells can be natural, naturally derived, engineered, doped, or otherwise altered.

[0031] Stimulating the region of the BBB can be accomplished by directing focused energy at the region. For example, directing focused energy can include one or more of directing focused ultrasound, transcranial magnetic stimulation, shockwave, or light therapy, alternatively, in sequence, or other combination.Attorney Docket No. 103366.0024PCT

[0032] A pressure device is used to generate a shockwave, a type of pressure wave. A shockwave differs from an acoustic or ultrasound wave in that a shockwave is a rapidly-rising (e.g., short rise period) and brief pressure wave administered in a repetitive fashion, in this case to the head of the patient. Indeed, the shockwave waveform is characterized by a rapid raise with an abrupt falloff of drop, and is taught in the art as dangerous when applied to the head or brain. Viewed from another perspective, while an acoustic or ultrasound wave is designed to be sinusoidal, a shockwave is not and is rather abrupt.

[0033] The pressure device is used to facilitate delivery of the stem cell or exosome across the BBB in a manner that is non-damaging and non-harmful to the BBB or brain tissue. For example, shockwaves applied to a targeted region of the brain can surprisingly be used to induce transcytosis delivery of micro vesicles, stem cells, or exosomes across the BBB at the targeted region. However, shockwaves used by the inventive subject matter are applied to the BBB and brain in a manner unharmful to the targeted area, and migration of microvesicles, exosomes, or stem cells across the BBB are likewise induced or facilitated by shockwaves in an unharmful manner.

[0034] Pressure devices generating the shockwaves are typically electrohydraulic, electromagnetic, and piezoelectric, or combinations thereof. In some embodiments, more than one pressure device, or more than one type of pressure device, can be used, whether concurrently or sequentially. Shockwaves are typically induced for at least 1 minute, at least 10 minutes, or up to two hours, and can be of different types (e.g., different peaks and drops, duration, etc.). Further, the period of induced or facilitated migration of microvesicles across the BBB (e.g., via transcytosis) is temporary and can last 1, 2, 5, 10 minutes up to 1, 2, 3, or many hours after applying shockwaves.

[0035] Apparatus, systems, and methods for delivering an exosome across the blood-brain barrier (BBB) of a (preferably human) patient are contemplated. A region of the patient’s brain, preferably a region of potential therapeutic interest, is targeted with a sonic transducer. The sonic transducer is used to sonicate the targeted region of the brain, which improves the targeted region by facilitating selective delivery of the exosome to the targeted region, for example by increasing VCAM, ICAM, or other endothelial receptor expression at the region to increaseAttorney Docket No. 103366.0024PCTexosome transit of the BBB. It should be appreciated such means minimize damage to the BBB or surrounding brain tissue while promoting delivery of allogenic exosomes or stem cells.

[0036] The region of the patient’s brain to be targeted is preferably associated with a disease condition but may also be used for brain enhancement or optimization. In some embodiments, the disease condition is associated with at least one of dementia, a learning disorder, an anxiety disorder, a motor disorder, a consciousness disorder, a movement disorder, an attention disorder, a stroke, a vascular disease, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, cancer, schizophrenia, depression, substance abuse, and traumatic brain injury. However, any disease or disease condition that is pathologically associated with a region of the brain is appropriate for the contemplated methods. For example, the targeted region of the patient’s brain can be the frontal lobe, parietal lobe, occipital lobe, temporal lobe, hippocampus, hypothalamus, brain stem, cerebellum amygdala, corticospinal tract, thalamus, substantia nigra, basal ganglia, a tumor, a lesion, necrotic tissue. Heschl’s gyrus. Brodmann area 25, a point of injury, or any other region of interest. In some embodiments more than one region of the brain is targeted, for example to treat more than one disease or to combat a disease associated with more than one region of the brain.

[0037] In some embodiments, the exosome is derived from full (or partial) term placental tissue, umbilical tissue, mesenchymal stem cell, is derived synthetically, or some combination thereof. For example, exosomes could be cultured in a supernatant solution, with the solution containing the exosomes administered to the patient. Viewed from another perspective, the administration of exosomes to the patient includes the administration of exosomes or media (e.g., fluids, solutions, plasmas, infusions, powders, inhalants, dehydrates, etc.) comprising, containing, or conveying the exosomes, or combinations thereof. In some embodiments it is favorable to deliver more than one exosome across the BBB of the patient, in some cases delivering multiple exosomes of multiple types or derivations. The exosomes can also contain therapeutic agents (e.g., medication, protein, antibody, etc), whether native, engineered, or synthetic.

[0038] The sonic transducer is preferably an ultrasonic transducer, though infrasonic and audible transducers are also contemplated. The ultrasonic transducer preferably has a working frequency of at least 20Hz, more preferably at least 200Hz, at least 1MHz, or at least 5MHz. In someAttorney Docket No. 103366.0024PCTembodiments, working frequencies of more than 1GHz are used. Likewise, the sonic transducer is operated at a power of at least 100 mW / cm2, though powers of more than 200 mW / cm2, more than 400 mW / cm2, and more than 800 mW / cm2are also contemplated. Typically, the region of the patient’s brain is sonicated for at least 1 minute, sonication can last 10 minutes, 20 minutes, 30 minutes, or more than 45 minutes, and optionally include pulses of 1 second, 5 seconds, 10 seconds, or more than 30 seconds. In some embodiments, therapeutic agents are also administered to the patient’s blood stream, whether before, after, or during sonication of the targeted region of the brain.

[0039] In certain embodiments, the methods described herein are based, at least in part, on the recognition that endogenous extracellular vesicles, including autologous exosomes circulating in a patient’s blood volume, may compete with introduced stem cells or exosomes for transport mechanisms at the BBB. Such endogenous exosomes may engage shared transcytotic pathways, adhesion molecules, receptors, or energy-dependent transport processes that otherwise could be utilized by therapeutic stem cells or exogenously administered exosomes.

[0040] Accordingly, in some implementations, the delivery efficiency of a stem cell or an exosome across the BBB is improved by reducing the concentration of endogenous exosomes prior to therapeutic introduction, thereby decreasing competition for transport across the BBB. This recognition departs from conventional approaches in which circulating exosomes are not considered to be limiting factors or competitors in BBB transport.

[0041] In some embodiments, a plasmapheresis process is applied not solely for removal of pathological substances, antibodies, or toxins, but rather for the intentional depletion of autologous exosomes from the blood volume as a preparatory step for enhancing central nervous system delivery. Unlike traditional uses of plasmapheresis, which are typically directed toward disease mitigation or immune modulation, the plasmapheresis processes described herein are configured to optimize conditions for subsequent transport of therapeutic stem cells or exosomes across the BBB. In certain embodiments, removal of a plurality, or a majority, of autologous exosomes shifts transport dynamics at the BBB in favor of the introduced therapeutic agent. In this manner, plasmapheresis is repurposed as an enabling step for biologic delivery, rather than as a primary therapeutic intervention.Attorney Docket No. 103366.0024PCT

[0042] In some embodiments, the methods include a deliberate sequencing of steps comprising plasmapheresis, stimulation of the BBB, and introduction of a stem cell or an exosome, where the steps function cooperatively rather than independently. For example, plasmapheresis may be performed to reduce competition in the circulation, followed by localized BBB stimulation to transiently increase permeability or transport activity at a selected region, and subsequently introducing the therapeutic stem cell or exosome during a temporal window in which BBB transport conditions are optimized. The coordination of these steps may result in synergistic enhancement of BBB crossing, exceeding what would be expected from performing any individual step alone. The methods are not limited to a particular order, and in some embodiments the steps may partially overlap, be repeated, or be customized based on patient-specific factors.

[0043] In certain embodiments, stimulation of a region of the BBB comprises applying focused energy, such as focused ultrasound, transcranial magnetic stimulation, shockwaves, or light energy, to transiently increase transport across the BBB without causing permanent structural damage. In contrast to approaches that rely solely on barrier disruption, the methods described herein may leverage BBB stimulation in combination with circulation-level optimization, such that lower stimulation intensities or reduced exposure times may be sufficient when endogenous transport competition has been reduced via plasmapheresis. Thus, BBB stimulation functions as one component of a multi-factor delivery system, rather than as the sole mechanism for enabling delivery.

[0044] In some embodiments, the methods include administering an integrin blocking agent, such as natalizumab or another integrin inhibitor, in a manner that temporally and functionally differs from conventional uses of such agents. While integrin blocking agents are commonly employed to inhibit leukocyte migration across the BBB or to reduce inflammation, the present methods may utilize integrin blockade to transiently modulate adhesion dynamics at the BBB, thereby influencing transport behavior of therapeutic stem cells or exosomes.

[0045] In certain embodiments, plasmapheresis is subsequently applied to remove free or circulating integrin blocking agents from the blood volume, allowing localized or transient effects at the BBB while reducing systemic exposure. This sequencing permits controlledAttorney Docket No. 103366.0024PCTmodulation of BBB transport properties without prolonged integrin inhibition, which would otherwise be expected to impede trafficking rather than facilitate delivery.

[0046] In some implementations, the plasmapheresis process removes free or unbound integrin blocking agents following their administration, enabling a decoupling of systemic pharmacokinetics from localized BBB effects. This approach allows integrin blockade to be used as a temporary conditioning tool, after which the circulating presence of the blocking agent is reduced. Such use contrasts with conventional teachings, which typically caution against removal of administered biologic agents during plasmapheresis. Accordingly, the methods described herein enable fine control over the duration, intensity, and localization of BBB modulation, supporting safer and more targeted CNS delivery.

[0047] In some embodiments, both a stem cell and an exosome are introduced to the patient, either concurrently or sequentially. For example, exosomes may be used to condition the BBB environment, modulate signaling pathways, or prime cellular uptake prior to or concurrent with stem cell delivery. The combination of stem cells and exosomes may provide complementary or cooperative effects, including enhanced transport, improved survival, or improved therapeutic efficacy within the central nervous system.

[0048] Taken together, the methods described herein represent a system-level approach to BBB delivery that integrates circulatory conditioning, barrier modulation, and therapeutic administration. Rather than relying on a single mechanism, the methods manipulate multiple biological layers — circulation, endothelial transport, and cellular adhesion — to achieve improved delivery outcomes. This integrated design departs from conventional approaches that treat plasmapheresis, BBB stimulation, exosome delivery, and integrin blockade as isolated or unrelated therapies. The inventive concepts arise from recognizing how these disparate techniques can be combined and timed to reinforce one another, yielding results not predictable from the individual components.

[0049] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.Attorney Docket No. 103366.0024PCT

[0050] As used herein, and unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously.

[0051] Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints, and open-ended ranges should be interpreted to include commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.

[0052] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range including endpoints, as well as interstitial ranges between each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0053] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0054] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, theAttorney Docket No. 103366.0024PCTinventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0055] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

Attorney Docket No. 103366.0024PCTCLAIMSWhat is claimed is:

1. A method of delivering a stem cell or an exosome across a blood-brain barrier (BBB) of a patient, comprising:applying a plasmapheresis process to the patient, wherein a plurality of autologous exosomes are removed from a blood volume of the patient;stimulating a region of the BBB to facilitate delivery of the stem cell or the exosome across the BBB at the region; andintroducing the stem cell or the exosome to the region of the BBB.

2. The method of claim 1, wherein the stem cell or the exosome is allogenic.

3. The method of claim 1 or claim 2, wherein a time between the steps of applying the plasmapheresis process and introducing the stem cell or the exosome is no more than 8 hours.

4. The method of any one of claims 1 to 3, wherein both the stem cell and the exosome are introduced to the region of the BBB.

5. The method of any one of claims 1 to 4, wherein a majority of autologous exosomes are removed from the blood volume during the plasmapheresis process, thereby limiting autologous exosomes in the blood volume that compete with the stem cell or the exosome to cross the BBB.

6. The method of any one of claims 1 to 5, wherein the plasmapheresis process comprises replacing a plasma volume of the blood volume with a donor plasma volume, an albumin volume, or a combined saline and albumin volume.

7. The method of any one of claims 1 to 6, wherein the stem cell or the exosome is introduced to the patient intravenously, intramuscularly, intrathecally, subcutaneously, orally, or inhaled8. The method of any one of claims 1 to 7, further comprising administering an integrin blocking agent to the patient.Attorney Docket No. 103366.0024PCT9. The method of claim 8, wherein the integrin blocking agent is natalizumab or other integrin blocker.

10. The method of claim 8, wherein the step of administering the integrin blocking agent precedes the step of applying the plasmapheresis process.

11. The method of any one of claims 1 to 8, wherein the step of stimulating the region of the BBB comprises directing focused energy at the region.

12. The method of claim 11, wherein directing focused energy comprises one of directing a focused ultrasound, a transcranial magnetic stimulation, or a shockwave.

13. A method of delivering a stem cell or an exosome across a blood-brain barrier (BBB) of a patient, comprising:administering a dose of integrin blocking agents to the patient;applying a plasmapheresis process to the patient;stimulating a region of the BBB to facilitate delivery of the stem cell or the exosome across the BBB at the region; andintroducing the stem cell or the exosome to the region of the BBB.

14. The method of claim 13, wherein the plasmapheresis process comprises removing a free integrin blocking agent of the dose from a blood volume of the patient.

15. The method of claim 13 or claim 14, wherein the plasmapheresis process comprises removing a plurality of autologous exosomes from a blood volume of the patient.

16. The method of any one of claims 13 to 15, wherein the plasmapheresis process comprises replacing a plasma volume of the blood volume with a donor plasma volume, an albumin volume, or a combined saline and albumin volume.

17. The method of any one of claims 13 to 16, wherein stimulating the region of the BBB comprises one of directing a focused ultrasound, a transcranial magnetic stimulation, a shockwave, or a light energy at the region.Attorney Docket No. 103366.0024PCT18. The method of any one of claims 13 to 17, wherein the exosome or stem cell is modified to improve adhesion of the exosome with the BBB.

19. The method of any one of claims 13 to 18, wherein the step of administering the integrin blocking agent precedes the step of applying the plasmapheresis process.

20. A method of delivering a stem cell or an exosome across a blood-brain barrier (BBB) of a patient, comprising:administering a dose of integrin blocking agents to the patient;stimulating a region of the BBB to facilitate delivery of the stem cell or the exosome across the BBB at the region; andintroducing the stem cell or the exosome to the region of the BBB.

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