Systems and methods for exosome-mediated targeted delivery of capsid-bound gene editing material

Abstract

Gene editing material transportation constructs consistent with embodiments of this disclosure are designed to enable transporting of proteins and large DNA segments to different cell types around the body including across the blood-brain barrier, for targeted delivery to a variety of intended cell types. The constructs include gene editing material-containing inner capsid structures surrounded by exosome structures. The exosome structures contain a plurality of first recombinant polypeptides including a tetraspanin, e.g., CD9, and photoreceptor cryptochrome 2 protein (CRY2) fusion protein, while the inner capsid structure contains a plurality of second recombinant polypeptides including an inner capsid protein, e.g., CA / p24, and truncated CRY-interacting basic helix-loop-helix 1 protein (CIBN) fusion protein. The CRY2 and CIBN portions of the constructs are a light-based dimerization system that enable reversible association of the first and second recombinant polypeptides to selectively assemble inner capsid constructs into exosomes for export.

Description

SYSTEMS AND METHODS FOR EXOSOME-MED1ATED TARGETED DELIVERY OF CAPSID-BOUND GENE EDITING MATERIALCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent ApplicationNo. 63 / 639,903, filed April 29, 2024, which is incorporated by reference as if disclosed herein in their entireties.BACKGROUND

[0002] There is an urgent need for transformative therapeutic approaches in the field of neurolog}'. Currently, Alzheimer’s and Parkinson’s disease alone affect 8 million Americans, the number of people with each disease is expected to double by 2050. While there have been some clinical successes in recent years, most of these, such as Lecanemab and Memantine, only amounted to a temporary slowing of the disease, not a curing of it. Clinical gene editing is still in its early days, and there have been very limited in vivo use cases. Further, the applications that can be targeted rely predominantly on introducing single base pair changes (base editors) or gene knockouts, but not insertions.

[0003] In recent years, research interest in the use of exosomes as a targeted delivers’ mechanism for potential therapeutics has exploded in popularity, primarily due to their ability to elicit little immune response even with high-dose administration of exosomes. Exosomes function as a means for the body to deliver packages of signaling molecules all across the body and are universal across many cell types. These natively produced vesicles can achieve targeted transport throughout the body, and even cross the blood-brain barrier (BBB), without generating an immune response since they contain all native cellular markers associated with the cell-type that created them. Typically measured between 30- 150 nm in diameter, exosomes have gained significant attention in recent years due to their role in intercellular communication. Exosomes have demonstrated their ability to be targeted to different cell types primarily by altering the proteins on their surface, which grants them the abi 1 i ty to be taken up by desired cell types with different producing cell types such as dendritic cells targeting immune function in the brain and blood exosomes being taken up more by brain endothelial cells. Exosomes can even be targeted to specific regions of the brain, and their targeting can change based on disease state of the targeted areas, such as what has been observed with human mesenchymal stem cells altering their targeting and retention time in Alzheimer's disease models compared to healthy models. Mesenchymal stem cell-derived exosomes have even been used with iron oxide nanoparticles tomanipulate exosomes to desired regions using strong magnets. Exosome targeting can be modified in several ways, such as by modifying the exosome proteins expressed, or by additional chemical modifications that can be introduced after production. Because of this, exosomes represent a strong candidate for advanced therapeutics, including for ferrying relatively large volumes of various compounds and molecules across the BBB.

[0004] One of the most effective markers for exosome uptake through the BBB is a fusion of the rabies viral glycoprotein (RVG) to a tetraspanin known to be included in exosome formation, Lamp2b. Previous research has shown a broad tropism to neurons, oligodendrocytes, and microglia in the brain for exosomes containing the RVG-Lamp2b fusion. RVG-Lamp2b fusion exosomes have been used previously to deliver gene editing technologies including miRNAs and siRNAs, where previous studies were able to knock down protein expression and mRNA levels of proteins related to Alzheimer’s disease like BACE1 by up to 60% while demonstrating very limited or no uptake by other cells and tissue types in the body. RVG- tagging alone gives exosomes broad targeting to microglia, oligodendrocytes, and neurons. Additional exosome tags can also bias exosomes to more specific cell types in the CNS, such as astrocytes, microglia, and oligodendrocytes.

[0005] The method by which exosomes normally cross the BBB is through adsorptive transcytosis, which allows for the intact exosome to be ferried across the BBB and released into the CNS while still containing relevant markers to target various cell types as desired. Prior research demonstrates that the type of cell that produces the exosomes influences the exosomes' targeting and uptake rate across the BBB. The rate of uptake for these unmodified exosomes can also be quite high, with a previous study using intranasal injection in mice showing that 60% of all microglia in the treated mice were PKH26-positive (a fluorescent marker contained in the exosomes) after just one hour post-intervention. Specific exosome types can even achieve preferential passage across the BBB in specific regions of the brain to give additional opportunities for cell-type targeting. Several other protein modifications have also been documented as enhancing targeting of specific cell types including several varieties of cancer, i.e., IRGD and GE11 both target breast cancer cells.

[0006] Crossing the BBB and entering the correct cell type is not the end of the road when it comes to performing targeted gene edits in vivo. Many ty pes of neurons in the brain are nondividing cells and have mechanisms to recognize and degrade foreign nucleic acids. Of particular concern are systems like cGAS-Sting and cGAMP, which identify foreign DNA and the RIG-I like receptor (RLR) family, which detects foreign RNA. CRISPR-Cas9 has also beenshown to activate the type-1 INF pathway, which could also result in a severe reaction. These systems have been verified to be active in many cell types outside of just immune cells, including neurons.

[0007] Once the gene-editing cargo has been delivered to the desired cell type, if any kind of new genetic information is being introduced or integrated, then these genes should be protected on their journey to the nucleus or else they run the risk of triggering the cell’s innate immune responses, as well as being degraded by nucleases expressed in the cytosol. Without protection and quick transport to the nucleus, most forms of DNA have a half-life of 50-90 minutes in the cytosol of human cells. Without active transport of the DNA there is no real chance to reach the nucleus, much less penetrate the nuclear membrane if the cell is not actively dividing. Aside from liposomes, there are currently no non-viral methods that can be used to pass large sections of DNA for integration past the BBB, with AAV vectors being the only method to deliver sections of DNA this way. However, liposomes generally perform worse than exosomes in terms of targeting, uptake, and have higher immune responses. Further, the small size of AAVs greatly limits their ability to transport genetic materials for editing since they can only transport DNA cargo up to 4.7 kB. Their efficiency when attempting to target different cell types also ranges widely between different cell types in the CNS and is cited as reducing desired protein concentrations by about 45% when performing knockouts (no additional integration) and with intracerebroventricular injection. Generally, AAVs as a delivery vehicle also show much lower uptake rates in neurons when administered via intravenous injection, with rates generally around -16% for AAV9s and at best -35% for modified MaCPNS2 AAVs. which are also less specific in their targeting. AAVs delivering CR1SPR-Cas9 have also demonstrated the ability to elicit significant immune response that prevents further treatments, and the high concentration of AAVs to achieve efficient transduction has also proven deadly in some clinical trials.

[0008] HIV-1 CA / p24 inner capsid proteins represent a strong candidate for the safe import of nucleotides into the nuclei of cells (both dividing and non-dividing) while minimizing the potential immunogenicity of the system. Lentiviral vectors have been used for more than a decade as a method to perform gene editing. One of the advantages to using lentiviral vectors comes from the inner capsid protein CA / p24, which provides protection and transport for genetic material while allowing for the reliable transfection of non-dividing cells. This inner capsid shell allows 10 kb of DNA (in native HIV-1) to enter the cell and be ferried into the nucleus without risk of generating an immune response or degradation of the nucleotides. Recent studies have demonstrated the effectiveness of lentiviral systems combined with Cas9 called LentiCRISPRsystems, suggesting that the inner capsid could also be used in CRISPR-Cas9 based insertional methods like twin prime editing.

[0009] The inner capsid of HIV- 1 is composed of 1,000-1,500 copies of CA / p24, arranged in about 250 hexamers and about 12 pentamers. Previous research conducted to verify the role of CA / p24 in the process of HIV-1 infection has verified that CA / p24 can be fused to relatively large protein constructs such as eGFP and still achieve correct assembly of the inner capsid and be successfully trafficked into the nucleus intact. It has also been shown that it is primarily the hexamers on the sides of the inner capsid that are responsible for binding to FEZ1 in the cells, which allows them to move unidirectionally along the microtubule network towards the nucleus of the cell.

[0010] The use of lentiviral virus-like particles (VLPs) generally results in a very high editing efficiency. Native HIV has an integration efficiency of upwards of 90%. Integrase deficient lentiviral vectors (IDLVs) demonstrated upwards of 80% editing when carrying Cas9 in vivo in the brain when using intracerebroventricular injection. This is almost twice the efficiency demonstrated by AAVs administered in the same way. A variety of different pseudotyping options have also demonstrated different neurotropism upon intracerebroventricular injection when incorporated into lentiviral vectors. However, normal lentiviral vectors cannot cross the BBB effectively, so while they are effective in transduction, they are lacking in delivery.SUMMARY

[0011] Aspects of the present disclosure are directed to modified proteins, e.g., produced in cells such as in HEK293F cells, which facilitate formation of inner capsid structures and their loading into exosome structures. Application of light was used to control assembly of inner capsid proteins within the exosome structures. Purified exosomes were injected into mice and deliver}' of the inner capsid structures across the blood-brain barrier (BBB) of those mice was confirmed.

[0012] Aspects of the present disclosure are directed to a gene editing material transportation system. In some embodiments, the gene editing material transportation system includes a first recombinant polypeptide including an exosome-associating complex. In some embodiments, the exosome-associating complex including an exosome-associating protein and a first light-controlled association domain. In some embodiments, the gene editing material transportation system includes a second recombinant polypeptide including an inner capsid complex. In some embodiments, the inner capsid complex includes an inner capsid protein and asecond light-controlled association domain. In some embodiments, the gene editing material transportation system includes an inner capsid structure including a plurality of the second recombinant polypeptides, and gene editing materials at least partially positioned within the inner capsid structure.

[0013] Tn some embodiments, the exosome-associating protein is a tetraspanin. In some embodiments, the tetraspanin includes CD63, CD81, CD82, CD9, or combinations thereof. In some embodiments, the first light-controlled association domain includes photoreceptor cryptochrome 2 protein (CRY2) and the second light-controlled association domain includes truncated CRY-interacting basic-helix-loop-helix 1 protein (CIBN). In some embodiments, the inner capsid protein includes an HIV-1 inner capsid protein. In some embodiments, the HIV-1 inner capsid protein includes CA / p24. In some embodiments, the tetraspanin includes CD9, the first light-controlled association domain includes CRY2, the second light-controlled association domain includes CIBN, and the inner capsid protein includes CA / p24. In some embodiments, the gene editing materials include one or more proteins, oligonucleotides, or combinations thereof.

[0014] Aspects of the present disclosure are directed to a gene editing material transportation system including an exosome structure including a plurality of first recombinant polypeptides, wherein the first recombinant polypeptide includes a tetraspanin and CRY2. In some embodiments, the gene editing material transportation system includes an inner capsid structure surrounded by the exosome structure, wherein the inner capsid structure includes a plurality of second recombinant polypeptides, and the second recombinant polypeptides includes an inner capsid protein and a truncated CRY-interacting basic-helix-loop-helix 1 protein (CIBN). In some embodiments, the gene editing material transportation system includes gene editing materials at least partially positioned within the inner capsid structure.

[0015] Aspects of the present disclosure include a method of editing genomic material in a target organism. In some embodiments, the method includes providing an assembly medium including a first recombinant polypeptide including an exosome-associating complex, the exosome-associating complex including a tetraspanin and a first light-controlled association domain; a second recombinant polypeptide including an inner capsid complex, the inner capsid complex including an inner capsid protein and a second light-controlled association domain; and gene editing materials. In some embodiments, the method includes contacting the assembly medium with light from a light source to assemble the first recombinant polypeptide with the second recombinant polypeptide via association of the first light-controlled association domainwith the second light-controlled association domain. In some embodiments, the method includes assembling a gene editing material transportation construct. In some embodiments, the method includes administering an effective amount of the gene editing material transportation construct to target cells. In some embodiments, the method includes fusing the exosome structure of the gene editing material transportation construct to membranes of the target cells to release the inner capsid structure into the target cells.

[0016] In some embodiments, assembling a gene editing material transportation construct includes assembling exosome structures including a plurality first recombinant polypeptides with bound second recombinant polypeptides positioned within the exosome structure, and assembling an inner capsid structure composed of second recombinant polypeptides within the exosome structure, wherein a concentration of gene editing materials is positioned within the inner capsid structure.

[0017] In some embodiments, contacting the assembly medium with light from a light source to assemble the first recombinant polypeptide with the second recombinant polypeptides includes alternating between a first phase where the light is applied and a second phase wherein the light is not applied, wherein the first phase is shorter than the second phase. In some embodiments, the first phase has a duration of at least 1 minute and the second phase has a duration of at least 2 minutes.

[0018] In some embodiments, administering an effective amount of the gene editing material transportation construct to target cells includes contacting an amount of the gene editing material transportation construct with the blood-brain barrier of a target organism and passing through it to reach targeted cell ty pes. In some embodiments, the outer exosome structure and its associated proteins are used for targeted delivery’ of gene editing contents to specific cell types.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings show embodiments of the disclosed subject matter for the purpose of illustrating the invention. How ever, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

[0020] FIGs. 1 A-1B are schematic representations of a gene editing material transportation system according to embodiments of the present disclosure;

[0021] FIG. 2 is a chart of a method of editing genomic material in a target organism according to embodiments of the present disclosure;

[0022] FIGs. 3A-3B are graphs confirming generation of gene editing material transportation constructs according to embodiments of the present disclosure; and

[0023] FIGs. 4A-4I are immunohistochemistry images of mouse brains with antibodies for HIV CA / p24 demonstrating treatment with gene editing material transportation constructs according to embodiments of the present disclosure. FIGs. 4A-4B show negative controls to establish background fluorescence; FIGs. 4C-4D show slices from brains 6 hours post injection; FIGs. 4E-4F show slices from brains 24 hours post injection; FIGs. 4G-4I show' slices from brains 30 hours post injection.DETAILED DESCRIPTION

[0024] Referring now to FIGs. 1 A-1B, some aspects of the disclosed subject matter are directed to a gene editing material transportation system 100. System 100 is configured to deliver gene editing materials G to target cells, e.g., cells in in vitro culture, non-dividing cells in vivo, etc. In some embodiments, system 100 is configured to transport gene editing materials G across the blood-brain barrier (BBB) of a target organism, e g., a human patient, as will be discussed in greater detail below. In some embodiments, gene editing materials G include one or more proteins, oligonucleotides, or combinations thereof.

[0025] In some embodiments, system 100 includes a gene editing material transportation construct 102. In some embodiments, gene editing material transportation construct 102 includes an exosome structure 104. In some embodiments, exosome structure 104 includes a plurality of phospholipids, proteins, etc., assembled into a membrane 104A defining an interior area 1041 and an exterior area 104E. In some embodiments, membrane 104A at least partially encloses interior area 1041. In some embodiments, membrane 104A completely encloses interior area 1041.

[0026] Still referring to FIG. 1 A, in some embodiments, system 100 includes a first recombinant polypeptide 106. In some embodiments, first recombinant polypeptide 106 includes an exosome-associating complex. In some embodiments, the exosome-associating complex includes an exosome-associating protein 106 A and a first light-controlled association domain 106B. In some embodiments, exosome-associating protein 106A is a tetraspanin. In some embodiments, the tetraspanin includes CD63, CD81, CD82. CD9, or combinations thereof. In some embodiments, first light-controlled association domain 106B is configured to associate w ith a corresponding second light-controlled association domain in the presence of light, and disassociate from the second light-controlled association domain in the absence of light, as willbe discussed in greater detail below. In some embodiments, first light-controlled association domain 106B includes photoreceptor cryptochrome 2 protein (CRY2), truncated CRY- interacting basic-helix-loop-helix 1 protein (CIBN), VfAUl-LOV, CrPHl-LOV, NcVVD-LOV, AtCRY2-PHR, AtCIBl, RsLP-LOV, MxCarH-CBD, TtCarH-CBD, ScPHl-S, AtPHYB-S. AtPIF6, AsLOV2-EcSsra, EcSspb. AsLOV2-pep, HsPDZlb, or combinations thereof.

[0027] In some embodiments, a plurality of first recombinant polypeptides 106 are associated with exosome structure 104. In some embodiments, first recombinant polypeptides 106 are at least partially incorporated into exosome structure 104, e.g., membrane 104A. In some embodiments, first recombinant polypeptides 106 are bound to exosome structure 104, e.g., via one or more linkers, direct binding, etc. In some embodiments, a plurality of first recombinant polypeptides 106 are associated with exosome structure 104 such that the first light-controlled association domains are positioned within interior area 1041.

[0028] In some embodiments, gene editing material transportation construct 102 includes an inner capsid structure 108. In some embodiments, inner capsid structure 108 is configured to mimic the function of viral capsids in facilitating cytoplasmic transport, nuclear transport, etc. In some embodiments, inner capsid structure 108 includes an interior area 1081 and an exterior area 108E. In some embodiments, inner capsid structure 108 at least partially encloses interior area 1081, i.e., the structure 108 is a partial capsid. In some embodiments, inner capsid structure 108 completely encloses interior area 1081 (see, e.g., FIG. IB).

[0029] Referring again to FIG. 1 A, in some embodiments, system 100 includes a second recombinant polypeptide 110. In some embodiments, second recombinant polypeptide 110 includes an inner capsid complex. In some embodiments, the inner capsid complex includes an inner capsid protein 110A and a second light-controlled association domain 110B. In some embodiments, second light-controlled association domain HOB is bound to the N-terminal end of inner capsid protein 110A. In some embodiments, second light-controlled association domain HOB is bound to the C-terminal end of inner capsid protein 110A. In some embodiments, second light-controlled association domain 110B includes CRY2, CIBN, VfAUl- LOV. CrPHl-LOV, NcVVD-LOV, AtCRY2-PHR, AtCIBl, RsLP-LOV. MxCarH-CBD, TtCarH-CBD, ScPHl -S, AtPHYB-S, AtPIF6, AsLOV2-EcSsra, EcSspb, AsLOV2-pep, HsPDZlb, or combinations thereof.

[0030] In some embodiments, inner capsid protein 110A includes an HIV-1 inner capsid protein. In some embodiments, the HIV-1 inner capsid protein includes CA / p24. In some embodiments, CA / p24 includes the wild-type structure, modified versions thereof that retain thefunctionality of the wild-type structure, etc. In some embodiments, inner capsid proteins 110A, e.g., CA / p24 units, include a mutation to force them into a pentameric configuration to allow these units to be placed more on the periphery of inner capsid structure 108.

[0031] As discussed above, in some embodiments, second light-controlled association domain HOB is configured to associate with first light-controlled association domain 106B in the presence of light, and disassociate from first light-controlled association domain 106B in the absence of light. In some embodiments, first light-controlled association domain 106B includes CRY2. In some embodiments, second light-controlled association domain 110B includes CIBN.

[0032] In some embodiments, first light-controlled association domain 106B and second light-controlled association domain 110B associate in the presence of light having a wavelength between about 480 nm and about 500 nm. In some embodiments, first light-controlled association domain 106B and second light-controlled association domain HOB associate in the presence of light having a wavelength of about 490 nm. In some embodiments, the applied light is alternated between a first phase where the light is applied and a second phase wherein the light is not applied. In some embodiments, the first phase is shorter than the second phase. In some embodiments, the first phase has a duration above about 15 seconds. In some embodiments, the first phase has a duration above about 1 minute. In some embodiments the second phase has a duration above about 30 seconds. In some embodiments the second phase has a duration above about 2 minutes. In some embodiments, the first phase has a duration of at least 1 minute and the second phase has a duration of at least 2 minutes.

[0033] In some embodiments, inner capsid structure 108 includes a plurality of second recombinant polypeptides 110. In some embodiments, inner capsid structure 108 is composed of hexamers, pentamers, etc. of second recombinant polypeptides 110. In some embodiments, inner capsid structure 108 is composed of hexamers, pentamers, etc. of inner capsid protein 110A, with the associated second light-controlled association domain 110B positioned in exterior area 108E. In some embodiments, gene editing materials G are positioned within inner capsid structure 108, e.g., in interior area 1081.

[0034] Referring again to FIG. 1A, gene editing material transportation construct 102 is assembled such that exosome structure 104 surrounds inner capsid structure 108 and a concentration of gene editing materials G is positioned within the inner capsid structure. In some embodiments, inner capsid structure 108 is first assembled such that second light- controlled association domains 110B are positioned in exterior area 108E. Subsequently, uponapplication of light, first recombinant polypeptides 106 associate with inner capsid structure 108 via the interaction between first light-controlled association domain 106B and second light- controlled association domain HOB, allowing inner capsid structure 108 to self-assemble at the surface of multi vesicular bodies and be encapsulated into exosome structure 104, with second recombinant polypeptides 110 positioned within the exosome structure and bound to first recombinant polypeptides 106. Upon removal of the light, first light-controlled association domain 106B and second light-controlled association domain HOB disassociate and result in the formation of gene editing material-containing capsids within an exosome. This exosome, e.g., gene editing material transportation construct 102. can then be used in processes to deliver gene editing material G to target cells, as will be discussed in greater detail below.

[0035] System 100 controllably associates inner capsid proteins with exosome-bound proteins to create virus-like particles containing protective capsid cores that leverages the high uptake efficiency, lack of immune response, specific targeting capabilities, and robust ability to cross the BBB of exosomes, along with the high transfection rates and large cargo capacity characteristics of lentiviral vectors. The gene editing material transportation constructs according to embodiments of the present disclosure, e g., gene editing material transportation construct 102, allow for the nuclear import of DNA of lOkB or higher while taking advantage of the ability of exosomes to both target a multitude of cell types as well as safely cross the BBB.

[0036] In an exemplary embodiment, 490 nm light was used to temporarily and quickly associate proteins for the loading of exosomes. CD9 has been shown to have the high yield in terms of inclusion of fusion proteins into exosomes and can be used in embodiments of the present disclosure. The optical binding domain CIBN was fused to the N-terminal of CA / p24, and its binding partner CRY2 was fused to the N-terminal of CD9. System 100 employs light- controlled association domains rather than directly fusing inner capsid proteins to tetraspanins, e.g.. CD9. because fusing to a membrane bound protein could tie the inner capsid to the cell membrane on entrance into the targeted cell which would inhibit trafficking of the capsid into the nucleus. Previous studies have also attempted to fuse proteins to luminal proteins in exosomes, however those studies showed very poor inclusion of these fusions in produced exosomes. The CIBN domain attached to the CA / p24 monomers is reasonably sized at 19 kDa, and smaller than eGFP that has been previously shown to be successfully ferried into the nucleus when fused with CA / p24. The CIBN domain is also quite flexible so it would not interfere with nuclear transport or capsid formation.[0037| In some embodiments, system 100 includes one or more exosome producing cell lines (not pictured). In some embodiments, the cell lines have been modified, e.g., via incorporation of one or more recombinant plasmids, with genomically integrated versions of gene editing material transportation constructs 102 and / or its components, e.g., 106B,110B, to increase production of lentiviral particles. In some embodiments, the cell lines also include the additional genetic elements including elements of the gag-pol, CRISPR domains, etc. to facilitate integration of gene editing material G into target cells. In an exemplary embodiment, plasmids coding for gag-pol are modified to replace matrix proteins with light-controlled association domains, such as those discussed above. In some embodiments, a copy of spCas9 (with dual nuclear localization signals) can be added between the light-controlled association domain and the rest of the gag-pol complex, e.g., with an inactivating mutation D64V in HIV IN, while adding an additional HIV protease cleavage site at both ends of the spCas9 as well. This same approach has also been used for dCas9 delivery.

[0038] Referring now to FIG. 2, some embodiments of the present disclosure are directed to a method 200 of editing genomic material in a target cell and / or organism, e.g., specific cell types in the central nervous system (CNS) of a human patient. In some embodiments, at 202, an assembly medium is provided in which to assemble gene editing material transportation constructs, e.g., gene editing material transportation construct 102 discussed above. In some embodiments, the assembly medium includes a first recombinant polypeptide including an exosome-associating complex. As discussed above, in some embodiments, the exosome- associating complex includes a tetraspanin and a first light-controlled association domain. In some embodiments, the first recombinant polypeptides include an exosome-associating protein and a first light-controlled association domain. In some embodiments, the exosome-associating protein is a tetraspanin. In some embodiments, the tetraspanin includes CD63, CD81, CD82, CD9, or combinations thereof. In some embodiments, the tetraspanin includes CD9. In some embodiments, the first light-controlled association domain includes CRY2.

[0039] In some embodiments, the assembly medium includes a second recombinant polypeptide including an inner capsid complex. In some embodiments, the inner capsid complex includes an inner capsid protein and a second light-controlled association domain. In some embodiments, the inner capsid protein includes an HIV-1 inner capsid protein. In some embodiments, the HIV-1 inner capsid protein includes CA / p24. In some embodiments, the second light-controlled association domain includes CIBN.[0040| In some embodiments, the assembly medium includes gene editing materials. In some embodiments, the gene editing materials include one or more proteins, oligonucleotides, or combinations thereof. These materials are produced in desired cell types by either temporary plasmid transduction or by more permanent genomic integration, whereupon the cells produce and package the gene editing material in a culture. In some embodiments, these materials are grown and recovered continuously or semi-continuously, e.g., with periodic centrifuging to harvest them.

[0041] Referring again to FIG. 2, in some embodiments, at 204, the assembly medium, e.g., the cells therein, is contacted with, e.g., exposed to, light from a light source. As discussed above, contacting 204 assembles the first recombinant polypeptide with the second recombinant polypeptide via association of the first light-controlled association domain with the second light- controlled association domain inside the cell. In some embodiments, the light used to contact 204 has a wavelength between about 480 nm and about 500 nm. In some embodiments, the light used to contact 204 has a wavelength of about 490 nm.

[0042] In some embodiments, at 206, a gene editing material transportation construct is assembled. As discussed above, in some embodiments, assembling the gene editing material transportation construct 206 assembles exosome structures and inner capsid structures. In some embodiments, the exosome structures include a plurality of first recombinant polypeptides. In some embodiments, the inner capsid structures include a plurality of second recombinant polypeptides. In some embodiments, the inner capsid structures are composed of second recombinant polypeptides.

[0043] In some embodiments, the inner capsid structures are assembled before, concurrently, or after assembling the exosome structure, or combinations thereof. In some embodiments, the inner capsid structure is formed by association of the inner capsid proteins, such that the second light-controlled association domains are disposed on the surface of the inner capsid structure. In some embodiments, the inner capsid structure is formed by association of the inner capsid proteins such that gene editing materials are encapsulated therein. Contacting 204 then associates a plurality of second recombinant polypeptides of the inner capsid with the surface of forming exosomes and facilitates their encapsulation by the outer exosome structure with integrated first recombinant polypeptides positioned within the exosome structure and a concentration of gene editing materials positioned within the inner capsid structure, thus assembling 206 the gene editing material transportation construct. The light can then be removed, such that the inner capsid structure can detach from the outer exosome structure. Insome embodiments, contacting 204 occurs concurrently or prior to assembly of the inner capsid structure, such that the assembly medium includes a concentration of first recombinant polypeptides associated with second recombinant polypeptides. Exosome structures can then be assembled with bound second recombinant polypeptides positioned within the exosome structure. The inner capsid structure can then assemble within the exosome structure, e.g., during or after contacting 204, to complete assembling 206 of the gene editing material transportation constructs.

[0044] Referring again to FIG. 2, at 208, an effective amount of the gene editing material transportation constructs are administered to target cells. In some embodiments, the gene editing material transportation constructs are administered 208 to cells during an in vitro process. In some embodiments, the gene editing material transportation constructs are administered 208 to cells in vivo, e.g., for transport across the BBB of a target organism such as a human patient. In some embodiments, at 210, the exosome structure of the gene editing material transportation construct is fused to membranes of the target cells to release the inner capsid structure into the target cells.EXAMPLES

[0045] HEK293F cells, a human embryonic kidney suspension cell line, were used to produce gene editing material transportation constructs according to embodiments of the present disclosure. The cells were transfected with 3 plasmids using the Expifectamine293 transfection system (Life Technologies Corporation, Carlsbad, CA). The three plasmids expressed a Rabies viral glycoprotein (RVG)-Lamp2b fusion protein, a CRY2-CD9 fusion, and a CIBN-CA / p24 fusion, each was transfected in equal amounts. The HEK293F cells were passaged 3 times post thaw and allowed to overgrow prior to transfection. The cells were grown 3 days following transfection, with shaking at 120 rpm and in the presence of a 490 nm LED from ThorLabs. An intensity curve was generated using a light intensity measurement tool to determine the distance at which to place the LED to achieve the desired intensity of 50 pW / cm2on the liquid in the flask. Two sets of gene editing material transportation constructs were produced and harvested under different light conditions to determine the effect on packing that occurred with a change in pulse frequency and length. The lighting was controlled by an Arduino UNO R4. The first set of exosomes was produced with the light being on for 1 minute and then off for 2 minutes. The second set was produced with the light on for 15 seconds and the light off for 30 seconds. In both cases, a volume of 50 ml of cells was cultured in 250 ml shake flasks.[0046| Referring now to FIGs. 3A-3B, after three days of growth with the light, the cultures were transferred to sterile 50 ml Falcon tubes, spun down at 2000 g, and the supernatant was collected and passed through a 0.22 pm filter. This filtrate was then ultracentrifuged for 70 minutes at 100,000 g, the supernatant was discarded and the pellet resuspended in 40 ml of sterile filtered PBS, a second ultracentrifugation under the same conditions was done, with the pellet being resuspended in 1 ml of sterile filtered PBS before being aliquoted into 100 pl amounts for freezing at -80°C. One aliquot of each sample was sent out for NTA analysis. The presence of the CA / p24 proteins in the gene editing material transportation constructs was verified by lysing with different concentrations of R1PA buffer before performing a western blot. Decreased packaging efficiency was observed in the set of exosomes produced with quicker light switching, which showed thinner bands in the western blots, decreased staining in the immunohistochemistry, and a decrease in mean exosome size from 151.7 pm with one minute on, 2 minutes off to 68.9 pm with 15 seconds on, 30 seconds off. Without wishing to be bound by theory, the calculated mean exosome size with the longer light cycle is well above the 120 nm size of a fully formed capsid, indicating that increased trafficking of the capsid proteins can cause full or at least partial capsids to form in or around budding exosomes, increasing their mean size.

[0047] After verifying the size and quantity of gene editing material transportation construct, they were injected into 6-8 week old male or female C57B1 / 6 mice. The mice were given an IV injection of 100 pL of solution including gene editing material transportation construct via the retro-orbital route while under general isoflurane anesthesia. All injections were performed in a BSL2 hood. All mice were allowed to recover and were euthanized by CO2 followed by cervical dislocation at 3 time points post injection, 6 hours, 24 hours, and 30 hours, with 3 mice allocated for each time point along with a control mouse. The brains were then harvested, washed in PBS, bisected in the median Sagittal plane, and flash frozen with dry ice in optimal cutting temperature medium (OCT). The brains were then sectioned along the sagittal plane using a Leica CM 1520 cryostat to a thickness of 15 pm. These slides were then fixed in 4% PFA for 10 minutes and washed in PBS three times for 5 minutes each. The slides were then blocked in a PBS solution with 5% BSA and 0.3% Triton X100 for 1 hour. Next, the slides were stained with anti-HlVl p24 antibody (ABCam, ab53841, Host:goat) in PBS at a concentration of 1 : 1000 and with 0.1% Triton X100 and 3% BSA overnight. The slides were then washed with PBS three more times, and then stained overnight with Donkey Anti-Goat IgG H&L (Alexa Fluor® 488, Molecular Probes, Inc., Eugene, OR) at 1:500 with 3% BSA blocking. The slides were then washed with PBS again three times before glycerol mounting media and acover glass were added. The slides were imaged using both a Leica TCS SP8 STED Microscope and a Laser Scanning Confocal Microscope - Zeiss LSM 510 META 1. The microscopes were set to excite the tags with 488 nm light and measured the corresponding fluorescence emitted at 520 nm. The mean intensity of the different areas was measured using ImageJ Fiji software. Additional negative controls were done for the different time points where no primary antibody was added to verify that no spurious binding of secondary antibody to tissue was observed

[0048] Referring now to FIGs. 4A-4I, the microscopy results for the brain tissues collected at different time points showed an increasing mean fluorescence intensity between all the observed time points in the more efficiently packed exosomes. FIGs. 4C, 4E, 4G, and 41) show slices from brains injected with exosomes produced with light applied for 1 minute on and 2 minute off, while FIGs. 4D, 4F, and 4H show slices from brains injected with exosomes produced with light applied for 15 seconds on and 30 seconds off. Previous literature has stated that exogenous exosomes do not typically circulate in the bloodstream for longer than 2-3 hours, meaning they should have already migrated to the epithelial cells of the BBB by the 6 hour time point. Without wishing to be bound by theory, what is likely being observed is that a large number of exosomes were taken up, passed through the BBB, and began diffusing through the CNS, which takes significantly longer given their larger size. This can also explain why fairly shallow penetration from the exposed surfaces of the brain is seen in the 6 hour time points, with a mean intensity of 55 in the first set (see FIG. 4C) and second set mean intensity of 20. A diffusion pattern through the CNS is seen in the 24 and 30 hour time points, with the 24 hour time points showing mean intensities of 110 and 40 for the first and second exosome sets with the greatest mean intensities being observed in the 30 hour time points with mean intensities of 170 and 60 for the first and second sets. Some regions were also observed to uptake exosomes more strongly than others, such as the bright region observed in one of the 30 hour slides, FIG. 41 (with a mean intensity of 220). The mean intensities for control samples were shown to be less than 10. Again, without wishing to be bound by theory, some images also appear to show what are assumed to be circular monocytes taking up a number of the exosomes, but this is likely incidental and not a cause of concern. No additional complications or changes in behavior were observed in any of the mice following injection and no signs of immune response were observed.

[0049] Systems and methods of the present disclosure advantageously transport gene editing materials, such as large sections of DNA, for insertion into dividing and non-dividing cell types in a targeted manner, without eliciting severe immune responses. HIV-1 CA / p24 has very low immunogenicity. Often the protein can be found in the bloodstream for months after initialinfection with little to no immune response resulting from its presence, this is due in large part to its recruitment of native cellular cofactors such as CPSF6 and CypA which help to shield it from immune detection. Another facet about the mechanism of the inner capsid is that the capsid cannot be made too stable as it is naturally made to come apart once the process of reverse transcription has proceeded to a point in which the larger DNA molecules force apart the capsid. The inner capsid structures according to embodiments of the present disclosure can be successfully enveloped into an exosome of a size capable of being ferried across the BBB.

[0050] Embodiments of the present disclosure associate inner capsid proteins, e.g., CA / p24, to the inside of exosomes. Specifically, light-based dimerization systems enable the reversible association of exosome proteins like tetraspanin, e.g., CD9 found in almost all exosomes with inner capsid proteins such as CA / p24, e.g., via the synthesis of CRY2-CD9 and CIBN-CA recombinant polypeptides. These recombinant polypeptides selectively assemble inner capsid constructs onto the surface of multivesicular bodies by binding them to tetraspanins for internalization into exosomes for export. Assembly can occur in response to shining 490 nm light on assembly media and allowing for that selective association (followed by dissociation after the exosomes have been successfully loaded). When compared with other studies demonstrating delivery of gene editing components across the BBB, the systems and methods of the present disclosure compare very favorably in terms of delivery, showing broad and very effective targeting of many cell types in the CNS.

[0051] An advantage of the systems and methods of the present disclosure, in contrast to numerous other gene editing approaches capable of breaching the BBB, lies in its proficiency in transporting relatively large DNA segments into intended cells. Furthermore, genetic cargo can be shuttled into the nucleus, significantly reducing the risk of DNA degradation by cytoplasmic nucleases and the activation of immune responses through the CGAS-Sting pathway. The use of lentiviral proteins enables low immunogenicity potential, meaning that there would be minimal risk of a major and very harmful immune response taking place in the CNS. Additionally, the ability to target non-dividing cells through the autonomous nuclear import enabled by the systems and methods of the present disclosure, enable targeting a variety of cell types, including many of the cells in the CNS.

[0052] By enabling insertions of large sections of DNA, the scope of diseases that can be treated can be greatly expanded, with opportunities to do such things as introduce recombinant proteins capable of digesting amyloid plaques to help treat Alzheimer’s or replacing the gene copy of the misfolding protein that allows the plaques to form. Genes damaged or silenced bythe normal processes of aging can also be reactivated, allowing treatment of a wide variety of rare conditions. In cases where efficient exosome targeting of a specific cell type has not yet been elucidated, this system has an additional measure to increase specificity by incorporating cell type specific promoters to reduce or eliminate expression of genes even in the event of exosomes being taken up by undesired cell types. This additional point of control can help to further improve the safety profile of the systems and methods of the present disclosure when used in vivo.

[0053] The targeting specificity for certain cell types can be further refined by identifying more fusion proteins that bias selection to particular cell-surface proteins for the targeted cell type without prompting immune responses. Physical and chemical methods are also used to modify exosomes to bias their targeting as well as improve the longevity and circulation of the exosomes in vivo. The systems and methods of the present disclosure also can allow for the more effective use of larger tools such as prime editors and transposons to integrate large sections of DNA into a variety of cell types, both dividing and non-dividing.

[0054] Although the invention has been described and illustrated with respect to exemplary embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions can be made therein and thereto, without parting from the spirit and scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A gene editing material transportation system, comprising: a first recombinant polypeptide including an exosome-associating complex, the exosome-associating complex including an exosome-associating protein and a first light-controlled association domain; and a second recombinant polypeptide including an inner capsid complex, the inner capsid complex including an inner capsid protein and a second light-controlled association domain.

2. The system according to claim 1, wherein the exosome-associating protein is a tetraspanin.

3. The system according to claim 2. wherein the tetraspanm includes CD63, CD81, CD82, CD9, or combinations thereof.

4. The system according to claim 1. wherein the first light-controlled association domain includes photoreceptor cryptochrome 2 protein (CRY2) and the second light-controlled association domain includes truncated CRY-interacting basic-helix-loop-helix 1 protein (CIBN).

5. The system according to claim 1, wherein the inner capsid protein includes an HIV-1 inner capsid protein.

6. The system according to claim 5, wherein the HIV-1 inner capsid protein includes CA / p24.

7. The system according to claim 2, wherein the tetraspanin includes CD9, the first light- controlled association domain includes CRY2, the second light-controlled association domain includes CIBN. and the inner capsid protein includes CA / p24.

8. The system according to claim 1, further comprising: an inner capsid structure including a plurality of second recombinant polypeptides, andgene editing matenals at least partially positioned within the inner capsid structure, wherein the gene editing materials include one or more proteins, oligonucleotides, or combinations thereof.

9. A method of editing genomic material in a target organism, comprising: providing an assembly medium including: a first recombinant polypeptide including an exosome-associating complex, the exosome-associating complex including a tetraspanin and a first light-controlled association domain; a second recombinant polypeptide including an inner capsid complex, the inner capsid complex including an inner capsid protein and a second light- controlled association domain; and gene editing materials, contacting the assembly medium with light from a light source to assemble the first recombinant polypeptide with the second recombinant polypeptide via association of the first light-controlled association domain with the second light- controlled association domain; assembling a gene editing material transportation construct, comprising: assembling exosome structures including a plurality' of first recombinant polypeptides with bound second recombinant polypeptides positioned within the exosome structure, and assembling an inner capsid structure composed of second recombinant polypeptides within the exosome structure, wherein a concentration of gene editing materials is positioned within the inner capsid structure, administering an effective amount of the gene editing material transportation construct to target cells; andfusing the exosome structure of the gene editing material transportation construct to membranes of the target cells to release the inner capsid structure into the target cells.

10. The method according to claim 9, wherein the tetraspanin includes CD63. CD81, CD82, CD9, or combinations thereof.

11. The method according to claim 9, wherein the first light-controlled association domain includes photoreceptor cryptochrome 2 protein (CRY2) and the second light-controlled association domain includes truncated CRY-interacting basic-helix-loop-helix 1 protein (CIBN).

12. The method according to claim 9, wherein the inner capsid protein includes an HIV-1 inner capsid protein.

13. The method according to claim 12, wherein the HIV-1 inner capsid protein includes CA / p24.

14. The method according to claim 9, wherein the tetraspanin includes CD9, the first light- controlled association domain includes CRY2, the second light-controlled association domain includes CIBN, and the inner capsid protein includes CA / p24.

15. The method according to claim 9, wherein contacting the assembly medium with light from a light source to assemble the first recombinant polypeptide with the second recombinant polypeptides includes: alternating between a first phase where the light is applied and a second phase wherein the light is not applied, wherein the first phase is shorter than the second phase.

16. The method according to claim 15, wherein the first phase has a duration of at least 1 minute and the second phase has a duration of at least 2 minutes.

17. The method according to claim 9, wherein administering an effective amount of the gene editing material transportation construct to target cells includes: contacting an amount of the gene editing material transportation construct with the blood-brain barrier of a target organism and allowing it to pass through.

18. A gene editing material transportation system, comprising:an exosome structure including a plurality of first recombinant polypeptides, the first recombinant polypeptide including a tetraspanin and photoreceptor cryptochrome 2 protein (CRY2); an inner capsid structure surrounded by the exosome structure, the inner capsid structure including a plurality of second recombinant polypeptides, the second recombinant polypeptides including an inner capsid protein and truncated CRY- interacting basic-helix-loop-helix 1 protein (CIBN); and gene editing materials positioned within the inner capsid structure.

19. The system according to claim 18, wherein: the tetraspanin includes CD63, CD81, CD82, CD9, or combinations thereof, and wherein the inner capsid protein includes an HIV-1 inner capsid protein.

20. The system according to claim 19, wherein the tetraspanin includes CD9 and the inner capsid protein includes CA / p24.

21. The system according to claim 18, wherein the gene editing materials include one or more proteins, oligonucleotides, or combinations thereof.

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