Low immunogen cells for generating bionic nanovesicles

Abstract

Disclosed herein are methods of producing low immunogen cells and methods of using low immunogen cells to treat mammalian diseases and to prepare biomimetic nanovesicles (BioNVs). The method comprises (a) reducing or eliminating the expression and / or activity of one or more immunogenic proteins in a cell; and (b) expressing or increasing the expression and / or activity of one or more immunoprotective proteins in the cell, thereby producing the low immunogen cell.

Description

Technical Field

[0001] The present disclosure provides, in part, methods of generating low immunogenic cells for producing biomimetic nanovesicles (BioNVs), as well as compositions and uses thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 345,669, filed May 25, 2022, and U.S. Provisional Application No. 63 / 387,516, filed November 14, 2022, the contents of which are incorporated herein by reference in their entirety. Description of Text Files Submitted Electronically

[0004] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer-readable XML formatted copy of the Sequence Listing (file name: "CAR-006PC_Sequence_Listing.xml"; record date: May 24, 2023; file size: 117,233 bytes). Background Art

[0005] Whole cell therapy is becoming an increasingly effective treatment. Cell therapy is administered by infusing whole cells from a donor into the patient and requires a strict immunogenicity match between the donor and the recipient. A variety of low immunogenicity engineering techniques for induced pluripotent stem cells (iPSCs) have been reported. Each approach aims to eliminate human leukocyte antigen (HLA) genes encoding multiple histocompatibility complex (MHC) membrane glycoproteins that produce immune responses associated with graft-versus-host disease (GVHD) rejection. HLA gene clusters can be divided into three categories: 1) MHC class I pathway, 2) MHC class II pathway, and 3) MHC class III pathway. Only the MHC class I and class II pathways express protein complexes that trigger immune responses in GVHD, while MHC class III complexes do not participate in immune activities.

[0006] While elimination or inhibition of these complexes can protect patients from harmful immune responses to some extent, other surface proteins and mechanisms need to be eliminated / inhibited to enhance the hypoimmune properties of the cells. For example, elimination of MHC class I / II protein complexes triggers NK cells and macrophages to enter an “active clearance mode”, followed by cell destruction.

[0007] While some systems are promising, they still express minor non-MHC antigens, may have genetic polymorphisms that lead to immune rejection, have significant teratoma potential, and may lead to eventual humoral immunity to the cells. In addition, even with low immunogenicity engineering, whole-cell therapeutic approaches are likely to suffer from cytokine release syndrome (CRS), cell exhaustion, tissue and / or tumor penetration issues, biological barrier crossing issues, payload packaging difficulties, and targeting issues (e.g., off-target effects), among others. In addition, low-immunogenic cells may require additional engineering to avoid persistence and initiate controlled cell death (e.g., a “suicide switch”). With each round of manipulation, the manufacturing cost of whole-cell therapies increases. There remains a need to combine the low immunogenicity properties of engineered whole-cell therapies with disease-specific targeting while circumventing issues and manufacturing costs to provide viable treatment options for the clinic. Summary of the invention

[0008] In embodiments, methods of producing low immunogenic cells are described herein, comprising reducing or eliminating the expression and / or activity of one or more immunogenic proteins in a cell, and expressing one or more immunoprotective proteins in a cell or increasing their expression and / or activity, thereby producing low immunogenic cells. In embodiments, low immunogenic cells produced using the present methods are described herein.

[0009] In embodiments, cell is stem cell, induced pluripotent stem cell (iPSC), reprogrammed multipotent or multipotent cell, embryonic stem cell, mesenchymal stem cell or the differentiated cell of any stem cell derived therefrom.In embodiments, differentiated cell is T cell, helper T cell, T memory cell or NK cell.In embodiments, differentiated cell is macrophage.In embodiments, differentiated cell is monocyte.In embodiments, differentiated cell is hepatocyte, cardiomyocyte, neuron, endothelial cell, pancreatic cell or retinal pigment epithelial (RPE) cell.

[0010] In embodiments, low immunogenic cells substantially lack one or more of MHC class I protein complexes, MHC class II complexes, T cell receptor (TCR) complexes and / or cytokine release syndrome (CRS) proteins.In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes β2-macroglobulin (B2M) gene disruption and / or reducing or eliminating MHC class I protein expression and / or active destruction.In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes CIITA gene disruption and / or reducing or eliminating MHC class II protein expression and / or active destruction.

[0011] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-A gene disruption and / or reducing or eliminating the destruction of HLA-A protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-B gene disruption and / or reducing or eliminating the destruction of HLA-B protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-C gene disruption and / or reducing or eliminating the destruction of HLA-C protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-E gene disruption or HLA-G gene disruption and / or reducing or eliminating the destruction of HLA-E or HLA-G protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-F gene disruption and / or reducing or eliminating the destruction of HLA-F protein expression and / or activity.

[0012] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises a T cell alpha constant (TRAC) gene disruption and / or a disruption that reduces or eliminates TRAC protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises a T cell beta constant (TRBC) gene disruption and / or a disruption that reduces or eliminates TRBC protein expression and / or activity.

[0013] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes PD-1 gene disruption and / or reducing or eliminating the destruction of PD-1 protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-4 gene disruption and / or reducing or eliminating the destruction of IL-4 protein expression and / or activity. In embodiments, wherein reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-6 gene disruption and / or reducing or eliminating the destruction of IL-6 protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-10 gene disruption and / or reducing or eliminating the destruction of IL-10 protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-16 gene disruption and / or reducing or eliminating the destruction of IL-16 protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises a disruption of the SerpinB9 gene and / or a disruption that reduces or eliminates the expression activity of the SerpinB9 protein.

[0014] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is achieved at the DNA level by one or more of the following: a transposase-based approach, a Cre / Lox-based approach, an endonuclease-based approach, a homologous recombination (HR)-based approach, a non-homologous end joining (NEHJ)-based approach, a microhomology-mediated end joining (MMEJ)-based approach, a homology-mediated end joining (HMEJ)-based approach, a small RNA, or a combination thereof. In embodiments, the small RNA is or includes one or more of the following: a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice switching oligonucleotide (SSO), a tRNA, a complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), an endonuclease, and a small non-coding RNA. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is achieved at the RNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA.

[0015] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing CD34 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing CCL2 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing PD-L1 genes and / or gene products or increasing their expression, wherein the cells are not activated. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing H2-M3 genes and / or gene products or increasing their expression.

[0016] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing a CD47 gene and / or gene product or increasing its expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing a CD24 gene and / or gene product or increasing its expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing a chimeric CD24 / CD47 gene and / or gene product or increasing its expression.

[0017] In embodiments, expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CTLA-4 gene and / or gene product.

[0018] In an embodiment, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing a CD200 gene and / or gene product or increasing its expression. In an embodiment, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing a chimeric CD24 / CD200 gene and / or gene product or a chimeric CD47 / CD200 gene and / or gene product or increasing its expression.

[0019] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing MFG-E8 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing NCAM genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing α-phagocytic integrin genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing antibodies or antibody-like molecules (anti-IL-6R) targeting IL-6 surface receptors or increasing their expression.

[0020] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expression of the FasL gene and / or gene product. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity does not comprise overexpression of the FasL gene and / or gene product.

[0021] In embodiments, the expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins of low immunogenic cells is reduced or eliminated. In embodiments, low immunogenic cells have expression of 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins, or 10 or more immunoprotective proteins, or their expression and / or activity is increased.

[0022] In embodiments, genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G of low immunogenic cells are reduced or eliminated. In embodiments, genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G of low immunogenic cells are reduced or eliminated. In embodiments, the expression and / or activity of genes and / or gene products of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G of low immunogenic cells, and IL-4, IL-10 and IL-16 are reduced or eliminated.

[0023] In embodiments, the low immunogenic cells express or increase expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1 and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200. In embodiments, the low immunogenic cells express or increase expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1 and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200.

[0024] In embodiments, low immunogenic cells express CD200 genes and / or gene products or increase their expression, and do not express and / or substantially lack CD24 or CD47 genes and / or gene products. In embodiments, low immunogenic cells express and do not have the expression and / or activity of SerpinB9 genes and / or gene products and CD200 genes and / or gene products.

[0025] In embodiments, expressing one or more immune protective proteins or increasing their expression is achieved by introducing exogenous genetic elements. In some embodiments, the introduction of exogenous genetic elements is achieved by stable integration into the cell genome. In embodiments, stable integration is achieved by one or more of the following: a method based on a transposase, a method based on Cre / Lox, a method based on an endonuclease, a method based on homologous recombination (HR), a method based on non-homologous end connection (NEHJ), a method based on microhomology-mediated end connection (MMEJ), a method based on homology-mediated end connection (HMEJ) or a combination thereof. In embodiments, stable integration is achieved by viral vectors. In embodiments, the introduction of exogenous genetic elements is achieved by transient transfection. In embodiments, expressing one or more immune protective proteins or increasing their expression is achieved by exogenous promoters and / or enhancers, and / or endogenous promoters and / or enhancers, or a combination thereof. In embodiments, expressing one or more immune protective proteins or increasing their expression is under the control of a constitutively active promoter.

[0026] In an embodiment, expression of one or more immunoprotective proteins or increased expression thereof is achieved at the DNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA. In embodiments, expression of one or more immune protective proteins or increased expression thereof is achieved at the RNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA. In embodiments, expression or overexpression of one or more immune protective proteins is achieved by one or more of small regulatory RNA, miRNA, IRES element, transcription factor, or a combination thereof.

[0027] In embodiments, the low immunogenic cells are allogeneic.In embodiments, the low immunogenic cells do not elicit an immune response in a patient to whom the cells or a biological composition derived therefrom is administered.

[0028] In embodiments, low immunogenic cells include one or more targeting agents. In embodiments, one or more targeting agents include chimeric antigen receptors (CAR). In embodiments, CAR is bispecific. In embodiments, CAR lacks an intracellular portion. In embodiments, CAR includes a targeting agent, a transmembrane domain and an intracellular domain, which includes a co-stimulatory domain and / or a signaling domain. In embodiments, the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α or ICOS, or a fragment thereof. In embodiments, the intracellular domain includes an intracellular signaling domain and / or one or more co-stimulatory molecules of a CD3ζ chain, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40.

[0029] In an embodiment, the one or more targeting agents include an antibody or antibody form. In an embodiment, the antibody or antibody form is selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), V NAR 、V H In some embodiments, the one or more targeting agents include antigen binding proteins, such as antigen binding proteins, ...

[0030] In embodiments, one or more targeting agents are operably linked to a regulatable expression element.

[0031] In embodiments, described herein are reduced immunogenic cells produced by any of the methods described herein. In embodiments, described herein are pharmaceutical compositions comprising reduced immunogenic cells produced by any of the methods described herein and one or more excipients.

[0032] In embodiments, described herein is a low immunogenic cell comprising (a) one or more membrane-embedded targeting agents targeting one or more cellular biomarkers, (b) expression or increased expression of immunoprotective proteins comprising (i) α-phagocytic integrin, CCL2, H2-M3, MFG-E8, and FasL, wherein FasL is not overexpressed, (ii) PD-L1 and / or CTLA-4, wherein the low immunogenic cell is not activated by expression of PD-L1, and (iii) CD24, CD4 7, one of CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200, and (c) substantially lacking expression and / or activity of immunogenic proteins comprising (i) HLA-A, HLA-B, HLA-C, HLA-F, CIITA, PD-1, IL-6, T cell alpha chain (TRAC) and T cell beta chain (TRBC), and (ii) HLA-E or HLA-G.

[0033] In embodiments, described herein is a low immunogenic cell comprising (a) one or more membrane-embedded targeting agents targeting one or more cellular biomarkers, (b) expression or increased expression of immunoprotective proteins comprising (i) α-phagocytic integrin, CCL2, H2-M3, MFG-E8 and FasL, wherein FasL is not overexpressed, (ii) PD-L1 and / or CTLA-4, wherein the low immunogenic cell is not activated by expression of PD-L1, (iii) CD24 and CD47, or chimeric CD24 / CD47, and (c) substantially lacking expression and / or activity of immunogenic proteins comprising (i) HLA-A, HLA-B, HLA-C, HLA-F, CIITA, PD-1, SerpinB9, IL-6, T cell alpha chain (TRAC) and / or T cell beta chain (TRBC), and (ii) HLA-E or HLA-G.

[0034] In embodiments, low immunogenic cells substantially lack expression and / or activity of one or more of IL-4, IL-10 and / or IL-16. In embodiments, low immunogenic cells further comprise expression or increased expression of antibodies or antibody-like molecules targeting IL-6 surface receptors (anti-IL-6R). In embodiments, low immunogenic cells further comprise expression or increased expression of NCAM.

[0035] In embodiments, one or more targeting agents include a chimeric antigen receptor (CAR). In embodiments, one or more targeting agents are antibodies or antibody forms. In embodiments, one or more targeting agents are antibodies or antibody forms. In embodiments, the antibodies or antibody forms are selected from one or more of the following: monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), V NAR 、V H H, afflilin, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen binding portion of antibodies. In embodiments, one or more targeting agents are viral epitope recognition receptors (VERRs) or viral ligands. In embodiments, one or more targeting agents are ligands of receptors or receptors of ligands.

[0036] In embodiments, the low immunogenic cells are allogeneic.In embodiments, the low immunogenic cells do not elicit an immune response in a patient to whom the cells or a biological composition derived therefrom is administered. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A non-limiting schematic diagram of T cell activation via interaction between MHC class I T cell receptor (TCR) synapses is depicted. Target peptides displayed by MHC class I complexes on virus-infected cells, cancer cells, etc. can be recognized by the hypervariable regions of TCR α / β on lymphocytes, resulting in conformational changes in the intracellular domain, thereby triggering signal transduction and producing cytotoxic cytokines.

[0038] Figure 2 A non-limiting illustration of activated immune cells containing anti-cytokines processed into biomimetic nanovesicles (BioNVs) is depicted. Anti-cytokines (and various cytotoxic peptides) can be loaded into BioNVs for potent delivery.

[0039] Figure 3 A non-limiting illustration of the process of activating cells with biomarker antigens through their CAR / TCR receptors is depicted. The biomarker antigen can be bound to magnetic beads or streptavidin beads (or other) and then added to the cell suspension to cause activation. After activation, the antigen can be separated from the cell suspension, leaving the activated cells with unbound CAR / TCR complexes.

[0040] Figure 4 Graphical representation of the B2M knockout strategy for developing hypoimmunogenic cell lines from human fibroblast reprogramming iPSCs is depicted. β2-macroglobulin (B2M) is a serum protein associated with major histocompatibility complex (MHC) class I heavy chains on the surface of nearly all nucleated cells involved in presenting peptide antigens to the immune system.

[0041] Figure 5 A graphical representation of the B2M gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the B2M gene.

[0042] Figure 6 Tabular representation of B2M gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0043] Figure 7 A graphical representation of the CIITA knockout strategy for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. The master control factor CIITA is an MHC class II transactivator involved in the transcriptional regulation of all MHCII genes.

[0044] Figure 8A graphical representation of the CIITA gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target the relevant regions of the CIITA gene.

[0045] Fig. 9 Tabular representation of CIITA gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0046] Fig.10 A graphical representation of the sequence analysis of B2M knockout clones used to develop low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. Results for two selected B2M / CIITA double knockout clones are shown. Sequencing data indicated successful B2M KO in the double KO clones.

[0047] Fig.11 A graphical representation of the sequence analysis of CIITA knockout clones used to develop low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. Results for two selected B2M / CIITA double knockout clones are shown. Sequencing data indicated successful CIITA KO in the double KO clones.

[0048] Fig.12 Depicted is a graphical representation of the B2M / CIITA double knockout clone sequence overview and clonal morphology of in vitro human fibroblast reprogrammed iPSC low immunogenicity cells. The data demonstrate that the clonal populations result in different overall knockouts.

[0049] Fig.13 A graphical representation of human CD47 (hCD47) isoform 2 knock-in used to develop low immunogenic cell lines from human fibroblast reprogramming iPSCs is depicted. The pcDNA3.1(+)XCC92 mammalian vector (6271 bp in length) was used with a selection marker. The CD47 3'UTR was deleted for stable clonal surface expression of CD47. The 3'UTR contains at least 6 microRNA binding sites that inhibit CD47 expression, in which a bGH poly A tail was used instead.

[0050] Fig.14 Depicted is a graphical representation of hCD47 isoform 2 knock-in clonal selection for development of low immunogenic cell lines in B2M / CIITA double KO human fibroblast reprogrammed iPSCs. The data demonstrates the success of hCD47 KI in double KO cell lines.

[0051] Fig.15A graphical representation of the TRAC knockout strategy for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. In embodiments, T cell receptor alpha chain (TRAC) elimination is performed to prevent interference with CAR targeting and off-target effects.

[0052] Fig.16 A graphical representation of the TRAC gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target the relevant regions of the TRAC gene.

[0053] Fig.17 Tabular representation of TRAC gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0054] Fig.18 A graphical representation of the TRBC1 knockout strategy for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. In embodiments, T cell receptor beta chain 1 (TRBC1) elimination is performed to prevent interference with CAR targeting and off-target effects.

[0055] Fig.19 A graphical representation of TRBC1 gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the TRBC1 gene.

[0056] Fig. 20 Tabular representation of TRBC1 gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0057] Fig.21 Graphical representation of sequence analysis of TRAC knockout clones depicting the development of a hypo-immunogenic cell line from B2M / CIITA double knockout, hCD47 knockin in human fibroblast-reprogrammed iPSCs. Results for two selected clones are shown. Sequencing data demonstrate successful knockout of TRAC in the double KO, CD47KI clones.

[0058] Fig. 22 Graphical representation of sequence analysis of TRBC1 knockout clones depicting the development of low immunogenic cell lines from B2M / CIITA double knockout, hCD47 knock-in, and TRAC knock-out in human fibroblast-reprogrammed iPSCs. Results for two selected clones are shown. Sequencing data demonstrate successful knockout of TRBC1 in double KO, CD47 KI, and TRAC KO clones.

[0059] Fig.23Graphical representation of B2M / CIITA double knockout, TRAC / TRBC1 double knockout, hCD47KI human fibroblasts reprogrammed into iPSCs with low immunogenicity. Data illustrate in vitro morphological analysis of clones.

[0060] Fig.24 Graphical representation of the IL-6 knockout strategy for developing hypoimmunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. Elimination of interleukin-6 (IL-6) is intended to prevent cytokine release syndrome (CRS).

[0061] Fig.25 A graphical representation of the IL-6 gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the IL-6 gene.

[0062] Fig.26 Tabular representation of IL-6 gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0063] Fig. 27 A non-limiting schematic diagram of the production of BioNV using continuous extrusion is depicted. DETAILED DESCRIPTION

[0064] The present disclosure relates in part to methods for producing low immunogenic cells for use in producing cell-derived biomimetic nanovesicles (BioNVs). BioNVs circumvent the caveats of whole cell therapies while retaining the functionality of the cells from which they are derived. Although nanovesicles (NVs) are less expensive to manufacture than whole cell therapies, NV therapies may have half-life and elimination issues, such as clearance by immune cells and kidneys. The approaches of Harding et al., Deuse et al., and Zhao et al. have direct implications for whole-cell therapy and partially achieve low immunogenicity, laying the foundation for true “off-the-shelf” therapy, but are useless for BioNV formation (Harding et al., “Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts.” BioRxiv716571 [preprint], July 30, 2019. doi:10.1101 / 716571.), (Deuse et al. “Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients.” Nat. Biotechnol. Vol. 37, No. 3, 2019: pp. 252-258. doi:10.1038 / s41587-019-0016-3), and (Zhao W et al. “Strategies for Genetically Engineering Hypoimmunogenic Universal Pluripotent Stem Cells.” iScience. Vol. 23, No. 6, 2020: 101162. doi: 10.1016 / j.isci.2020.101162.).

[0065] In embodiments, methods for generating low immunogenic cells for producing BioNV overcome the shortcomings of other cells used for BioNV, for example, 1) expression of SerpinB9 inhibits the function of granzymes, thereby limiting the functionality of the BioNV mechanism of action; 2) overexpression of FasL is unnecessary because the density of naturally expressed FasL on the surface of BioNV increases during cell processing (e.g., after extrusion); 3) overexpression of CD200 may inhibit granulocyte function necessary for efficacy in the microenvironment of solid tumors (if used for cancer treatment purposes); 4) overexpression of too many anti-phagocytic tags may cause the cell product to be too stable and difficult to clear from the body after therapeutic efficacy.

[0066] In embodiments, methods for producing low immunogenic cells improve upon other methods for producing low immunogenic cells that focus on reducing or eliminating the expression and / or activity (e.g., knocking out) of MHC class I / II genes and / or HLA genes, followed by overexpression of CD47. For example, overexpression of CD47 can inhibit BioNV function for a variety of reasons. (1) Knockout of the B2M and CIITA genes combined with overexpression of the CD47 tag may inhibit the NK cell response required for the BioNV target site. Whole cell therapies generally do not require this requirement, but BioNV lacks genetic material that helps trigger NK cells to respond to cells lacking all HLA genes. (2) Overexpression of CD47 in some differentiated cell subsets may be inhibited, so reliance on CD47 limits the cell types that can be used for BioNV. CD47 is a highly regulated surface protein due to its importance to cell stability. If CD47 expression is too low, the cells (and BioNV) are at risk of premature immune clearance. In contrast, if CD47 expression is too high (as occurs in cancer cells, for example), the cells may not be cleared by the immune system in a timely manner. Expression of the CD47 isoform 2 gene is tightly regulated at the level of transcription (via multiple transcription factors, including STAT3, NF-kβ, Hif-1, Myc), RNA translation by microRNAs (miR-708, miR-192, miR-222, miR-133a, miR-155miR-200a, and miR-340), and post-translational protein modification (forming an N-terminal pyroglutamate that facilitates interaction with SIRPα). In the cellular context, the tight regulation of CD47 may inhibit its overexpression as a protective antiphagocytic marker in cells differentiated from engineered iPSC sources, even if iPSCs are derived from syncytiotrophoblasts that naturally overexpress CD47 (i.e., lineage differentiation may result in downregulation). (3) TCR genes remain intact. In iPSCs differentiated into the T cell (naive) lineage, the presence of the TCR may contribute to GVHD in recipient patients. Many studies have reported TCR-mediated rejection of non-MHC antigens in patients.

[0067] In an embodiment, a method for generating a low immunogenic cell line that is engineered to be allogeneic and does not cause adverse immune responses in patients. Low immunogenic cells can be engineered to express CAR, as well as other targeting agent formats (e.g., VERR, V NAR 、V HH, scFv, bispecific T cell engager (BiTE), engineered T cell receptor (TCR), dual affinity redirection (DART) antibody, etc.). Low immunogenic cells can retain the ability to be activated to express a variety of intracellular therapeutic biomolecules (e.g., cytokines, perforins, granzymes, interferons (INF), tumor necrosis factor (TNF), interleukins (IL), etc.), which can be packaged into BioNV. Low immunogenic cells can be engineered to produce gene editing payloads, including nucleic acids (e.g., plasmids and gene cassettes expressing tracer RNA (trRNA), guide RNA (gRNA), miRNA, tRNA, RNAi, small RNA and endonucleases, such as CRISPR, TALEN, ZFN, etc.) and other gene editing formats that can be packaged into the resulting BioNV. Low immunogenic cells can be engineered to produce and package therapeutic fusion proteins, antibodies and antibody fragments, nucleic acids, etc., which can be packaged into BioNV or naturally shed as exosomes. Low immunogenic cells can be differentiated into, for example, T cells, macrophages, cardiomyocytes, etc. while retaining their properties, which can then give the cells new properties, such as the ability to cross biological barriers, recruit cells, etc. The BioNVs derived from low immunogenic cells described herein can retain these properties of cells and payloads, representing an effective alternative to whole cell therapy.

[0068] In embodiments, described herein are cells with reduced immunogenicity. In embodiments, described herein are cells with reduced immunogenicity produced using the present methods.

[0069] Low immunogenic cells can be modified to express one or more targeting agents (e.g., CAR) targeting one or more cellular biomarkers. Low immunogenic cells can be modified to express or increase the expression of one or more membrane-embedded immune protective surface markers, including α-phagocytic integrin, CCL2, PD-1, CTLA-4, H2-M3, SerpinB9, CD24, CD47, CD24 / CD47 chimeric forms, CD200, CD200 chimeras with CD24 or CD47, MFG-E8, anti-IL-6R, NCAM and / or FasL. Low immunogenic cells can be modified to substantially lack the expression and / or activity of one or more immunogenic proteins, including MHC class I, MHC class II, HLA-A, HLA-B, HLA-C, HLA-E or HLA-G, HLA-F, CIITA, PD-1, SerpinB9, IL-4, IL-6, IL-10, IL-16, T cell alpha chain (TRAC) and / or T cell beta chain (TRBC). Low immunogenic cells may or may not express CD200 and / or SerpinB9, depending on the function of the desired BioNV derived therefrom, such as for delivery of granzymes.

[0070] In embodiments, "increased expression and / or activity" as used herein refers to an increase in expression and / or activity in a low immunogenic cell compared to a native or wild-type homologous cell. For example, in embodiments, increased expression and / or activity of one or more biomolecules described herein can confer low immunogenicity to an iPSC relative to an iPSC that does not have the same protein expression pattern or expression level. In embodiments, the "increased expression and / or activity" is due to a genetic modification, such as a knock-in.

[0071] In embodiments, compositions of low immunogenic cells and kits comprising low immunogenic cells are described herein. Compositions may include whole cells suspended in a solution compatible with cell cryopreservation or administration of cells to a subject (e.g., intravenous, intraperitoneal, intramuscular, etc.). Low immunogenic cell compositions may include additional therapeutic agents for use as whole cell therapy. Kits may include any low immunogenic cell compositions described herein, e.g., packaged into syringes, IV bags, and include instructions for use and diagnostic materials.

[0072] Methods for generating low immunogenic cell lines

[0073] In some aspects, the present disclosure includes methods of producing hypoimmunogenic cells, the methods comprising reducing or eliminating the expression and / or activity of one or more immunogenic proteins in a cell, and expressing one or more immunoprotective proteins in the cell or increasing their expression and / or activity, thereby producing hypoimmunogenic cells.

[0074] In embodiments, low immunogenic cells can be derived from any cell that can be differentiated into a specific cell type. In embodiments, cell is stem cell, induced pluripotent stem cell (iPSC), reprogrammed multipotent or multipotent cells, embryonic stem cell, mesenchymal stem cell or the differentiated cell derived from any stem cell thereof. In embodiments, differentiated cell is lymphoid lineage cell, such as T cell, helper T cell, T memory cell, NK cell etc. In embodiments, differentiated cell is myeloid cell, such as macrophage, monocyte, neutrophil etc. In embodiments, differentiated cell is tissue-specific cell, such as hepatocyte, cardiomyocyte, neuron, endothelial cell, pancreatic cell, retinal pigment epithelium (RPE) cell etc. In an embodiment, the low immunogenic cell can be any terminally differentiated cell, such as, but not limited to, a muscle cell (satellite cell), an adipocyte, a bone cell, a cardiomyocyte, a hepatocyte, a blood cell (including erythrocytes, thrombocytes, and all immune cell types), a glial cell (and other neuronal cell types), an epithelial cell, an epidermal cell, a mesenchymal cell (e.g., an airway mesenchymal cell), a fibroblast (e.g., a dermal fibroblast), an endothelial cell (e.g., a bronchial endothelial cell), an oral cell, a stromal cell, or a germ cell. In an embodiment, the low immunogenic cells can be any function-specific cell type, such as but not limited to exocrine secretory epithelial cells, hormone-secreting cells (e.g., enteroendocrine cells, thyroid cells, pancreatic islet cells, etc.), sensory transduction cells, autonomic nerve cells, sensory organ cells (e.g., columnar cells, olfactory cells, Schwanncell cells, satellite glial cells, etc.), barrier cells (e.g., lung cells, ductal cells, kidney cells, podocytes, etc.), extracellular matrix cells (e.g., tendon fibroblasts, osteoblasts, connective tissue cells, etc.) or contractile cells (e.g., skeletal muscle cells, cardiomyocytes, myoepithelial cells, etc.).

[0075] In embodiments, low immunogenic cells (or cells for producing low immunogenic cells) are human female cells (e.g., from female donors or sources). In embodiments, low immunogenic cells (or cells for producing low immunogenic cells) are human male cells (e.g., from male donors or sources). In embodiments, low immunogenic cells (or cells for producing low immunogenic cells) are human female fibroblast iPSCs. In embodiments, low immunogenic cells (or cells for producing low immunogenic cells) are human male fibroblast iPSCs.

[0076] In embodiments, the allogeneic and low immunogenic properties of cells (e.g., derived from iPSC) are produced by knocking out, silencing, inactivating, blocking or otherwise negating the expression, transcription efficiency and / or activity of one or more immunogenic molecules. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins include β2-macroglobulin (B2M) gene disruption and / or reducing or eliminating the destruction of MHC I class protein expression and / or activity, such as in the case of CD8+T cell lineage. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins include CIITA gene disruption and / or reducing or eliminating the destruction of MHC II class protein expression and / or activity, such as in the case of CD4+T cell lineage. Not wishing to be bound by theory, these proteins contribute to human leukocyte antigen (HLA) immunogenicity, which requires HLA allele matching in donor-recipient to be treated by cell-based therapy. In embodiments, allogeneic and / or low immunogenicity is achieved by reducing or eliminating the expression and / or activity of genes encoding T cell receptor (TCR) proteins, including, for example, α and β chains (such as αβT cells) or γ and δ chains (such as γδT cells) that form ligand binding sites, and signaling modules CD3δ, CD3γ, CD3ε, and CD3ζ. In embodiments, this is done to reduce foreign T cell receptor types other than those of the CAR box, and further improve the homogeneity of the CAR of interest and reduce off-target effects in BioNV formation.

[0077] In embodiments, low immunogenic cells substantially lack one or more of MHC class I protein complexes, MHC class II complexes, T cell receptor (TCR) complexes and / or cytokine release syndrome (CRS) proteins. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes β2-macroglobulin (B2M) gene destruction and / or reducing or eliminating the destruction of MHC class I protein expression and / or activity. In embodiments, knocking out the B2M gene can reduce the potential dose number that needs to be administered, because there is a risk of preventing the recipient from receiving BioNV for a long time, such as the situation observed in the above-mentioned whole cell-based method. In order to solve this problem, in embodiments, HLA-E or HLA-G genes remain intact, so that the immune system can adapt to the BioNV produced. In embodiments, HLA-A, HLA-B, HLA-C, HLA-F and HLA-E or HLA-G (but not both HLA-E or HLA-G) are knocked out in sequence.

[0078] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-A gene disruption and / or reducing or eliminating the destruction of HLA-A protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-B gene disruption and / or reducing or eliminating the destruction of HLA-B protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-C gene disruption and / or reducing or eliminating the destruction of HLA-C protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-E gene disruption or HLA-G gene disruption and / or reducing or eliminating the destruction of HLA-E or HLA-G protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises HLA-F gene disruption and / or reducing or eliminating the destruction of HLA-F protein expression and / or activity.

[0079] In embodiments, low immunogenic cells include CIITA gene disruption and / or the disruption that prevents MHC class II protein expression. In embodiments, the CIITA gene of allogeneic iPSC is disrupted so that the resulting differentiated cell line (e.g., DC, mononuclear phagocyte, endothelial cell, thymic epithelial cell, B cell, etc.) does not express MHC class II protein or its expression is reduced.

[0080] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises T cell alpha constant (TRAC) gene disruption and / or reduction or elimination of TRAC protein expression and / or activity disruption. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises T cell beta constant (TRBC) gene disruption and / or reduction or elimination of TRBC protein expression and / or activity disruption. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises PD-1 gene disruption and / or reduction or elimination of PD-1 protein expression and / or activity disruption.

[0081] CRS is a major problem with whole cell therapies, and despite the presence of engineered low immunogenicity, the effector functions and other consequences of interaction with cells after infusion may lead to the release of biomolecules, resulting in systemic inflammatory syndrome characterized by fever, multiple organ dysfunction, etc. In embodiments, low immunogenic cells are engineered to destroy one or more proteins that cause CRS. In embodiments, low immunogenic cells have reduced or eliminated expression and / or activity (e.g., knockout or silencing) of CRS-related cytokines.

[0082] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-4 gene destruction and / or reducing or eliminating the destruction of IL-4 protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-6 gene destruction and / or reducing or eliminating the destruction of IL-6 protein expression and / or activity. In embodiments, IL-6 knocks out to prevent undesirable IL-6 from being packaged into BioNV, and reduces the contribution of BioNV to local (and due to biomarker targeting and concentration) and / or potential systemic CRS events. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-10 gene destruction and / or reducing or eliminating the destruction of IL-10 protein expression and / or activity. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-16 gene destruction and / or reducing or eliminating the destruction of IL-16 protein expression and / or activity. In embodiments, the reduction or elimination of interleukins reduces the possibility of CRS.

[0083] SerpinB9 is a member of the serine protease inhibitor superfamily. It is reported that SerpinB9 protects cells from the immunological killing effect of granzyme B. In embodiments, low immunogenic cells express SerpinB9 or increase their expression. In embodiments, SerpinB9 of low immunogenic cells is knocked out and / or silenced. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes SerpinB9 gene disruption and / or reducing or eliminating the disruption of SerpinB9 protein expression activity.

[0084] In embodiments, overexpression of SerpinB9 isolates the function of granzyme B associated with immune stimulation responses (e.g., apoptosis of target cells and / or diseased cells). In embodiments, granzyme B is inhibited in activated lymphocytes, NK cells, macrophages, and follicular DCs, as well as other cell types. In embodiments, for example, when BioNV is intended to deliver a non-granzyme payload (e.g., a gene editing payload), low immunogenic cells may express and / or overexpress SerpinB9.

[0085] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is achieved at the DNA level by one or more of the following: a transposase-based approach, a Cre / Lox-based approach, an endonuclease-based approach, a homologous recombination (HR)-based approach, a non-homologous end joining (NEHJ)-based approach, a microhomology-mediated end joining (MMEJ)-based approach, a homology-mediated end joining (HMEJ)-based approach, a small RNA, or a combination thereof. In embodiments, the small RNA is or includes one or more of the following: a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice switching oligonucleotide (SSO), a tRNA, a complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), an endonuclease, and a small non-coding RNA. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is achieved at the RNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA.

[0086] In embodiments, site-specific nucleases for any gene modification herein may include CRISPR / Cas endonucleases, TALENS, ZFNs, or any other site-specific nuclease system for gene silencing and / or knockout. In embodiments, gene disruption is due to a gene editing system comprising one or more proteins and / or nucleic acids working in concert, such as TALENs, ZFNs, RNase P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposases, and / or any orthologs or homologs of any of these editing agents.

[0087] In embodiments, gRNA sequences compatible with the CRISPR / Cas9 system can be designed to target at least a portion of the B2M gene (including coding and non-coding sequences that control gene expression) (e.g., as described in Table 8). For example, in non-limiting embodiments, SEQ ID NO:22-23 lists exemplary B2M gene sequences for designing gRNAs for knockout purposes. In non-limiting embodiments, a series of illustrative gRNA sequences (listed in DNA format) are described in SEQ ID NO:21, 24, 26-29, and 133-134, which target sequences in SEQ ID NO:22-23. In non-limiting embodiments, such gRNAs can produce allelic knockouts (including, for example, biallelic knockouts) of B2M, thereby producing cloned DNA sequences, such as those listed in SEQ ID NO:43-49. In embodiments, the resulting genomic DNA sequences modified by CRISPR / Cas from B2M can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., in the space on both sides of SEQ ID NO:43-49 for B2M knockouts).

[0088] In embodiments, gRNA sequences compatible with CRISPR / Cas9 systems can be designed to target at least a portion of the CIITA gene (including coding and non-coding sequences controlling gene expression) (e.g., as described in Table 8). For example, in non-limiting embodiments, SEQ ID NO:32-33 lists exemplary CIITA gene sequences for designing gRNA for knockout purposes. In non-limiting embodiments, a series of illustrative gRNA sequences (listed in DNA format) are described in SEQ ID NO:31, 34, and 36-42, which target the sequences in SEQ ID NO:32-33. In non-limiting embodiments, such gRNAs can produce allelic knockouts of CIITA (including, for example, biallelic knockouts), thereby producing cloned DNA sequences, such as listed in SEQ ID NO:50-55. In embodiments, the resulting genomic DNA sequence modified from CRISPR / Cas of CIITA can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., in the space on both sides of SEQID NO:50-55 for CIITA knockouts).

[0089] In embodiments, gRNA sequences compatible with the CRISPR / Cas9 system can be designed to target at least a portion of the TRAC gene (including coding and non-coding sequences that control gene expression) (e.g., as described in Table 8). For example, in non-limiting embodiments, SEQ ID NO:56-57 lists exemplary TRAC gene sequences for designing gRNAs for knockout purposes. In non-limiting embodiments, a series of illustrative gRNA sequences (listed in DNA format) are described in SEQ ID NO:60-75, which target sequences in SEQ ID NO:56-57. In non-limiting embodiments, such gRNAs can produce allelic knockouts of TRAC (including, for example, biallelic knockouts), thereby producing cloned DNA sequences, such as those listed in SEQ ID NO:102-108. In embodiments, the resulting genomic DNA sequence modified by CRISPR / Cas from TRAC can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., in one or more spaces on both sides of SEQ ID NO:102-108 for TRAC knockout).

[0090] In an embodiment, a gRNA sequence compatible with the CRISPR / Cas9 system can be designed to target at least a portion of the TRBC1 gene (including coding and non-coding sequences that control gene expression) (e.g., as described in Table 8). For example, in a non-limiting embodiment, SEQ ID NOs: 76-77 list exemplary TRBC1 gene sequences for designing gRNAs for knockout purposes. In a non-limiting embodiment, a series of illustrative gRNA sequences (listed in DNA format) are described in SEQ ID NOs: 79-101, which target sequences in SEQ ID NOs: 76-77. In a non-limiting embodiment, such gRNAs can produce allelic knockouts (including, for example, biallelic knockouts) of TRBC1, thereby producing clonal DNA sequences, such as those listed in SEQ ID NOs: 109-110 and 112-116. In embodiments, the resulting genomic DNA sequence from CRISPR / Cas modification of TRBC1 can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., within one or more spaces flanking SEQ ID NOs: 109-110 and 112-116 for TRBC1 knockout).

[0091] In an embodiment, a gRNA sequence compatible with the CRISPR / Cas9 system can be designed to target at least a portion of the IL-6 gene (including coding and non-coding sequences that control gene expression) (e.g., as described in Table 8). For example, in a non-limiting embodiment, SEQ ID NO: 117-118 lists exemplary IL-6 gene sequences for designing gRNA for knockout purposes. In a non-limiting embodiment, a series of illustrative gRNA sequences (listed in DNA format) are described in SEQ ID NO: 119-130, which targets the sequences in SEQ ID NO: 117-118. In a non-limiting embodiment, such gRNA can produce allelic knockouts of IL-6 (including, for example, double allelic knockouts), thereby producing cloned DNA sequences. In an embodiment, the resulting genomic DNA sequence from CRISPR / Cas modified IL-6 can be used to insert one or more genes, coding sequences, DNA sequences, etc.

[0092] In embodiments, low immunogenic cells can be generated by knocking out one or more genes described herein using one or more gRNAs described herein. For example, in embodiments, the gRNA has about or at least about 85% sequence identity, about or at least about 86% sequence identity, about or at least about 87% sequence identity, about or at least about 89% sequence identity, about or at least about 90% sequence identity, about or at least about 91% sequence identity, about or at least about 92% sequence identity, about or at least about 93% sequence identity, about or at least about 94% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to one or more of SEQ ID NOs: 31, 24, 26-29, 31, 34, 36-42, 60-75, 79-101, 119-130, and 133-134.

[0093] In embodiments, low immunogenic cells can be produced by targeting one or more genes as described herein, for knocking out and / or inserting one or more proteins, to express and / or increase expression and / or activity by targeting one or more DNA sequences, as described herein. For example, in embodiments, the DNA sequence comprises one or more of SEQ ID NO:22-23, 32-33, 56-57, 76-77 and 117-118, including sequences having one or more substitutions, deletions, insertions, variants and / or nucleotide polymorphisms (e.g., single nucleotide polymorphisms SNPs).

[0094] In embodiments, low immunogenic cells (and BioNV derived therefrom) express one or more immunoprotective proteins or increase their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing CD34 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing CCL2 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing PD-L1 genes and / or gene products or increasing their expression, wherein low immunogenic cells are not activated by the expression of PD-L1. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing H2-M3 genes and / or gene products or increasing their expression.

[0095] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing a CD47 gene and / or gene product or increasing its expression. In embodiments, the potential inhibitory phenotype of CD47 expression across cells is prevented by interfering with the inhibitory action mechanism of a series of microRNAs on the 3'UTR of the CD47 gene, by deleting this region in a stable construct or by eliminating / inhibiting the expression of microRNAs. In embodiments, this can solve the inhibitory problem caused by microRNAs.

[0096] In the embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing CD24 genes and / or gene products or increasing their expression. CD24 is a sialic acid glycoprotein expressed on mature granulocytes and B cells, and is also an anti-phagocytic protein. CD24 prevents phagocytosis by interacting with Siglec-G / 10 on macrophages. In the embodiments, low immunogenic cells express or overexpress CD24 proteins and / or gene products. In the embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing chimeric CD24 / CD47 genes and / or gene products or increasing their expression. In the embodiments, low immunogenic cells include CD24 / CD47 with tethered transmembrane domains. In the embodiments, the domains of CD47 isotype 2 and CD24 can be expressed or tethered separately to form bilobed chimeric proteins. In the embodiments, low immunogenic cells are iPSCs from fibroblasts rather than ABO cells.

[0097] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity includes expressing the CD200 gene and / or gene product or increasing its expression. In embodiments, the CD200 tag minimizes phagocytosis of macrophages and also prevents the activation of granulocytes. In embodiments, when it is not desired to prevent granulocytes, such as in a solid tumor microenvironment (TME), low immunogenic cells do not express CD200 because the activation of granulocytes will complement the mechanism of action of BioNV intended to release granzymes and perforins. However, in embodiments, if CD47 or CD24 tags are used, or a combination of CD24 / CD47 chimeric bilobed protein tags (each preventing phagocytosis) and overexpressed H2-M3 (inhibiting NK responses) is used, stability can be achieved without CD200 while allowing BioNV clearance. In embodiments where granzymes and perforins are not preferred therapeutic biomolecules, CD200 can be expressed to prevent the activation of granulocytes while eliminating the CD47 tag or CD24 tag, but not both tags. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products or increasing their expression. In embodiments, these CD200 strategies represent low immunogenic cell lines for generating BioNVs for targeting non-cancerous cells (e.g., targeting liver, kidney, heart cells and / or tissue regeneration pathways).

[0098] In an embodiment, the low immunogenic cells (or cells differentiated therefrom) do not express and / or overexpress all three of CD47, CD24, and CD200. In an embodiment, the low immunogenic cells are engineered so that the BioNV produced by the low immunogenic cell line is stable, but not to the extent that the BioNV is difficult to clear from the body. Too stable BioNV may eventually trigger a humoral response, thereby limiting the dose or number of treatments that can be administered.

[0099] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing CTLA-4 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing MFG-E8 genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing NCAM genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing α-phagocytic integrin genes and / or gene products or increasing their expression. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing antibodies or antibody-like molecules (anti-IL-6R) targeting IL-6 surface receptors or increasing their expression.

[0100] In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expression of a FasL gene and / or gene product. In embodiments, expressing one or more immunoprotective proteins or increasing their expression and / or activity does not comprise overexpression of a FasL gene and / or gene product. In embodiments, overexpression of FasL is avoided because enrichment of the native expression level of FasL is observed in the membrane of BioNV after processing (e.g., by continuous extrusion). Too high a concentration of FasL may be counterproductive and prevent T cell recruitment to solid tumors and / or cause premature death of T cells.

[0101] In embodiments, low immunogenic cells may express one or more fusion proteins of one or more portions of any of the immunoprotective proteins described herein. For example, in embodiments, constructs may be prepared in which the appropriate portion of a selected ligand is tethered to a transmembrane domain. In embodiments, constructs may be prepared in which biologically relevant portions of two or more proteins are tethered together and / or to a transmembrane domain.

[0102] In embodiments, the low immunogenic cells have reduced or eliminated expression and / or activity of one or more immunogenic proteins, e.g., proteins that cause an immune response, donor-recipient mismatch, HLA alloimmunization, inflammation, CRS, etc. in a subject, e.g., MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In embodiments, the low immunogenic cells have reduced or eliminated expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins.

[0103] In embodiments, the modified cells have expression or increased expression and / or activity of one or more immunoprotective proteins, such as proteins that prevent or reduce the immune response of a subject, prevent or reduce premature clearance of a subject's BioNV, prevent or reduce phagocytosis, confer barrier function, etc., such as CD47, CD24, CD200, CD34, CCL2, H2-M3, MFG-E8, PD-L1, CTLA-4, etc. In embodiments, the low immunogenic cells are not activated, or are in an activated state, and express PD-L1. In embodiments, the low immunogenic cells have expression of 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins, or 10 or more immunoprotective proteins, or increase their expression.

[0104] In an embodiment, the genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0105] In an embodiment, the genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0106] In an embodiment, the expression and / or activity of genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G, and one or more of IL-4, IL-10 and IL-16 of the low immunogenic cells is reduced or eliminated.

[0107] In embodiments, the low immunogenic cells express or increase expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1 and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200.

[0108] In embodiments, the low immunogenic cells express or increase expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1 and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200.

[0109] In embodiments, low immunogenic cells express CD200 genes and / or gene products or increase their expression, and do not express and / or substantially lack CD24 or CD47 genes and / or gene products. In embodiments, low immunogenic cells do not have expression and / or activity of SerpinB9 genes and / or gene products and CD200 genes and / or gene products.

[0110] Immunocompetent HLA complex

[0111] In embodiments, the low immunogenic cells used to produce BioNV express one or more immunocompetent complexes or increase their expression. In embodiments, BioNV has one or more immunocompetent complexes. In embodiments, the one or more immunocompetent complexes include one or more human leukocyte antigens (HLA), and when the complexes interact with one or more T cells, the HLA is inhibited from eliciting a T cell response. In embodiments, the immunocompetent complexes include fusion proteins that are inhibited from activating T cells, but maintain structural integrity to allow a certain degree of effector function.

[0112] In embodiments, the low immunogenic cells used to produce BioNV express or increase the expression of one or more T cell receptors (TCRs), instead of or in addition to a CAR construct, as described herein.

[0113] In an embodiment, the construct of an immunocompetent HLA includes one or more nucleic acids encoding an immunocompetent HLA. In an embodiment, the complex optionally includes an amino acid sequence or a nucleic acid sequence encoding an amino acid sequence in an order from N-terminal to C-terminal, the amino acid sequence including at least a portion of a peptide and a human HLA class 1 heavy chain sequence. In an embodiment, an immunocompetent HLA is a fusion protein with a peptide that binds to HLA, interfering with one or more interactions of HLA molecules with cell surface complexes on immune cells. In an embodiment, when the peptide interacts with one or more T cells, the peptide does not cause a substantial T cell response. In an embodiment, the peptide cannot activate one or more T cells (e.g., CD4+T cells, CD8+T cells, etc.). In an embodiment, the peptide is capable of binding to one or more T cell receptors, and the binding is insufficient to activate one or more T cells. In an embodiment, the peptide binds to one or more HLA binding groove domain residues of a human HLA class 1 heavy chain sequence. In an embodiment, the peptide regulates the conformation of a human HLA class 1 heavy chain sequence. In an embodiment, the conformation prevents one or more T cells from binding to a human HLA class 1 heavy chain sequence.

[0114] MHC class I molecules typically bind peptides of about 8-10 amino acids in length, but may also bind non-canonical longer peptides (e.g., greater than about 13 amino acids in length). In embodiments, the length of the peptide is about or at least about 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 or more amino acids. In some embodiments, the human HLA class 1 heavy chain sequence comprises one or more class 1 HLAs.

[0115] In an embodiment, the human HLA class 1 heavy chain sequence is HLA-A, HLA-B, HLA-C, or any combination thereof. In an embodiment, the human HLA class 1 heavy chain sequence comprises multiple forms of HLA-A, HLA-B, HLA-C, or any combination thereof. In an embodiment, the human HLA class 1 heavy chain sequence comprises HLA-A, wherein HLA-A is translocated between HLA-B and HLA-C.

[0116] In an embodiment, the complex has one or more linkers between the peptide and the human HLA class 1 heavy chain sequence. In an embodiment, the one or more linkers are configured to resist or reduce proteolytic cleavage. In an embodiment, the peptide is coupled to the complex via a disulfide bond. In an embodiment, the one or more linkers comprise a conformation configured to not block one or more killer cell immunoglobulin-like receptor (KIR) binding sites on the human HLA class 1 heavy chain sequence.

[0117] In embodiments, the complex is a fusion protein with one or more immune checkpoint agonists. In embodiments, the one or more immune checkpoint agonists include CD47, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, or a combination thereof.

[0118] In embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof, HLA-F or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof. In embodiments, when the complex interacts with one or more T cells, at least one of HLA-E or a fragment thereof, HLA-F or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof is inhibited from eliciting a T cell response.

[0119] In embodiments, the complex has a regulatory peptide. In embodiments, the regulatory peptide is an apoptosis-inducing peptide, for example, acting as a "kill switch" to control the complex. In embodiments, BioNV does not require the use of a regulatory peptide in its complex, but low immunogenic cells intended for use as cell therapy use a regulatory peptide in its complex.

[0120] In embodiments, the complex has an epitope configured to allow detection of the complex. In embodiments, the epitope is or comprises 3,5-dinitrosalicylic acid.

[0121] In embodiments, the complex comprises a human β2M amino acid sequence. In embodiments, the complex has one or more linkers between the peptide sequence and the human β2M sequence, or between the human β2M sequence and the human HLA class 1 heavy chain sequence, or between both.

[0122] In embodiments, the complex has one or more joints. In embodiments, one or more joints are placed between the peptide and the HLA class I sequence; alternatively, one or more joints are located between the HLA class I sequence and one or more of β2M, a checkpoint agonist sequence, and / or a second HLA sequence. In non-limiting embodiments, one or more joints have a sequence having at least about 70%, 80%, 90%, 95%, or 99% sequence identity with an influenza A virus M1 peptide or a histone M3 peptide. In non-limiting embodiments, one or more joints have a sequence consisting essentially of glycine and / or serine residues, such as Gly-Gly-Ser, Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, Gly-Ser-Ser, and the like.

[0123] In an embodiment, the complex includes a fusion construct from N-terminus to C-terminus, a peptide (e.g., binding to the HLA groove), one or more linkers, a human β2M sequence, one or more linkers, and a human HLA class 1 heavy chain sequence. In an embodiment, the complex includes one or more HLAs, wherein when the complex interacts with one or more T cells, the one or more HLAs are inhibited from eliciting a T cell response. In an embodiment, the HLA fusion has one or more linkers that adopt a conformation that is capable of interacting with one or more killer cell immunoglobulin-like receptor (KIR) binding sites on a human HLA class 1 heavy chain sequence (e.g., not blocking binding).

[0124] In embodiments, one or more HLAs have one or more mutations that reduce or inhibit HLA-induced T cell responses when the complex interacts with one or more T cells, including cytotoxic T lymphocytes (e.g., CD4+ and / or CD8+ T cells).

[0125] In an embodiment, the complex has one or more proteins or fragments thereof that reduce or inhibit the immune response of the complement system. In an embodiment, the one or more proteins or fragments thereof include CD48, CD59, or a combination thereof.

[0126] In embodiments, the immunocompetent HLA is in the form of a nucleic acid molecule that is optionally used to replace one or more sequences encoding native HLA genes in a low immunogenic cell. In embodiments, the low immunogenic cell has a genetic element configured to receive one or more sequences for deletion / disruption in the HLA locus. In embodiments, the one or more sequences encode a human HLA class 1 heavy chain sequence fusion complex described herein.

[0127] BioNV for neoantigen presentation

[0128] In some aspects, the present disclosure includes BioNVs with one or more surface-exposed neoantigens. In embodiments, the neoantigen is or includes a protein (or a peptide derived therefrom) of one or more proteins in Table 5 and / or Table 6, including variants or mutants thereof. In embodiments, the neoantigen is one or more peptides in Table 1. Without being bound by theory, one or more biomarkers stimulate whole cells expressing homologous CARs in co-therapeutic applications while also retaining the CAR-directed lethality of BioNV.

[0129] Table 1. Illustrative neoantigen amino acid sequences.

[0130]

[0131] In embodiments, BioNV (and cells derived from BioNV) express MHC class I / II receptors to display neoantigens. In embodiments, MHC class I / II receptors are immunocompetent HLA molecules, as described herein. In embodiments, MHC class I / II receptors display neoantigens through direct binding interactions. In embodiments, MHC class I / II receptors are fused to neoantigens (e.g., via a flexible peptide bond). In embodiments, the fusion comprises one or more amino acid linker sequences, as described herein.

[0132] In embodiments, "expression or increased expression" of "genes and / or gene products" encompasses increased expression at the DNA level (e.g., stable expression of gene knock-ins, insertion of a transgenic cassette with a gene and enhancer, an internal ribosome entry site (IRES) element, etc.), or changes external to the gene itself, resulting in an increase in the gene product (e.g., increased transcription factor concentration, insertion / replacement of a more active promoter upstream of a gene, etc.). In embodiments, expression or overexpression of one or more immunoprotective proteins is achieved at the RNA level using one or more of small regulatory RNAs, IRES elements, regulating the concentration of activated transcription factors in cells, or a combination thereof.

[0133] In embodiments, expressing one or more immune protective proteins or increasing their expression is achieved by introducing exogenous genetic elements. In some embodiments, the introduction of exogenous genetic elements is achieved by stable integration into the cell genome. In embodiments, stable integration is achieved by one or more of the following: a method based on a transposase, a method based on Cre / Lox, a method based on an endonuclease, a method based on homologous recombination (HR), a method based on non-homologous end connection (NEHJ), a method based on microhomology-mediated end connection (MMEJ), a method based on homology-mediated end connection (HMEJ) or a combination thereof. In embodiments, stable integration is achieved by viral vectors. In embodiments, the introduction of exogenous genetic elements is achieved by transient transfection. In embodiments, expressing one or more immune protective proteins or increasing their expression is achieved by exogenous promoters and / or enhancers, and / or endogenous promoters and / or enhancers, or a combination thereof. In embodiments, expressing one or more immune protective proteins or increasing their expression is under the control of a constitutively active promoter.

[0134] In an embodiment, expression of one or more immunoprotective proteins or increased expression thereof is achieved at the DNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA. In embodiments, expression of one or more immune protective proteins or increased expression thereof is achieved at the RNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA. In embodiments, expression or overexpression of one or more immune protective proteins is achieved by one or more of small regulatory RNA, miRNA, IRES element, transcription factor, or a combination thereof.

[0135] The expression or overexpression of one or more immune protective proteins is achieved by knocking in genetic elements. In embodiments, the expression or overexpression of one or more immune protective proteins is achieved by Cre / Lox recombinase system, transposase-based system or endonuclease system, as described herein. In embodiments, the "knock-in" of "genetic elements" may refer to an engineered promoter / enhancer element upstream of an existing gene, or a new transgenic box is inserted, the transgenic box having a gene copy consistent with one or more promoters, enhancers, introns, IRES sequences or any other elements that can be used to increase gene and / or gene product expression. In embodiments, expression or overexpression is achieved by exogenous promoters or enhancers (for example, from plasmids, or promoters or newly added cis-acting DNA elements). In embodiments, expression or overexpression is achieved by endogenous promoters or enhancers, for example, due to an increase in the concentration of transcription factors acting on endogenous promoters (for example, without genome manipulation). In embodiments, expression or overexpression is under the control of a constitutively active promoter (e.g., for mammalian systems, SV40, CMV, UBC, EF1A, PGK CAGG, etc.) and / or an inducible promoter (e.g., tetracycline-controlled, etc.).

[0136] In embodiments, expression or overexpression is achieved by stable integration of genes into cells. In embodiments, stable integration is achieved by viral vectors. In embodiments, iPSC (and other cells) are genetically engineered for gene cassette integration (e.g., CAR or any other element described herein). In embodiments, gene cassette integration may include both integrative and non-integrative transgenic insertions. Non-limiting examples of non-integrative transgenic insertions include mRNA, non-integrative lentivirus, and endonuclease targeting methods. Integrative gene cassette insertion methods include stable retroviral vector insertion and transposase-based integration systems. For example, stable gene cassette transduction can be achieved using retroviral vectors that enable iPSC to maintain genetic elements encoding genes throughout differentiation, amplification, and activation. In embodiments, brexucabtagene autoleucel (brexucabtagene autoleucel) has been achieved using GRV vectors. KitePharma Inc.) and axicabtagene ciloleucel ( Kite Pharma Inc.) CAR cassette into T cells, and tisagenlecleucel ( Novartis International AG) (Labbé, RP, et al. "Lentiviral Vectors for TCell Engineering: Clinical Applications, Bioprocessing and Future Perspectives." Viruses 13 (2021); 1528. doi:10.3390 / v13081528).

[0137] In embodiments, low immunogenic cells can express one or more immunoprotective proteins by transient transfection (e.g., electroporation, lipid reagents, etc.). In embodiments, low immunogenic cells can be genetically modified to contain one or more knockouts of the above-mentioned immunogenic proteins, and the expression or overexpression of one or more immunoprotective proteins can be controlled by transient expression in subsequent steps of the manufacturing process.

[0138] In embodiments, the low immunogenic cells (and BioNV derived therefrom) are allogeneic. In embodiments, allogeneic refers to a "ready-made" quality of cells derived from a single source, used to create clonal cell populations suitable for treating a variety of diseases, and can be administered to multiple patients regardless of the immunological characteristics of the subjects to be treated.

[0139] In embodiments, the low immunogenic cells do not cause an immune response in a patient to whom the cells or biological compositions derived therefrom are administered. For example, in embodiments, the low immunogenic cells do not cause an inflammatory response and / or an immune response when administered. In embodiments, upon administration to a subject, the low immunogenic cells cause less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of an inflammatory or immune response, as described herein. The measurement is performed as a function of the concentration of a cytokine, chemokine, or immunomodulatory enzyme, such as IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20, IFN-α / β / γ, TNFα / β, IDO, HLA-G, HGF, PGE2, etc., or any combination thereof, such as compared to a homologous whole cell therapy counterpart.

[0140] In embodiments, low immunogenic cells include one or more targeting agents. In embodiments, cells include one or more targeting agents. In embodiments, one or more targeting agents include a chimeric antigen receptor (CAR). In embodiments, CAR is bispecific. In embodiments, CAR lacks an intracellular portion. In embodiments, CAR includes a targeting agent, a transmembrane domain, and an intracellular domain, which includes a co-stimulatory domain and / or a signaling domain. In embodiments, the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α or ICOS, or a fragment thereof. In embodiments, the intracellular domain includes an intracellular signaling domain and / or one or more co-stimulatory molecules of the CD3ζ chain, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40. In embodiments, one or more targeting agents include an antibody or an antibody form. In embodiments, the antibody or antibody form is selected from one or more of the following: monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), V NAR 、V H In some embodiments, the one or more targeting agents include antigen binding proteins, such as antigen binding proteins, ...

[0141] In embodiments, low immunogenic cells express one or more targeting agents through adjustable expression elements. In embodiments, adjustable expression elements include adjustable promoters (e.g., Tet on / off promoters) or CRISPRa / i regulatory systems, and other adjustable expression elements. In embodiments, CAR expression is controlled by adjustable expression elements. Simple overexpression of the construct of CMV (or other types) promoter may cause the surface density of CAR to be too high, which may cause many problems, such as 'overactivation', leading to in vitro exhaustion and cell death after activation. Cell death leads to the loss of the cell line producing BioNV. In embodiments, the surface density of CAR protein constructs is adjusted. For example, in embodiments, the typical concentration range of CAR protein in each microgram of T cell is between 0.20ng-0.70ng. Too little CAR expression can lead to poor biomarker targeting. However, the density cannot exceed the cell limit. Too high CAR density may lead to exhaustion of mother cells during in vitro activation before BioNV derivation, and may also lead to poor quality of BioNV due to high protein concentration in the plasma membrane. The upper limit allows the improvement of the targeting efficiency of low biomarker expression on cancer cells. For example, density limits for cell therapy are listed in published U.S. non-provisional application US20220040106, which is hereby incorporated by reference.

[0142] In an embodiment, the stable cell integration (safe harbor genetic position) of any genetic element described herein in a cell (e.g., iPSC) can be controlled by implementing a Tet-regulated CRISPRa+ targeting 3x transcription factor targeting gRNA system. The activation of three upstream transcription factors by the CRISPR activation system can trigger a signal cascade event, enhancing the production of CARs that replace endogenous antibody ORFs at specified sites. This system can be 'tuned' by including a Tet-regulated promoter, thereby being able to change the concentration of cell surface CARs. Next, Cpf-1 directed homology-directed repair (HDR) can be achieved by replacing CDRs and heavy and light chain antibody regions with CAR cassettes. Finally, the stably integrated CAR cassette may contain flanking gRNA binding sites, which allow scFV (and other antibody forms) or VERR / viral ligands to be repeatedly exchanged or changed to quickly and consistently insert the desired sequence.

[0143] In embodiments, a variety of transcriptional control elements, such as tetracycline on / off promoters (or similar drug-regulated promoters) can be used to regulate the CAR concentration on the surface of iPSC basal cell lines or any downstream differentiated cells (and the resulting BioNV), to drive the expression of CRISPR activation / gRNA (CRISPRa) systems. Then, the CRISPRa system can activate antibody regulatory transcription factors, such as Drm2, Fr5, and Bxp2, which regulate the expression of engineered CAR boxes integrated into antibody gene loci (positions where antibody genes have been replaced). In addition, similar transcriptional control elements can be provided to control the overexpression of genes (such as CD47) at defined manufacturing stages, drive genes that control differentiation, etc.

[0144] In embodiments, regulatory points within cells may be engineered to express a particular therapeutically relevant protein of interest into a stable cell line (eg, stable integration for constitutive expression).

[0145] In embodiments, transcription factors can be activated in cells by providing small molecules, (de) phosphorylation events or nucleic acids, etc. In embodiments, transcription factors can be expressed transiently by plasmids. In embodiments, transcription factors can be stably expressed by integration (e.g., with constitutively active promoters) to produce stable cell lines with constitutive signaling of signaling pathways associated with therapeutic biomolecules. In embodiments, therapeutic biomolecules expressed in low immunogenic cells include cytokines (e.g., for functions such as pro-inflammatory, anti-antigen, and recruitment), perforins, granzymes, chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines, tumor necrosis factors (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) or a combination thereof.

[0146] In an embodiment, the promoter region or enhancer region of a gene can be regulated by overexpression of a specific transcription factor, regulated by microRNA, tRNA, activated or inhibited using a (g)RNA-guided nuclease with an activation or inhibition domain associated therewith (e.g., CRIPSRa / CRISPRi).

[0147] In embodiments, therapeutically relevant genes of interest can be integrated (stably or transiently) into cells of interest (based on desired properties, such as membrane proteins that are inferred to cross a barrier). In embodiments, stably integrated genes can be activated by any of the methods described herein.

[0148] In embodiments, background (unwanted) mRNA can be silenced with interfering RNA (e.g., siRNA, RNAi, etc.) to enhance the presence / expression of the desired mRNA for the protein of interest. In embodiments, mRNA can also be regulated by IRES elements. In embodiments, specific splice variants can be enhanced by adding IRES enhancing and / or inhibiting biomolecules to produce desired therapeutically relevant peptides / proteins that can be packaged into BioNV during post-activation cellular processing.

[0149] In embodiments, unwanted genes may be knocked out to enhance expression of desired therapeutically relevant genes.One skilled in the art will appreciate methods that can be used to transiently and stably knock out unwanted genes and / or entire signaling pathways during BioNV processing.

[0150] In embodiments, low immunogenic cells are derived from iPSCs that have been engineered as described herein. In embodiments, iPSCs are recovered from somatic cell states using microRNA technology instead of small molecule transactivators. A tighter differentiation system can be provided using microRNAs, thereby producing higher quality iPSCs. Without wishing to be bound by theory, these high-quality iPSCs are less prone to expression inhibition (rear engineering proteins, such as CD47) and genetic drift, and have higher culture division quality / quantity (before cell integrity problems occur, cultures can be divided more times than other methods).

[0151] In embodiments, BioNVs derived from iPSC-derived low immunogenic cells retain the functions of low immunogenic cells, such as, but not limited to, the ability to cross the blood-brain barrier, such as in the case of macrophages / monocytes, or tissue-specific factors, such as in the case of cardiomyocytes, hepatocytes, etc.

[0152] In an embodiment, the MHC class I and MHC class II complexes of allogeneic iPSCs are disrupted by knocking out key proteins involved in their expression, such as B2M, which is a serum protein found associated with MHC class I heavy chains on the surface of nearly all nucleated cells and is involved in presenting peptide antigens to the immune system.

[0153] In the embodiments, once B2M knockout (KO), CIITAKO, IL-6KO and CD47tg knock-in (KI) are engineered into iPSC, TRAC and TRBC genes can be knocked out. In the embodiments, only one gene of each is knocked out, rather than two genes on a single allele. In the embodiments, TRAC and TRBC genes can be knocked out as described herein. The purpose of knocking out TRAC and TRBC genes is to eliminate T cell receptors. In the embodiments, modified cells are differentiated into T cell subsets lacking T cell receptors to derive BioNV. Cells are genetically modified so that they are substantially lacking TCR, reducing the chance of competing ligands for CAR constructs that can non-specifically target alternative tissues. Therefore, in the embodiments, TCR genes are knocked out as a strategy to reduce the off-target effects of BioNV. In the embodiments, TRAC / TRBC knockouts typically reduce the possibility of CRS and BioNV toxicity.

[0154] In embodiments, the modified cells are expanded following engineering; any small-scale expansion or large-scale feeder system expansion method known in the art may be used.

[0155] In embodiments, after constructing B2M KO, CIITA KO, IL-6KO, CD47tg KI and IL-2 promoter-driven green fluorescent protein (GFP) (IL-2p GFP) reporter genes, CAR constructs can be integrated / engineered into cells. In embodiments, CAR constructs can be knocked into TRAC / TRBC genes, while knocking out the remaining TRAC / TRBC genes, thereby producing CAR+ and TRAC / TRBC- / - cells. In embodiments, CAR constructs can knock into the TRAC / TRBC gene positions on two loci at the same time, thereby producing CAR+ / + and TRAC / TRBC- / - cells.

[0156] In the embodiments, once B2M KO, CIITA KO, IL-6KO, CD47tg KI, IL-2p GFP KI and CAR modified cells (e.g., iPSC) are engineered, the quality of the immune synapse (IS) is measured between the CAR recognition domain and the biomarker. In the embodiments, the IS quality of BioNV can be directly related to the efficacy of whole cell therapy.

[0157] In embodiments, BioNV or low immunogenic cells derived therefrom include nucleic acids encoding GFP (and other fluorescent proteins). In embodiments, once B2M KO, CIITA KO, CD47tg KI, IL-6KO, TRAC / TRBC single KO are engineered into iPSC, GFP molecules are engineered into modified cell lines. In embodiments, this serves as a control cell line. In embodiments, non-control cell lines (therapeutic cell lines) do not have GFP. In embodiments, nucleic acids encoding GFP are operably connected to promoters from one or more of the following: IL-2, perforin, granzyme, alarmin, TNF, INF, combinations thereof and / or any other cell-specific genes or reporter genes. When lymphocytes are widely / overall activated due to various stimuli, the IL-2 promoter will be constitutively activated. In embodiments, more concentrated activation / inhibition (regulation) is used. In embodiments, IL-2p GFP reporter gene serves as an indicator (as part of BioNV derivatization process) of the extensive / overall activation degree of cells. In embodiments, GFP signaling combined with immunoblot analysis of cytokine levels (e.g., perforin, granzymes, alarmins, TNF, and INF) can effectively modulate the extent of widespread / overall activation of lymphocytes upon exposure to activation antigens. In embodiments, GFP is used to compare activation levels between manufacturing batches and ensure consistency in therapeutic development.

[0158] In embodiments, broad or global regulation of cells results in the expression of multiple genes to produce cytokines, chemokines, regulatory nucleic acids, and other therapeutically relevant biomolecules that cause cells to enter an 'activated' state, thereby enhancing the ability of the cells to achieve their metabolically predetermined purpose / phenotype. In embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can reproducibly, for example, T lymphocytes enter an activated state, in which T cell receptor (TCR) binding to antigen presenting cells (APCs) occurs ( Figure 1 In embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can mimic the interaction between antigenic peptides presented in the major histocompatibility complex (MHC) and the TCR of T cells, which induces conformational changes in the TCR, thereby triggering intercellular signaling cascades and the expression of a large number of genes for cytokines, including perforins, granzymes, alarmins, interleukins, and interferons, etc. ( Figure 1). Typically, cytokines 'activate' cells into a specialized mode that enables the cells to clear the host's antigens and recruit immune cells to the site of infection to aid in the clearance of antigens / infected cells and repair of surrounding tissues. In embodiments, these cellular processes occur without the provision of exogenous cytokines, which can result in deleterious effects in cell lines, such as exhaustion, competing signaling pathways, etc. In embodiments, the activation state of cells (e.g., T cells) is exploited in BioNV / exosomes by processing methods that capture cytokines or transmembrane ligands in their lumen and / or membrane, such as Figure 2 shown.

[0159] In embodiments, low immunogenic cells are activated and / or purified, e.g. Figure 3 In embodiments, in addition to activating pathways, methods for broad or global cell regulation also include binding the antigen (in addition to other activating molecules) to magnetic or streptavidin-biotin beads to ensure that the antigen is separated from the cell activation receptor ( Figure 3 ). For example, in embodiments, biomarker antigens that form immune synapses (IS) with CAR constructs can be conjugated to magnetic or streptavidin-biotin beads and then added to CAR-containing cells in vitro to activate the cells. In embodiments, once activated, biomarker antigens can be mechanically removed from the cell suspension.

[0160] In embodiments, by manipulating regulatory points within signal transduction sites, therapeutically relevant biomolecules (e.g., cytokines) can be selectively expressed from within any given cell. In embodiments, a point (or points) within a single signal transduction pathway (or pathways) can be activated, for example, activating a kinase, (de)activating a phosphorylase, activating a hydrolase (e.g., a GTPase), introducing an inhibitor (to diffuse or block a signal from one pathway to another), or accelerating an activator / agonist. In embodiments, these signal transduction pathways can be selectively activated by providing proteins, peptides, small molecules, nucleic acids, carbohydrates, chimeric molecules, viral ligands, inorganic elements / compounds (e.g., calcium), etc., to cells alone or in combination to drive the expression of therapeutically relevant biomolecules within the cell, which can then be packaged into the lumen of the BioNV / exosome ( Figure 3). In embodiments, regulatory points may include, but are not limited to, one or more cell surface receptors that are targeted alone or in combination to focus intracellular signals to express biomolecules of interest. In embodiments, cells may be genetically modified to be particularly sensitive to activation of these pathways, thereby generating metabolic / phenotype regulatory cells (of any kind) that can produce BioNV / exosomes. In embodiments, cells may be made "particularly sensitive" to activation of certain stimuli by overexpressing cell surface receptors, overexpressing intracellular signaling molecules (e.g., STAT, NF-κB, MAPK / ERK / ATM kinases, etc.), and / or constitutively active mutants thereof.

[0161] In the embodiments, the low immunogenic cells are CD34+, or derived from CD34+ cells, such as human CD34+ umbilical cord blood. In the embodiments, CD34+ umbilical cord blood-derived cell lines can be used as the basic cell lines developed, produced and manufactured by BioNV for delivery of gene editing therapeutics. In the embodiments, low immunogenic cell lines derived from CD34+ umbilical cord blood have been experimentally confirmed to have low expression of HLA1 / 2 and overexpression of CD47 (Deuse T. et al. "Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fullyimmunocompetent allogeneic recipients." Nat Biotechnol. 2019; 37 (3): 252-258).

[0162] In embodiments, hypoimmunogenic cells can be engineered using a variety of hypoimmunogenic engineering techniques, for example, as described in Deuse et al., Han et al., Xu et al., Harding et al., and as described in published U.S. patent applications US20190376045, US20190376045, US20210308183, and US20210292715 (to Deuse), US20210161971 (to Nagy), US20180141992 (to Strominger), and published European patent application 3693384 (to Poirot), each of which is incorporated herein by reference in its entirety (Han X et al., "Generation of hypoimmunogenic human pluripotent stem cells"). cells." PNAS. Vol. 116, No. 21, 2019: pp. 10441-10446. doi: 10.1073 / pnas.1902566116.) (Xu H et al. "Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility." Cell Stem Cell. Vol. 24, No. 4, 2019: pp. 566-578. doi: 10.1016 / j.stem.2019.02.005.).

[0163] In an embodiment, BioNV is derived from cells that have been eliminated from HLA genes that encode MHC membrane glycoproteins that confer immune responses associated with GVHD rejection. HLA gene clusters can be divided into three categories: 1) MHC class I pathway, 2) MHC class II pathway, and 3) MHC class III pathway. Only MHC class I and class II pathways express protein complexes that trigger immune responses in GVHD, while MHC class III complexes do not participate in immune activities.

[0164] The elimination of MHC class protein complexes can trigger NK cells and macrophages to enter active clearance mode, and then the cells are destroyed. In order to avoid this killing mechanism, in an embodiment, CD47 isotype 2 transmembrane molecule protein tags can be added to the cell membrane of the modified cell to avoid NK and macrophage-mediated killing reactions, for example, as described in Willingham et al., Deuse et al. and Han et al. (Willingham SB et al. "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors." PNAS. Vol. 109, No. 17, 2012: 6662-7 pages. doi: 10.1073 / pnas.1121623109.).

[0165] In embodiments, cells may be engineered to use additional mechanisms to prevent these killing responses, such as those described in the following literature: 1) CD24 transmembrane molecule protein tag (e.g., performed in Zhao et al.), 2) membrane-bound surfactant protein-D (SP-D) (e.g., performed in Jiaravuthisan et al.) (Jiaravuthisan P et al. "Amembrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells." Transpl Immunol. Vol. 47, 2018: pp. 44-48. doi: 10.1016 / j.trim.2018.02.003.), and 3) a molecule PD-L1 tag for preventing T cell responses. In embodiments, BioNV derived from 'activated' cells will encapsulate and / or release perforin and / or granzymes, resulting in targeted cell death. In embodiments, activated cells will produce perforin and / or granzymes to be packaged into BioNV. In an embodiment, the low immunogenic cells to be activated do not express PD-L1 to avoid the resulting BioNV being targeted to PD-1 on T cells. In an embodiment, this reduces the possibility of releasing perforin and / or granzymes, resulting in unwanted T cell death. In an embodiment, PD-L1 is overexpressed in BioNVs derived from cells that have not yet been activated and are not loaded with apoptotic cytokines. In an embodiment, the PD-L1 of low immunogenic cells to be activated is downregulated, knocked out, or otherwise silenced. In an embodiment, unactivated low immunogenic cells have upregulated PD-L1, i.e., for BioNVs for gene editing agent delivery. In an embodiment, CD47 isotype 2 can be engineered into cells to prevent macrophage and NK cell-mediated cytotoxicity because it acts as a "don't eat me" tag through SIRP-α receptors expressed on these cells and other cells.In an embodiment, CD47 can be used in genetically engineered iPSCs to achieve immune tolerance to innate immune cells, such as in studies by Chhabra et al., Han et al., and Jaiswal et al. (Chhabra A et al. "Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy." Sci Transl Med. Vol. 8, No. 351, 2016: 351ra105. doi: 10.1126 / scitranslmed.aae0501.) and (Jaiswal S et al. "CD47 is upregulated oncirculating hematopoietic stem cells and leukemia cells to avoid phagocytosis." Cell. Vol. 138, No. 2, 2009: pp. 271-85. doi: 10.1016 / j.cell.2009.05.046.). In embodiments, cells may be modified as described in U.S. Patent No. 8,562,997 to Jaiswal et al., which is incorporated herein by reference in its entirety.

[0166] In embodiments, some methods that do not completely knock out all HLA genes can be used, for example, as done by Xu et al. and Han et al., only HLA genes that are highly associated with immune response are knocked out, while HLA genes that inhibit macrophage or NK response remain intact (e.g., HLA-E, HLA-F, and HLA-G). In embodiments, this method does not require the addition of a CD47 tag; modified cells can be engineered to produce BioNVs with or without CD47.

[0167] In an embodiment, the method improves upon the low immunogenicity method of Table 2.

[0168] Table 2 Three methods of modifying cells using HLA knockout combined with CD47 isoform 2 tag and PD-L1 transmembrane tag (Zhao et al.) and (Gornalusse GG et al. "HLA-E-expressing pluripotent stem cells "Escape allogeneic responses and lysis by NK cells." Nat Biotechnol. Vol. 35, No. 8, 2017: pp. 765-772. doi:10.1038 / nbt.3860.).

[0169]

[0170]

[0171] In embodiments, developing allogeneic modified cells involves removing MHC class I and MHC class II protein complexes by destroying certain HLA genes or B2M knockouts, and then knocking out the CIITA gene. In embodiments, due to its rapid mechanism of action, CRISPR gene editing methods can be used for knockout. In embodiments, zinc finger nucleases (ZFNs) and / or TALENS are used for knockout. In embodiments, Cre / Lox recombinase system is used to produce modified cells. In embodiments, RNA silencing (RNAi, shRNA, microRNA, CRISPR Cas13a-d, etc.) is used to produce modified cells.

[0172] In the embodiments, the method for developing allogeneic, low-immunogenic modified cells is different from the method of Harding et al. to produce allogeneicity. The method of Harding et al. includes an alternative method based on immune escape mechanisms in nature, rather than deleting MHC class I / II genes and risking long-term acceptance of receptors. The method relies on the biomimicry of Harding et al. based on horizontally transmitted cancer DFTD type 2 (dominant in Tasmanian devils). In the embodiments, the development of allogeneic modified cells includes expressing immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200 and / or MFG-E8 or increasing their expression to protect cell derivatives from long-term immune rejection in mice (and humans) without deleting MHC class I / II proteins. In the embodiments, the modified cells express one or more proteins shown in Table 3, including any splice variants and / or isomers (e.g., CD200 splice variants) of any of the proteins shown. In the embodiments, this system can be used to interfere with the activity of APCs, macrophages, NK cells, and T lymphocytes. In an embodiment, the modified cell line may also contain a safe cell system developed by Liang et al. in 2018, in which a cell division gene is linked to a suicide gene to prevent uncontrolled teratomas from causing cancer (Liang Q et al. "Linking a cell-division gene and a suicide gene to define and improve cell therapy safety." Nature. Vol. 563, No. 7733, 2018: pp. 701-704. doi: 10.1038 / s41586-018-0733-7.).

[0173] In an embodiment, the method improves upon the low immunogenicity method of Table 3.

[0174] Table 3. Expression or increased expression of illustrative proteins used to generate allogeneic modified cells.

[0175]

[0176] In an embodiment, the low immunogenic cells from which BioNV are derived are engineered to knock out one or more of the following: HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, CIITA, IL-6, IL-4, IL-10, IL-16, TRAC, TRBC, SerpinB9 and / or any combination thereof; and knock in one or more of the following: CCL2, PD-L1 (in BioNV from unactivated cell sources), CTLA-4, H2-M3, CD24, CD47 (minus the 3'UTR region or an alternative 3'UTR region that does not contain inhibitory microRNA binding sites), MFG-E8, CD200 and / or any combination thereof.

[0177] In embodiments, BioNV is produced by low immunogenic cells having one or more modifications of Table 4.

[0178] Table 4 Illustrative engineered cell expression profiles for BioNV formation for human use (Fife BT and Bluestone JA."Control ofperipheralT-cell toleranceand autoimmunityviatheCTLA-4 and PD- 1pathways.” Immunol Rev. Vol. 224, 2008: pp. 166-82. doi:10.1111 / j.1600- 065X.2008.00662.x.) and (Rong Z et al. "An effective approach topreventimmunerejectionof humanESC-derivedallografts.” CellStemCell. Volume 14, No. Issue 1 2014: Pages 121-30. doi:10.1016 / j.stem.2013.11.014.).

[0179]

[0180] In embodiments, the inactivation / activation of genes is controlled by inducible promoters during the entire differentiation, activation and manufacturing process of BioNV. In embodiments, the destruction of MHC, TCR and CRS genes will produce - / -CRS and - / -TCR allogeneic iPSC, resulting in it having a plasma membrane showing low immunogenicity characteristics when infused into a subject. The CRS genes related to CRS pathogenesis include IL-6, IL-10, IFN-γ, monocyte chemotactic protein 1 (MCP-1), granulocyte macrophage colony stimulating factor (GM-CSF), and other cytokines, including tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor-α and IL-8. In embodiments, one or more of these genes are inactivated, for example, in cells derived from BioNV.

[0181] In the embodiments, BioNV is formed by destroying the cell membrane of engineered iPSC. In the embodiments, hypo-iPSC is characterized by B2M- / -, CIITA- / -, CD47+ / +, PD1- / - plasma membrane spectrum, and can be used to produce BioNV. Low immunogenicity BioNV can be produced from parental iPSC cell lines by methods such as ultrasonic treatment, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis and electroporation. In the embodiments, continuous extrusion is a method for producing low immunogenicity BioNV. In the embodiments, continuous extrusion of iPSC can produce HLA1 / HLA2 negative (low immunogenicity) and tgCD47+ BioNV, showing PD1 resistance elimination.

[0182] BioNV produced by low immunogenic cells

[0183] In some aspects, the present disclosure includes BioNVs of about 20-1200 nm in size, and in embodiments, may contain outward-facing, membrane-embedded targeting agents (e.g., CARs) capable of binding one or more target molecules. In embodiments, BioNVs are biomimetic due to nanovesicle compositions derived from the plasma membrane of allogeneic, low-immunogenic modified cells. In embodiments, BioNVs comprise a plasma membrane-derived lipid bilayer that completely encapsulates an aqueous core that can accommodate a variety of cell-derived molecules, including perforins, granzymes, cytokines, gene editing payloads, and the like. In embodiments, the aqueous core of BioNVs may further encapsulate exogenous biologics, fluorescent proteins, tracer dyes, radionuclides, and small molecules, as well as other therapeutic agents, which may be synthesized in cells before cell disruption or added in cell processing steps.

[0184] In embodiments, BioNV can inherit CAR constructs from low immunogenic cells, and the cells may include a variety of structural molecules. The structural function of prototype CAR includes a fusion protein, which includes an extracellular (or outward-facing) binding portion (e.g., scFv), which is connected to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.) by a hinge peptide (e.g., CH2 / CH3 domains from IgG Fc regions, Gly-Gly-Ser peptide bonds, CD28 peptides, CD8α peptides, etc.), followed by a variety of intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.). In embodiments, BioNV lacks the intracellular mechanism of whole cells, so CAR design does not require any intracellular signaling molecules (primary CAR constructs). In embodiments, CAR constructs include an extracellular scFV binding portion fused to a CD28 transmembrane domain with an IgG CH2 / CH3 linker, and substantially lack any intracellular domain or function.

[0185] In embodiments, the CAR construct has a prototype intracellular domain exchanged or otherwise fused with an anchor protein, such as a PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., which may facilitate fusion with target cells and / or packaging and release of therapeutic payloads. In embodiments, BioNV expresses one or more factors that increase absorption, including but not limited to one or more membrane-embedded proteins, surface functionalization and / or anionic or cationic binding lipids and / or viral ligands / receptors, which improve or promote the absorption of BioNV.

[0186] In embodiments, the CAR antigen binding molecule comprises a variety of binding moieties, including binding domains based on antibodies or antibody forms. In embodiments, BioNV comprises an antibody or antibody form binding moiety selected from one or more of the following: monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), double antibodies, nano antibodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising antigen binding moieties of antibodies. In embodiments, the CAR construct comprises a fusion protein with a bispecific T cell engager (BiTE), a viral epitope recognition receptor (VERR) or a viral ligand, a variable heavy chain IgG fragment V H H.V NAR or by engineering the binding portion of a T cell receptor (TCR).In an embodiment, the BioNV comprises a ligand of a receptor or a receptor of a ligand as a targeting agent.

[0187] To ensure the correct directionality of the CAR and eliminate BioNVs lacking the CAR, in embodiments, HPLC-based affinity chromatography techniques can be used to select and concentrate only the BioNVs exposed to the solvent CAR with sufficient surface concentrations. HPLC-based affinity chromatography techniques can be used to reduce the concentration of contaminating cell material and NVs containing immunogenic cell surface markers by positive or negative selection.

[0188] In embodiments, BioNVs may include NVs with only an outer plasma membrane leaflet, only an inner plasma membrane leaflet, and / or both leaflets of the plasma membrane lipid bilayer intact. In embodiments, iPSC-derived NVs include additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, phosphatidylcholine, etc.), nonionic surfactants (e.g., sorbitol monostearate, octadecylamine, etc.), sterols (e.g., cholesterol, bile salt derivatives, etc.), polyols (e.g., maltodextrin, sorbitol, sucrose, mannitol, etc.), and proteins (e.g., serum albumin, Fc, etc.) to improve physicochemical properties such as thermal stability, clearance, and therapeutic payload packaging / release. The cholesterol content and the length and saturation of the hydrocarbon chain of the phospholipids affect the rigidity and stability of the bilayer, and thus the ability of the NV to load and release drugs, biomolecules, and other therapeutic payloads. In embodiments, BioNV also incorporates zwitterionic lipids and methods of using zwitterionic lipids, for example, as described in U.S. Patent Publication No. US20130216607, the contents of which are incorporated herein by reference in their entirety. Accordingly, functionalizing the hydrophilic head of the lipid with a polymer or biomolecule can provide additional features to the vesicle surface, thereby shaping its interaction with blood components, tissues, and the immune system in vivo.

[0189] In embodiments, low immunogenic cells can synthesize one or more therapeutically relevant biomolecules for packaging into BioNV. In embodiments, the one or more therapeutically relevant biomolecules are cytokines, proinflammatory cytokines, anti-antigen cytokines, perforins, granzymes, chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines and tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), or a combination thereof.

[0190] In embodiments, low immunogenic cells can be used to encapsulate payloads; for example, "lumen loads", or the ability of BioNV derived from cells to load payloads into the lumen (space in biomimetic nanovesicles). In embodiments, payloads are one or more of biologics, nucleic acids, fusion proteins, fluorescent proteins, tracer dyes, radionuclides, and / or small molecules. In embodiments, payloads are therapeutic payloads for the disease types targeted by CAR. In embodiments, payloads include one or more of the following: alkylating agents, anthracyclines, antimetabolites, antitumor antibiotics, antibodies or antibody forms, corticosteroids, plant alkaloids, topoisomerase inhibitors, checkpoint inhibitors, anti-infective agents, and / or growth factors.

[0191] In an embodiment, the nucleic acid payload encodes one or more of the following: a CRISPR / Cas component, a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and / or a small non-coding RNA.

[0192] In an embodiment, the payload includes gene editing nucleic acids and / or proteins, such as TALEN, ZFN, RNaseP RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposases, and / or any orthologs or homologs of any of these editing agents. In an embodiment, the gene editing agent may also include gRNA, which refers to guide RNA as used herein. In an embodiment, the gRNA may be a sequence complementary to a coding or non-coding sequence and may be customized according to a specific sequence to be targeted. In an embodiment, the gRNA may be a sequence complementary to a protein coding sequence, such as a sequence encoding one or more viral structural proteins (e.g., gag, pol, env, and tat). In an embodiment, the gRNA sequence may be a sense sequence or an antisense sequence. In an embodiment, when a gene editing agent composition is administered herein, it is preferably but not limited to including two or more gRNAs; however, a single gRNA may also be used.

[0193] In embodiments, BioNV delivers a gene editing payload comprising a transcription activation response region (TAR) loop system. In embodiments, BioNV encapsulates a plasmid expressing a gene editor and contains a TAR loop sequence between the 5' end of the promoter and the gene editor / guide box and acts as a barrier to block transcription. In embodiments, transcription will only be triggered in cells that are infected and contain the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, relaxes it, and releases the promoter for transcription, thereby expressing the editor and its guide.

[0194] In an embodiment, the primary targeted BioNV is used to deliver a small molecule therapeutic payload. In an embodiment, a second generation (or third or fourth generation) of BioNV containing CAR derived from activated lymphocytes may contain cytokines and other cytotoxic peptides. In an embodiment, BioNV can be formatted to encapsulate and deliver plasmid DNA, for example, to express gene editing nucleases and gRNA in target cells. Alternatively or additionally, in an embodiment, BioNV can encapsulate nucleases and gRNA. In an embodiment, a targeted second generation (or third or fourth generation) BioNV can be designed to encapsulate and deliver additional therapeutic proteins or peptides of interest.

[0195] In embodiments, BioNV comprises one or more of CD34, CCL21, PD-L1 (non-activated cell source) and / or CTLA-4, FasL, SerpinB9, H2-M3; one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200; or both of CD24, CD47, and CD200; MFG-E8, NCAM, α-phagocytic integrin, anti-IL-6R antibody or antibody form, and / or fusions or portions thereof. In embodiments, BioNV may have NCAM or any other protein that promotes fusion of BioNV with the plasma membrane of target cells. In embodiments, BioNV encapsulating at least one granzyme may lack SerpinB9 and / or CD200. In embodiments, BioNV expressing PD-L1 is derived from non-activated cells and BioNV is substantially lacking perforin and / or granzymes.

[0196] In embodiments, BioNV is substantially lacking one or more of the following: MHC class I complex, MHC class II complex, HLA-A complex, HLA-B complex, HLA-C complex, HLA-E or HLA-G complex, HLA-F complex, T cell receptor alpha chain (TRAC) protein, T cell receptor beta chain (TRBC) protein, PD-1, SerpinB9, IL-4, IL-6, IL-10 and / or IL-16. In embodiments, BioNV is substantially lacking one or more of the above, thereby being allogeneic and / or low immunogenic. In embodiments, BioNV does not produce an adverse immune response when infused into a subject to treat a disease.

[0197] In embodiments, BioNV comprises one or more targeting agents. In embodiments, cells from which BioNV is derived may be modified to express one or more targeting agents. In embodiments, one or more targeting agents are CARs. In embodiments, one or more targeting agents may be any antibody or antibody form described herein.

[0198] In embodiments, BioNV targets one or more biomarkers of Table 5 and / or Table 6.

[0199] Table 5. Illustrative checkpoint biomarkers and checkpoint biomarker ligands.

[0200]

[0201]

[0202] Table 6. Illustrative cancer cell biomarkers.

[0203]

[0204]

[0205]

[0206] In embodiments, BioNV is formed by destroying activated cells by one or more of the following: sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, enzymatic disruption of cells (e.g., trypsin digestion), detergent cell lysis and / or electroporation. In embodiments, cell disruption is performed by continuous extrusion.

[0207] In embodiments, the size uniformity of BioNV can be analyzed by dynamic light scattering (DLS), flow cytometry, mass spectrometry, and other methods for determining particle size. In embodiments, BioNV can be filtered according to particle size or size range to optimize renal clearance and other clinically relevant NV characteristics. In embodiments, the size of BioNV is about 20nm to 1200nm. In embodiments, the size of BioNV can be about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 120nm, about 140nm, about 160nm, about 180nm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, about 1000nm, about 1100nm, or about 1200nm. In embodiments, the BioNV has a size range of about 10 nm to 20 nm, about 20 nm to 30 nm, about 30 nm to 40 nm, about 40 nm to 50 nm, about 50 nm to 60 nm, about 60 nm to 70 nm, about 70 nm to 80 nm, about 80 nm to 90 nm, about 90 nm to 100 nm, about 10 nm to 100 nm, about 100 nm to 200 nm, about 200 nm to 400 nm, about 400 nm to 600 nm, about 600 nm to 800 nm, about 800 nm to 1000 nm, or about 1000 nm to 1200 nm.

[0208] Methods of treating diseases in mammals

[0209] In embodiments, the low immunogenic cells can be used to treat mammalian diseases. In embodiments, the BioNV derived from low immunogenic cells can be used to treat mammalian diseases. In embodiments, the mammalian disease is cancer, infectious disease, genetic disease and / or orphan disease.

[0210] In embodiments, the method for treating a mammalian disease herein comprises co-administering a second whole cell therapy (e.g., T cells, NK cells, TIL, macrophage therapy). In embodiments, supplementing whole cell therapy with low immunogenic cells and / or BioNV can be used to reduce the effective dose of the whole cell therapy required, reduce CRS, off-target effects, teratoma potential, etc.

[0211] In embodiments, the method of treating a mammalian disease comprises administering an additional therapeutic agent. In embodiments, the additional therapeutic agent may be any additional anti-cancer agent, anti-infective agent, analgesic agent and / or non-steroidal anti-inflammatory agent (NSAID).

[0212] In embodiments, the low immunogenic cells may be frozen at about -80°C or suitable for storage at -80°C. In embodiments, the BioNV may be frozen at about -80°C or suitable for storage at -80°C and / or lyophilized (e.g., for reconstitution in a buffer). In embodiments, the low immunogenic cells and / or BioNV may be stable at about ambient temperature, about -20°C, about 4°C, about 25°C, or about 37°C for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about a week, or at least about a month or longer.

[0213] In embodiments, treatment of mammalian disease can be achieved within about 2 weeks, within about 4 weeks, within about 6 weeks, within about 12 weeks, within about 18 weeks, within about 24 weeks, within about 6 months, within about 1 year, or within about 2 years or more following administration of the compositions and methods employing such compositions.

[0214] Dosage and Administration

[0215] The dosage and dosing schedule of any low immunogenic cells (or BioNV) disclosed herein may depend on various parameters and factors, including but not limited to the specific low immunogenic cells, the disease being treated, the severity of the condition, whether the condition is being treated or prevented, the age, weight and general health of the subject, and the judgment of the attending physician. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy spectrum of the therapeutic agent) information about a particular subject may affect the dosage used. In addition, the exact individual dosage may be adjusted to a certain extent based on a variety of factors, including the specific combination of drugs administered, the time of administration, the route of administration, the nature of the formulation, the excretion rate, the disease being treated, the severity of the condition, and the anatomical location of the condition. Some dosage changes may be expected.

[0216] In embodiments, delivery of BioNV can be similar to delivery of vesicles, particularly liposomes (see Langer, 1990, Science 249: 1527-1533; Treat et al., Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0217] In embodiments, the methods described herein for treating mammalian diseases using low immunogenic cells include a dosage range in units of low immunogenic cell number concentration per kilogram (kg) of subject body weight. In embodiments, a suitable dosage range for the methods described herein may include about 103 cells / kg (or BioNV / kg) to about 10 9 In one embodiment, the low immunogenic cells are present at about 10 3 cells / mL (or BioNV / mL) to about 10 9 In some embodiments, the low immunogenic cells (or BioNV) composition is present in the composition at a concentration of about 5 cells / mL (or BioNV / mL). Alternatively, in embodiments, the low immunogenic cells (or BioNV) composition is present in the composition at a weight / volume in the range of about 5 ng / mL to about 500 mg / mL. In embodiments, the BioNV dosage is based on the size of the BioNV used in the treatment, for example, at an equivalent dose, 1000 nm BioNV is provided in an amount that is about 5 to 10 times less than the amount of 100 nm BioNV.

[0218] In an embodiment, the BioNV (or low immunogenic cells) disclosed herein are administered by controlled or sustained release means or by delivery by devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be suitable for providing controlled or sustained release of one or more active ingredients using, for example, hydroxypropyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof, to provide a desired release profile in varying proportions. Controlled or sustained release of an active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, changes in temperature, stimulation by light of an appropriate wavelength, concentration or availability of an enzyme, concentration or availability of water, or other physiological conditions or compounds.

[0219] In an embodiment, a polymeric material is used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

[0220] In an embodiment, a controlled release system is placed adjacent to the target area to be treated, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249: 1527-1533 may be used.

[0221] In embodiments, methods of using low immunogenic cells (or BioNV) include applying low immunogenic cells to the surface of a device (e.g., a catheter) or containing them in a pump, patch, or other drug delivery device. Excipients or carriers can be selected based on the mode and route of administration. Suitable pharmaceutical carriers and pharmaceutical necessities for pharmaceutical formulations are described in the well-known reference books in the art, Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopeia and National Formulary).

[0222] In embodiments, low immunogenic cells (or BioNV) can be administered at a dose consistent with that of whole cells, for example, based on CAR concentration. In embodiments, the typical concentration range of CAR protein per microgram of T cell is between 0.20ng-0.70ng, and the total number of CARs in a single BioNV may be 5 to 10,000 times less than that of whole cells, resulting in the conversion of BioNV mass into CAR concentration, wherein it can be assumed that the CAR concentration is equal to the cell (e.g., T cell) from which it is derived (e.g., the case in exosomes) or increases (e.g., the case in BioNV). In embodiments, compared with the whole cell from which it is derived, the concentration and / or surface density of the targeting agent (e.g., CAR) on BioNV increases. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) is enriched by continuous extrusion processing of the whole cell. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) and other cell surface molecules on BioNV increases by 2 to 100 times relative to the whole cell. In embodiments, because exosomes are naturally shed, their concentration and / or surface density of targeting agents (e.g., CARs) and other cell surface molecules are substantially the same as whole cells.

[0223] The dosage regimen using any of the low immunogenic cells (or BioNVs) disclosed herein can be selected based on a variety of factors, including the type of cancer, species, age, weight, sex, and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or liver function of the subject; the pharmacogenomic makeup of the individual; and the specific composition employed in the present disclosure. Any of the low immunogenic cells (or BioNVs) disclosed herein can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times, or four times a day. In addition, any of the low immunogenic cells (or BioNVs) disclosed herein can be administered continuously rather than intermittently throughout the dosing regimen.

[0224] In embodiments, the low immunogenic cells (or BioNV) are administered in continuous doses about once per hour, about once every 2 hours, about once every 6 hours, about once every 12 hours, about once every 24 hours, about once every 2 days, about once every 4 days, about once every 7 days, about once every 2 weeks, about once every 4 weeks, or about once a month.

[0225] Additional therapeutic agents

[0226] In embodiments, the compositions or methods described herein further comprise a therapeutically effective amount of one or more additional therapeutic agents. In embodiments, the therapeutically effective amount of one or more additional therapeutic agents may be in solution with the BioNV, adsorbed onto the surface of the NV, or as a payload encapsulated within the BioNV. In embodiments, the additional therapeutic agent is one or more of a checkpoint inhibitor, an analgesic, and / or an anti-infective agent.

[0227] In embodiments, the compositions or methods of the invention contemplate other additional therapeutic agents, such as analgesics to help treat inflammation or pain at the site of application, or anti-infective agents to prevent infection at the site treated with the composition. Non-limiting examples of additional therapeutic agents include analgesics such as nonsteroidal anti-inflammatory drugs, opioid agonists, and salicylates; anti-infective agents such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other beta-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterial drugs, antituberculosis antimycobacterial drugs, antiprotozoal drugs, antimalarial antiprotozoal drugs, antivirals, antiretrovirals, scabicides, anti-inflammatory drugs, corticosteroids, Steroidal anti-inflammatory drugs, antipruritic / local anesthetics, topical anti-infectives, antifungal topical anti-infectives, antiviral topical anti-infectives; electrolyte and renal agents, such as acidifiers, alkalizers, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium sparing diuretics, thiazide diuretics, electrolyte substitutes, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylic acid gastrointestinal anti-inflammatory agents, antacids and anti-ulcer agents, acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents , H2-blockers, antiulcers, gallstone dissolving agents, digestives, emetics, laxatives and stool softeners and prokinetics; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics and opioid agonist intravenous anesthetics; hormones and hormone modulators, such as abortifacients, adrenal drugs, corticosteroid adrenal drugs, androgens, antiandrogens; immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids and vaccines; local anesthetics, such as amide local anesthetics and ester local anesthetics Anesthetics; musculoskeletal agents, such as anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, non-steroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents; minerals; vitamins, such as water-soluble or fat-soluble vitamins, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and / or vitamin K; and radionuclides, such as yttrium-90, iodine-131, samarium-153, lutetium-177, astatine-211, lead-212 / bismuth-212, radium-223, actinium-225 and thorium-227.

[0228] Additional non-limiting examples of useful therapeutic agents in the above categories include: (1) general analgesics, such as lidocaine or its derivatives, and NSAID analgesics, including diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opioid agonist analgesics, such as codeine, fentanyl, hydromorphone, and morphine; (3) salicylate analgesics, such as aspirin (ASA) (enteric-coated ASA); (4) Hi-blocker antihistamines, such as clemastine and terfenadine; (5) sedative analgesics, such as ... enadine); (5) anti-infectives, such as mupirocin; (6) anti-anaerobic anti-infectives, such as chloramphenicol and clindamycin; (7) anti-fungal antibiotic anti-infectives, such as amphotericin B, clotrimazole, fluconazole and ketoconazole; (8) macrolide antibiotic anti-infectives, such as azithromycin and erythromycin; (9) other β-lactam antibiotic anti-infectives, such as aztreonam and imipenem; (10) penicillin antibiotic anti-infectives, such as nafcillin, oxacillin, penicillin G and penicillin V; (11) quinolone antibiotic anti-infectives, such as ciprofloxacin ) and norfloxacin; (12) tetracycline antibiotics, such as doxycycline, minocycline and tetracycline; (13) anti-tuberculosis and anti-mycobacterial drugs, such as isoniazid (INH) and rifampin; (14) antiprotozoal drugs, such as atovaquone and dapsone; (15) antimalarial and antiprotozoal drugs, such as chloroquine and pyrimethamine; (16) antiretroviral drugs, such as ritonavir and zidovudine; (17) antiviral drugs, such as acyclovir, ganciclovir, interferon α , remdesivir and rimantadine; (18) antifungal topical anti-infective drugs, such as amphotericin B, clotrimazole, miconazole and nystatin; (19) antiviral topical anti-infective drugs, such as acyclovir; (20) electrolyte and renal drugs, such as lactulose; (21) loop diuretics, such as furosemide; (22) potassium-sparing diuretics, such as triamterene; (23) thiazide diuretics, such as hydrochlorothiazide (HCTZ); (24) uricosuric drugs, such as probenecid; (25) enzymes, such as RNAse and DNAse; (26) antiemetics, such as prochlorperazine; (27) salicylic acid gastrointestinal anti-inflammatory drugs, such as sulfasalazine; (28) gastric acid pump inhibitor anti-ulcer drugs, such as omeprazole;(29) H2-receptor blocker anti-ulcer drugs, such as cimetidine, famotidine, nizatidine and ranitidine; (30) digestive agents, such as pancreatic lipase; (31) prokinetic drugs, such as erythromycin; (32) ester local anesthetics, such as benzocaine and procaine; (33) musculoskeletal corticosteroid anti-inflammatory drugs, such as beclomethasone, betamethasone, cortisone, etc. sone), dexamethasone, hydrocortisone, and prednisone; (34) musculoskeletal anti-inflammatory immunosuppressants, such as azathioprine, cyclophosphamide, and methotrexate; (35) musculoskeletal nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketoprofen, and naproxen; (36) minerals, such as iron, calcium, and magnesium; (37) vitamin B compounds, such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) vitamin C compounds, such as ascorbic acid; and (39) vitamin D compounds, such as calcitriol.

[0229] Compositions of Low Immunogenic Cells and / or BioNV

[0230] In some aspects, the disclosure relates to compositions useful for treating mammalian diseases, comprising allogeneic, low immunogenic cells and / or BioNV. In embodiments, the low immunogenic cells and / or BioNV comprise one or more therapeutically relevant biomolecules.

[0231] In embodiments, the composition comprises low immunogenic cells. In embodiments, the composition may comprise BioNV. In embodiments, the composition comprises low immunogenic cells and BioNV. In embodiments, the composition comprises at least one or more of an anti-cancer therapeutic, an anti-infective therapeutic, or a gene editing payload. In embodiments, the composition comprises low immunogenic cells (or BioNV) that can adsorb therapeutic molecules onto the surface of a therapeutic payload and / or encapsulate a therapeutic payload. In embodiments, the composition comprises a therapeutically effective amount of low immunogenic cells and / or BioNV.

[0232] In embodiments, the composition is allogeneic and / or hypoimmunogenic.In embodiments, the composition is derived from iPSCs (and other cell types) that have been modified to reduce expression of immunogenic molecules and / or increase expression of immunoprotective molecules.

[0233] In embodiments, the composition of low immunogenic cells and / or BioNV is low immunogenic. For example, in embodiments, the composition does not cause an inflammatory response and / or an immune response when administered. In embodiments, after administration to a subject, the composition (optionally the low immunogenic cells therein) causes less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% of inflammation or An immune response measured as a function of the concentration of a cytokine, chemokine, or immunomodulatory enzyme, such as IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20, IFN-α / β / γ, TNFα / β, IDO, HLA-G, HGF, PGE2, etc., or any combination thereof, e.g., compared to a homologous whole cell therapy counterpart.

[0234] In an embodiment, the low immunogenic cells are present in an amount of about 10 3 cells / mL (or BioNV / mL) to about 10 9 Alternatively, in embodiments, the cells and / or BioNV composition are present in the composition at a weight / volume in the range of about 5 ng / mL to about 500 mg / mL.

[0235] In embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasmas, pyrogens, and in more specific embodiments, substantially free of all of the foregoing. In embodiments, the BioNV composition is substantially free of whole cells and intracellular cellular components, including organelles such as nuclei, mitochondria, Golgi bodies, etc., and / or substantially free of non-CAR-expressing NVs and / or substantially free of ruptured, damaged NVs. In embodiments, the composition is substantially free of extracellular chromatin, nucleosomes, and other genetic material and non-therapeutic nucleic acids. In embodiments, the BioNV composition is substantially free of cellular genomic DNA.

[0236] In the embodiments, due to the lack of immunogenicity of engineered iPSC, low immunogenic cells are modular and allogeneic (ready-made). In the embodiments, the lack of whole cell signaling components in BioNV enables it to easily regulate target specificity and resistance to immunosuppressive signals. In the embodiments, low immunogenic cells lack genetic elements that cause uncontrolled cytokine storms, thereby minimizing the risk of CRS in patients. In the embodiments, the amount of active cytokines, perforins, granzymes, interferons, interleukins, etc. encapsulated in BioNV is regulated in the process upstream of the cell (before BioNV is derived). In the embodiments, low immunogenic cells are derived from cells that can pass through biological barriers and / or are known to promote viral receptors that pass through.

[0237] Without wishing to be bound by theory, the low immunogenic cells produced by the iPSC engineered allogeneic basal cell line represent immune stealth cells, which means that low immunogenic cells have the potential for multiple dosing, and antibody-mediated neutralization is minimized, and immune cell-mediated clearance is evaded (T cells and macrophages). In embodiments, BioNV derived from low immunogenic cells does not contain live genetic material from cells derived therefrom to cause CRS or teratoma. In embodiments, the expression of certain cytokines in low immunogenic cells increases and is encapsulated in BioNV that can recruit natural T cells. In embodiments, BioNV can be derived from improved cell types with or without barrier-penetrating ligands to further control activity after infusion.

[0238] Pharmaceutical compositions and formulations of low immunogenic cells and / or BioNV

[0239] In some aspects, the composition is a pharmaceutical composition. In an embodiment, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of low immunogenic cells as an active ingredient. In an embodiment, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of one or more anti-cancer therapeutic agents as the effective load within the BioNV as an active ingredient. Typically, the pharmaceutical composition also includes one or more pharmaceutically acceptable excipients, carriers (including inert solid diluents and fillers), diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers, and adjuvants.

[0240] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When applied to a subject, pharmaceutically acceptable excipients are usually sterile. When any agent disclosed herein is administered intravenously, water is a useful excipient. Saline solutions and dextrose aqueous solutions and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, any composition disclosed herein can also be formulated with a wetting agent or emulsifier or pH buffer. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro ed., 19th ed. 1995), which is incorporated herein by reference.

[0241] In an embodiment, the composition comprises an excipient or carrier. In an embodiment, the diluent may be a pharmaceutically acceptable excipient or carrier.

[0242] In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerol, and various combinations thereof, and inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In embodiments, the diluent includes one or more of the following: saline, phosphate buffered saline, Dulbecco's modified Eagle's medium (DMEM), α modified minimum essential medium (αMEM), Roswell Park Memorial Institute medium 1640 (RPMI medium 1640), HBSS, human albumin, Ringer's solution, etc., or any combination thereof.

[0243] In embodiments, the active ingredient is typically mixed with an excipient, diluted by an excipient, or encapsulated in a carrier in the form of, for example, a capsule, tablet, pouch, paper, or other container. When an excipient is used as a diluent, it may be a solid, semisolid, or liquid substance (e.g., saline) as a vehicle, carrier, or medium for the active ingredient. In embodiments, the composition may be in the form of a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), lotion, cream, ointment, gel, soft and hard gelatin capsules, suppository, sterile injection solution, and sterile packaged powder. As known in the art, the type of diluent may vary according to the intended route of administration. In embodiments, the resulting composition may include additional agents, such as preservatives, cryopreservatives (e.g., DMSO), and / or lyoprotectants (e.g., polyols, salts). In embodiments, the carrier may be or may include a lipid-based or polymer-based colloid. In embodiments, the carrier material may be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. In embodiments, the carrier material may form a capsule, and the material may be a polymer-based colloid.

[0244] In embodiments, the pharmaceutical composition comprising low immunogenic cells includes a solubilizing agent. In embodiments, the pharmaceutical composition comprising low immunogenic cells includes a cryoprotectant or an agent that improves thermal stability, such as DMSO or glycerol. In embodiments, the pharmaceutical composition can be delivered with a suitable vehicle or delivery device known in the art.

[0245] In embodiments, the composition comprises a scaffold. In embodiments, the scaffold comprises a biomaterial. In a non-limiting example, the three-dimensional biomaterial comprises low immunogenic cells embedded in an extracellular matrix that is attached to, dispersed within, or captured within the scaffold. In embodiments, the biomaterial is biodegradable and / or synthetic.

[0246] In embodiments, the scaffold comprises a biodegradable biomaterial. Non-limiting examples of biodegradable biomaterials include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstructed basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, sugar, hyaluronic acid, poly (lactic acid), poly (glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, derivatives and mixtures thereof. Other useful biodegradable polymers or polymer types include, but are not limited to, polydioxanone, polycarbonate, polyoxalate, poly(α-ester), polyanhydride, polyacetate, polycaprolactone, poly(orthoester), polyamino acid, polyamide and mixtures and copolymers thereof, L-lactic acid and D-lactic acid stereopolymers, copolymers of bis(p-carboxyphenoxy) propionic acid and sebacic acid, sebacic acid copolymers, caprolactone copolymers, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, polyurethane and poly(lactic acid) copolymers, polyurethane and poly(lactic acid) copolymers, α-amino acid copolymers, α-amino acid and caproic acid copolymers, A-glutamic acid benzyl ester and polyethylene glycol copolymers, succinate and poly(ethylene glycol) copolymers, polyphosphazenes, polyhydroxyalkanoates and mixtures thereof. Binary and ternary systems are also contemplated. In embodiments, the scaffold includes one or more of collagen, various proteoglycans, alginate-based matrix and chitosan. In embodiments, the scaffold comprises one or more of a hydrogel, silk, matrigel, acellular and / or decellularized scaffolds, poly-ε-caprolactone scaffolds, absorbable scaffolds, and nanofiber-hydrogel composites.

[0247] In embodiments, the support comprises a synthetic biomaterial. Non-limiting examples of synthetic biomaterials include lactone-based polyesters or copolyesters, such as polylactide, polycaprolactone, polyorthoesters, polyanhydrides, polyamino acids, polysaccharides, polyphosphazenes, poly(ether-ester) copolymers (e.g., PEO-PLLA); polydimethylsiloxane, poly(ethylene-vinyl acetate), acrylate-based polymers or copolymers (e.g., polyhydroxyethyl methyl methacrylate, polyvinyl pyrrolidone), fluorinated polymers (e.g., polytetrafluoroethylene) and cellulose esters.

[0248] In embodiments, the composition can be prepared in any manner known in the pharmaceutical art, and can be administered by a variety of routes (e.g., subcutaneous, intravenous, etc.), depending on whether local or systemic treatment is required and the area to be treated. In embodiments, administration can be local (including ocular and mucosal, including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. In embodiments, methods may include ocular delivery, topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by surgical placement of a balloon catheter or ophthalmic insert in the conjunctival sac. In embodiments, parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration. In embodiments, parenteral administration may be in the form of a single bolus dose, or may be, for example, by a continuous infusion pump.

[0249] In embodiments, pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. In embodiments, methods of treating and / or preventing cancer include the use of pharmaceutical carriers; aqueous, powder or oily bases; thickeners, etc.

[0250] In an embodiment, the pharmaceutical composition contains nucleic acids and vectors described herein as active ingredients and one or more pharmaceutically acceptable carriers. In an embodiment, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce side effects, allergies or other adverse reactions when applied to animals or humans, as appropriate. The methods and compositions disclosed herein can be applied to a variety of species, such as humans, non-human primates (e.g., monkeys), horses or other livestock, dogs, cats, ferrets, or other mammals, rats, mice or other experimental animals raised as pets. In an embodiment, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents and absorption delaying agents, buffers, excipients, adhesives, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0251] In embodiments, the composition can be applied to the surface of a device (e.g., a catheter) or contained in a pump, patch or other drug delivery device. In embodiments, the composition can be applied alone or in the presence of a pharmaceutically acceptable excipient or carrier (e.g., normal saline) in the form of a mixture. Excipients or carriers are selected based on the mode of administration and approach. Suitable pharmaceutical carriers and pharmaceutical necessities for pharmaceutical formulations are described in the well-known reference books Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopoeia and National Formulary) in the art.

[0252] In an embodiment, a composition disclosed herein, e.g., a pharmaceutical composition, is suspended in a saline buffer (including but not limited to TBS, PBS, etc.).

[0253] The present technology includes the disclosed low immunogenic cells in various formulations of pharmaceutical compositions. In embodiments, the low immunogenic cells (or BioNV) disclosed herein can be in the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained release formulations, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0254] The pharmaceutical composition comprising the low immunogenic cells described herein can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. Such methods generally include the step of combining the therapeutic agent with a carrier consisting of one or more additional components. Typically, the pharmaceutical composition is prepared by uniformly and closely combining the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, molding the product into a dosage form of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).

[0255] In embodiments, any of the low immunogenic cells disclosed herein are formulated according to routine procedures into pharmaceutical compositions suitable for the modes of administration disclosed herein.

[0256] Subjects and / or Animals

[0257] In embodiments, the subject and / or animal intended to be used together with low immunogenic cells and / or BioNV are mammals, such as humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, rabbits, sheep or non-human primates. In embodiments, subject and / or animal are non-mammals, such as zebrafish. In embodiments, subject and / or animal are transgenic animals, which include fluorescent cells, such as RPE cells and / or immune cells with GFP. In embodiments, subject and / or animal are humans. In embodiments, low immunogenic cells are derived from fluorescently labeled cells and / or are packaged with fluorescently labeled proteins or labels (e.g., GFP). In embodiments, humans are pediatric humans, adults, elderly, infants or children. In other embodiments, the people refer to patients or subjects.

[0258] In an embodiment, the method of treatment comprises administering to a human whose age ranges are about 0 months to about 6 months, about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 36 months, about 1 year to about 5 years, about 5 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, about 20 years to about 25 years, about 25 years to about 30 years, about 30 years to about 35 years, about 35 years to about 40 years, about 40 years to about 45 years, about 45 years to about 50 years, about 50 years to about 55 years, about 55 years to about 60 years, about 60 years to about 65 years, about 65 years to about 70 years, about 70 years to about 75 years, about 75 years to about 80 years, about 80 years to about 85 years, about 85 years to about 90 years, about 90 years to about 95 years, or about 95 years to about 100 years.

[0259] In embodiments, the subject is a non-human animal, and thus the disclosure relates to veterinary uses. In embodiments, the non-human animal is a domestic pet. In embodiments, the non-human animal is a livestock animal.

[0260] In embodiments, serum and / or immune cells and / or tumor cells are assessed and / or affected. In embodiments, immune cells include cells of the innate immune system of a subject and / or animal. In embodiments, such cells include but are not limited to NK cells, monocytes, DCs, B cells, macrophages, CD4+T cells, and CD8+T cells. In various embodiments, the disclosure provides for detecting the presence, absence, or measurement of tumor volume, tumor cells, metastasis, cDNA, or RNA in a sample derived from a subject.

[0261] Reagent test kit

[0262] In an embodiment, the present disclosure provides a kit that can simplify the administration of any agent described herein. An exemplary kit of the present disclosure includes any agent / composition described herein in unit dosage form. In an embodiment, the unit dosage form is a container, such as a prefilled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient or vehicle. In an embodiment, the kit also includes a label or printed instructions indicating the use of any agent described herein. In an embodiment, the kit also includes an eyelid speculum, a local anesthetic, and a cleaning agent for injection surface. In an embodiment, the kit further includes one or more additional agents described herein.

[0263] In aspects, the disclosure includes syringes comprising one or more compositions of the disclosure. In embodiments, the syringe is pre-filled with a certain volume of the composition. In embodiments, the volume of the syringe pre-filled is about 1 mL to about 10 mL. In embodiments, the syringe is pre-filled with a volume of about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, or about 1.0 mL or less of the composition.

[0264] In an embodiment, the syringe comprises a composition having a shelf life in the range of about 1 hour to about 1 week. In an embodiment, the syringe comprises a composition having a shelf life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about -85°C to about 25°C. In an embodiment, the syringe comprises a composition having a shelf life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about 15°C to about 25°C.

[0265] In embodiments, the storage temperature is about -80°C. In embodiments, the storage temperature is about -20°C. In embodiments, the storage temperature is about 4°C. In embodiments, the storage temperature is about 21°C. In embodiments, the kit comprises lyophilized BioNV.

[0266] In one embodiment, the kit includes a container containing a composition comprising a CDV / exosome of the present disclosure, a therapeutically effective amount of an additional therapeutic agent (eg, a therapeutic agent described herein), and instructions for use.

[0267] Example

[0268] Example 1: Construction of a low immunogenic cell line for producing BioNV

[0269] Low immunogenic cells will be made from human fibroblast induced pluripotent stem cells (iPSCs). Human fibroblast iPSCs will be genetically corrected using lentiviral transduction with the CRISPR Cas9 system. The design of each gRNA with minimal off-target effects for each target locus will be engineered. The lentiviral approach is suitable for generating BioNV because the resulting product is not a cell-based therapy.

[0270] The following knockouts (KO) will be performed in sequence:

[0271] Option 1:

[0272] HLA-A, HLA-B, HLA-C, HLA-E / G (chosen by design to correspond to the gRNA most suitable for CRISPR Cas9), and HLA-F;

[0273] Option 2:

[0274] Use current CRISPR Cas9 shotgun-based approaches to simultaneously (or in two steps) knock out HLA A, HLA B, HLA C, HLAE / G, and HLA F. Knock out HLA simultaneously to ensure that permanent silencing of the B2M locus does not produce lethality or phenotypic effects (beyond MHC I expression);

[0275] MHC Class II Number:

[0276] CIITA; and

[0277] CRS-related cytokines:

[0278] IL-6, IL-4, IL-10 and / or IL-16. Starting with IL-6, cytokines will be knocked out sequentially. IL-6 KO will be used to evaluate the CRS effect, and if IL-6 KO leads to CRS or CRS-related phenotypes, more cytokines will be eliminated.

[0279] The following knock-ins (KI) will be implemented in sequence:

[0280] CCL2, CTLA-4, H2-M3, MFG-E8;

[0281] CD24, CD200, and / or CD47 antiphagocytic transmembrane domains; and

[0282] IL-2p GFP construct (as reporter gene for diagnostic and preclinical experiments). Each KI will use a single integration with drug / reporter gene selection marker.

[0283] Table 7 below details the steps for executing the cell line library and engineering sequence.

[0284] Table 7: Cell line pool and engineering order. KO = knockout; KI = knockin.

[0285]

[0286]

[0287] All cell lines will be screened for mycoplasma, HBV, HIV, and other standardized contaminants during engineering. Clonal evaluation of cell lines for gene editing will be performed between gene corrections, including suitability of growth medium type, conditions such as division time, cell viability, etc.

[0288] CRISPR and / or TUNR (AMSBIO) design, assembly, and validation will first determine if any HLA and / or CIITA gene KO is lethal. TUNR is a product that integrates a DNA fragment into the gene of interest to reduce its expression to levels just above lethality (if necessary for cell line development).

[0289] Transfection / nucleofection and transduction of lentiviral systems are used for knock-in and knock-out engineering, respectively. KO and KI will be performed in the form of pools at each step, followed by single cell dilution, cloning, drug selection / genetic testing, and maintenance of single cell-derived clones. Clones at each step will be expanded for subsequent steps.

[0290] Single cell derived clone pools will be screened by deep sequencing to prepare a gene library for deep sequencing runs and analysis for additional clonal analysis. Data will be collected and made available at each step. For KO using CRISPR, cells with the lowest incidence of indels will be selected for further steps in the engineering process.

[0291] The final positive clone expansion (after KO and KI, before the CAR platform intersection line) will be performed for final genotype confirmation and clone cryopreservation.

[0292] An example of genetic modification used to generate human fibroblast reprogrammed iPSC lines is described in Figure 4-26 In, they collectively illustrate CRISPR / Cas9 gRNA design, knockout efficacy, and off-target analysis for serial B2M and CIITA KO, human CD47 (hCD47) isoform 2 knock-in, TRAC, and TRBC1 KO in human fibroblast reprogramming iPSCs. Overall, the clonal sequencing data showed that B2M, CIITA, TRAC, and TRBC1 (one of the TRBC genes) were all successfully targeted by gRNA, and CD47 had been successfully knocked in, as demonstrated by RNA extraction.

[0293] The B2M knockout strategy is described in e.g. Figure 4 , which depicts a graphical representation of the B2M knockout strategy for developing hypoimmunogenic cell lines from human fibroblast reprogramming iPSCs. β2-macroglobulin (B2M) is a serum protein associated with major histocompatibility complex (MHC) class I heavy chains on the surface of nearly all nucleated cells involved in presenting peptide antigens to the immune system. Figure 5 A graphical representation of the B2M gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the B2M gene (e.g., SEQ ID NOs: 22-23), such as gRNAs of SEQ ID NOs: 21, 24, 26-29, and 133-134. Figure 6 Tabular representation of B2M gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0294] For example, Figure 7 The strategy for CIITA knockout is illustrated in Figure 2, which depicts a graphical representation of the B2M gene knockout strategy for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts. The master control factor CIITA is an MHC class II transactivator that is involved in the transcriptional regulation of all MHC II genes. Figure 8 A graphical representation of the CIITA gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the CIITA gene (e.g., SEQ ID NOs: 32-33), such as gRNAs of SEQ ID NOs: 31, 34, and 36-42. Fig. 9 Tabular representation of CIITA gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0295] Fig.10 A graphical representation of the sequence analysis of B2M knockout clones used to develop low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. Results of two selected B2M / CIITA double knockout clones are shown. Sequencing data illustrate successful B2M KO in the double KO clones, and the resulting genomic sequences are SEQ ID NOs: 43-44. Fig.11 Depicted is a graphical representation of the sequence analysis of CIITA knockout clones for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts. Results of two selected B2M / CIITA double knockout clones are shown. Sequencing data illustrate successful CIITA KO in double KO clones, resulting in genomic sequences of SEQ ID NOs: 50-55.

[0296] Morphological analysis was performed by light microscopy (e.g. Fig.12 and Fig.23 ) showed that the clonal cell population appeared to have normal morphology and was able to replicate. Fig.12 Depicted is a graphical representation of the B2M / CIITA double knockout clone sequence overview and clonal morphology of in vitro human fibroblast reprogrammed iPSC low immunogenicity cells. The data demonstrate that the clonal populations result in different overall knockouts. Fig.23 Graphical representation of B2M / CIITA double knockout, TRAC / TRBC1 double knockout, hCD47KI human fibroblasts reprogrammed into iPSCs with low immunogenicity. Data illustrate in vitro morphological analysis of clones.

[0297] Fig.13 A graphical representation of human CD47 (hCD47) isoform 2 knock-in used to develop low immunogenic cell lines from human fibroblast reprogramming iPSCs is depicted. The pcDNA3.1(+)XCC92 mammalian vector (6271 bp in length) was used with a selection marker. The CD47 3'UTR was deleted for stable clonal surface expression of CD47. The 3'UTR contains at least 6 microRNA binding sites that inhibit CD47 expression, in which a bGH poly A tail was used instead. Fig.14 Depicted is a graphical representation of hCD47 isoform 2 knock-in clonal selection for development of low immunogenic cell lines in B2M / CIITA double KO human fibroblast reprogrammed iPSCs. The data demonstrates successful hCD47 KI in double KO cell lines. The data demonstrates that hCD47 at the exon 1-2 and 3-4 junctions is increased approximately two-fold.

[0298] Fig.15 A graphical representation of the TRAC knockout strategy for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. In embodiments, T cell receptor alpha chain (TRAC) elimination is performed to prevent interference with CAR targeting and off-target effects. Fig.16 A graphical representation of TRAC gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the TRAC gene (e.g., SEQ ID NOs: 56-57), such as gRNAs of SEQ ID NOs: 60-75. Fig.17 Tabular representation of TRAC gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0299] Fig.18A graphical representation of the TRBC1 knockout strategy for developing low immunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. In embodiments, T cell receptor beta chain 1 (TRBC1) elimination is performed to prevent interference with CAR targeting and off-target effects. Fig.19 A graphical representation of TRAC gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of TRAC genes (e.g., SEQ ID NOs: 76-77), such as gRNAs of SEQ ID NOs: 79-101. Fig. 20 Tabular representation of TRAC gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0300] Fig.21 A graphical representation of the TRAC knockout clone sequence analysis for the development of a low immunogenic cell line from B2M / CIITA double knockout hCD47 knock-in human fibroblast reprogrammed iPSCs is depicted. Results and resulting sequences for two selected clones are shown, e.g., SEQ ID NOs: 102-108. The sequence data demonstrates successful TRAC knockout in the double KO / CD47 KI clone. Fig. 22 Graphical representation of TRBC1 knockout clone sequence analysis depicting the development of low immunogenic cell lines from B2M / CIITA double knockout, hCD47 knock-in, and TRAC knockout in human fibroblast reprogrammed iPSCs. Results and resulting sequences for two selected clones are shown, e.g., SEQ ID NOs: 109-110 and 112-116.

[0301] Fig.23 The results showed that biallelic disruption of B2M, CIITA, TRAC and TRBC1 was successful, as demonstrated by sequencing data. In addition, the normal cell morphology of the clones was maintained.

[0302] Fig.24 Graphical representation of the IL-6 knockout strategy for developing hypoimmunogenic cell lines from iPSCs reprogrammed from human fibroblasts is depicted. Elimination of interleukin-6 (IL-6) is intended to prevent cytokine release syndrome (CRS). Fig.25 A graphical representation of the IL-6 gRNA design for low immunogenic cell line development is depicted. In embodiments, the strategy involves using CRISPR / Cas9 and specially designed gRNAs to target relevant regions of the IL-6 gene (e.g., SEQ ID NOs: 117-118), such as gRNAs of SEQ ID NOs: 119-120 and 122-130. Fig.26 Tabular representation of IL-6 gRNA off-target analysis depicting the development of low immunogenic cell lines.

[0303] Table 8: Genes used to generate B2M KO, CIITA KO, TRAC KO, TRAC KO, TRBC1 KO, IL-6 KO, and The sequence of one or more of the hCD47 isoform 2KIs.

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] Example 2: Production of BioNV by continuous extrusion

[0310] Biomimetic nanovesicles (BioNV) can be produced from the above-mentioned low immunogenic cell lines, such as Fig. 27 As described in the scheme depicted in .

[0311] A combination of flow cytometry and iodixanol density gradients can be used to measure CAR expression levels in poorly immunogenic cell lines (e.g., Fig. 27 Step 1).

[0312] Differentiation of surface CAR expressing iPSCs into CAR lymphocytes can be analyzed by identification of lymphocyte markers, including, for example, CD4 / CD8 (T cells) or CD56 / CD16 (natural killer cells) as well as other cell surface markers (e.g., Fig. 27 Step 2). The expression profile can be determined by flow cytometry, RT-PCR and / or CRISPR-based analysis.

[0313] Next, activation of CAR lymphocytes can be achieved using low predetermined concentrations of biomarker antigen-coated beads in two stages over two weeks (e.g., Fig. 27 This process also allows analysis of the quality of the immunological synapse (IS) between the CAR and the antigen-coated beads, using well-established protocols to measure i) quantification of F-actin accumulation at the site of synapse formation, ii) distribution of pZeta at the synapse, iii) clustering of antigen across the IS site, and / or iv) polarization of lytic granules (LGs) containing perforin and granzymes.

[0314] After lymphocyte activation, the cells are expanded using a defined protocol (e.g. Fig. 27After expansion, each cell population is analyzed for levels of perforin and granzymes (or other luminal payload, if applicable) to ensure that concentration levels are consistent across batches. This is accomplished using a series of qPCR, immunoblotting, flow cytometry, and / or mass spectrometry. If a sufficiently large cell population is available in step 3, the expansion step may not be necessary.

[0315] Once the cells are activated and producing the desired therapeutic protein, they are expanded, harvested, washed several times, and then placed into a buffered extrusion medium. The cells are then fully processed (e.g., Fig. 27 Step 5). In the initial extrusion step of the continuous extrusion process, the nucleus (along with nuclear components, including nuclear pores, genomic material, and transcription factors) and mitochondria are eliminated. The sample is then treated with an endonuclease, such as BENZONASE. BENZONASE is a non-specific recombinant endonuclease that cuts all types of DNA and RNA variants into non-functional fragments of <8 soluble base pairs. This minimizes the amount of nucleic acid loaded per sample and on a scalable basis without interfering with the BioNV membrane chemistry. The lysis process also eliminates nucleic acid viscosity, allowing the material to be subsequently loaded and passed through the next set of extrusion filters. The FDA has developed regulatory guidance applicable to the BioNV extrusion process for the use of BENZONASE in vaccine production.

[0316] The continuous extrusion process will avoid eliminating other organelles, such as the Golgi or endoplasmic reticulum. The membrane systems of these organelles are highly evolved to transport vesicles (release and absorption) between folded membranes. For example, the cis and trans faces of the Golgi contain unique lipid compositions that contribute to low energy barrier absorption and release in vesicle transport. These components are relatively low in the cytoplasmic membrane. Therefore, it is not so advantageous to separate the cytoplasmic membrane for BioNV derivatization. When BioNVs pass through polycarbonate filters during continuous extrusion, they are disrupted and spontaneously formed depending on the pore size. The BioNVs produced by this process contain membranes with a uniform mixture of cytoplasmic, Golgi and ER lipid content and protein components, which can significantly improve their affinity for cell and tissue delivery absorption compared to BioNVs that have been treated to eliminate these organelles. These features can be translated into better and more consistent absorption of BioNVs into target cells at much lower doses than systems that do not have these characteristics.

[0317] After the extrusion step, the BioNV was passed through an α-CD3 HPLC (FPLC under expansion) column to remove a low percentage (approximately 0.05%) of reversed BioNV (e.g. Fig. 27This is done to ensure that the resulting BioNV has homogenous orientation relative to the membrane. Yield loss in this step is lower because it is a flow-through process that captures impurities. After the BioNV is collected after the HPLC / FPLC step, it will be tested through a standardized process.

[0318] The standardization process includes one or more of the following measurements:

[0319] BioNV Homogeneity : Nanoparticle flow cytometry (NanoFCM) can be used to confirm the homogeneity of BioNV concentration, size, density of BioNV and / or homogeneity of BioNV luminal components.

[0320] Luminal payload concentration NanoFM technology can be used to determine the type and concentration of nucleic acids / proteins packaged into the BioNV lumen. These data can be confirmed in parallel by one or more methods including immunoblotting, mass spectrometry and BCA analysis to determine the nucleic acid and protein content of BioNV.

[0321] BioNV stability A combination of nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy (EM) can be used in combination with immunoblotting and mass spectrometry to determine the physical and biochemical characteristics of BioNV over 8 to 10 months. Data from these assays can include protein expression profiles, the extent of intact BioNV membrane / packaging, and / or the extent of aggregation.

[0322] Membrane Integrity: The integrity of the BioNV membrane was assessed using a calcein release assay in combination with NanoFCM to evaluate membrane permeability. The results provided insight into the leaky properties of the BioNV relative to a standardized set of BioNVs.

[0323] Mass of lumen payload: Depending on the nature of the payload, a variety of analytical assays can be used to determine the quality of the lumen-encapsulated payload. If the delivery is a nucleic acid, qPCR and / or sequencing at 8 to 10 months can be used to check the integrity and quantity of the nucleic acid payload. For proteins, BioNV components can be analyzed using one or more of NanoFCM, mass spectrometry, and immunoblot analysis to analyze protein payloads.

[0324] CAR mass and surface density:NanoFCM, mass spectrometry and / or immunoblotting analysis can be used to determine the CAR surface density. Compared with the whole cell surface density, the CAR surface density in BioNV is expected to be at least about 5 times to at least about 10 times higher. Compared with whole cells, this can significantly enhance the targeting of antigens. CAR quality can be determined at the cell stage, as described above (e.g., as described in step 3). Mathematical models can be used to infer cell quality data and apply them to BioNV related to efficacy study data results.

[0325] BioNV features: BioNVs can be tested for basic functionality, including multiple well-defined standardized assays, such as in vitro cellular uptake into target cells with or without expressed antigens, and their ability to cross dense tissues, such as those in a human retina model. These basic functional assays can be performed immediately following the continuous extrusion process, after which preclinical studies will address the remaining quality and functional properties of the BioNVs.

[0326] definition

[0327] The following definitions are used in conjunction with the disclosure herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0328] An "effective amount" or "therapeutically effective amount" is an amount effective for treating, preventing or ameliorating a disease in a mammal.

[0329] As used herein, "a", "an" or "the" may mean one or more than one.

[0330] As used herein, the word "include" and variations thereof are intended to be non-limiting, such that the enumeration of items in a list does not exclude other similar items that may also be used in the materials, compositions, devices, and methods of the present technology. Likewise, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that a statement that an embodiment may or may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0331] Although the open-ended term "comprising," as a synonym for terms such as including, containing, or having, is used herein to describe and claim the disclosed methods and compositions, alternative terminology such as "consisting of" or "consisting essentially of" may optionally be used to describe the disclosure or embodiments thereof.

[0332] In embodiments, "BioNV" refers to a biomimetic nanovesicle (NV) that encapsulates an aqueous compartment. In embodiments, BioNV is allogeneic and / or hypoimmune. In embodiments, BioNV may refer to NVs made by rupturing / destroying cells (e.g., cell-derived vesicles), or nanovesicles (e.g., exosomes) that are naturally shed from cells. In embodiments, BioNV comprises at least one surface-oriented, membrane-embedded CAR. In embodiments, the "nanovesicle (NV)" mentioned herein is a lipid-bound vesicle having a size of about 10nm to about 1200nm, which encapsulates an aqueous core. In embodiments, lipid-bound NVs can be formed using a lipid monolayer, a lipid bilayer, or maintain a multilayer form. In embodiments, BioNV refers to a bio-derived nano-sized vesicle that can have a designed biological functionalization. In embodiments, BioNV is "biomimetic" because it is derived from endogenous cell material, more specifically, it substantially reproduces the plasma membrane material found in cells. In embodiments, the cells from which BioNV is derived may include any kind of stem cells, including cell types differentiated from the stem cells. In embodiments, the BioNV is substantially free of encapsulated cellular debris, including nucleic acids, organelles, or organelle portions. In embodiments, the BioNV is characterized by having one or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more of the following:

[0333] a. A size of about 10 nm to about 1200 nm;

[0334] b. Total volume is about 500nm 3 to about 5 μm 3 (assuming a spherical shape);

[0335] c. having a content of at least one phospholipid and cholesterol;

[0336] d. having a surface membrane, the surface membrane having one or more of CD34, CCL21, PD-L1 (in BioNV derived from non-activated cell sources), FasL, SerpinB9, H2-M3, CD47, CTLA-4, CD24, CD200, MFG-E8, NCAM, α-phagocytic integrin, and / or anti-6R antibody or antibody form, or a chimera of one or more thereof; having a surface membrane, the surface membrane substantially lacking T cell receptor components (TRAC and / or TRBC), MHC class I components and / or MHC Class II components lacking proteins of one or more of HLA-A, HLA-B, HLA-C, HLA-E or HLA-G (but not both HLA-E and HLA-G), HLA-F and / or CIITA, SerpinB9, and substantially lacking proteins of one or more of IL-4, IL-6, IL-10 and / or IL-16 within the vesicles;

[0337] e. Encapsulation of one or more therapeutically relevant biomolecules, including, for example, cytokines, including chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), proinflammatory cytokines, anti-antigen cytokines, perforin granzymes (e.g., granzymes A, B, H, K, and M), gene editing payloads, fusion proteins, antibodies, or antibody-format constructs, or combinations thereof.

[0338] f. having a membrane embedded targeting agent comprising a target binding portion, wherein the target binding portion may include an antibody or antibody format selected from one or more of the following: CAR, monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, fusion protein comprising an antigen binding portion of an antibody, bispecific T cell engager (BiTE), viral epitope recognition receptor (VERR) or viral ligand, variable heavy chain IgG fragment V H H or V NAR or through a T cell receptor (TCR), wherein the CAR can target a single biomarker or multiple biomarkers, or multiple portions of a single biomarker, and the one or more targeting agents can include a ligand for the receptor or a receptor for the ligand;

[0339] g. payloads capable of adsorbing and / or encapsulating: one or more perforins, granzymes, cytokines, cytotoxic proteins, non-naturally occurring cellular agents, checkpoint inhibitors, recombinant gene editing payloads, antibodies or antibody fragments, small molecule inhibitors, biologics, radionuclides, tracers, dyes, fluorescent proteins, and other therapeutic payloads and / or any combination thereof; and

[0340] h. be capable of not causing a harmful immune response in the subject.

[0341] In embodiments, "induced pluripotent stem cells" or "iPSCs" refer to stem cells that can be generated directly from adult cells. iPSCs can be derived from differentiated cells that are reprogrammed back to an embryonic-like pluripotent state. iPSCs can generally propagate indefinitely and become any cell type of the organism from which they originated.

[0342] In embodiments, "allogeneic" as used herein refers to biological material, tissue or cells that are genetically different and initially immunologically incompatible, although derived from the same species. For example, allogeneic CDV / exosomes are material derived from a first subject (iPSC donor) and can be provided to any number of different genetically non-identical subjects.

[0343] In embodiments, "low immunogenicity" or "low immunity" used herein for cells and / or BioNV refers to a reduced ability to produce an immune response. In embodiments, cells and BioNV may have low immunogenicity due to reduced or eliminated expression of one or more specific cell surface proteins and / or secretory proteins (e.g., T cell receptor (TCR) proteins, cytokine response syndrome proteins, MHC class I or class II proteins, etc.). In embodiments, cells and BioNV may have low immunogenicity due to increased immunoprotective cell surface proteins (e.g., CD47, CD34, CD24, CD200, α-phagocytic integrins, etc.). In embodiments, BioNV and / or cells may have low immunogenicity due to not triggering CRS in a subject and / or not inducing HLA incompatibility.

[0344] In embodiments, "knockout", "silencing", "inactivation", "destruction" or "blocking" and equivalents thereof, with respect to transcription, gene expression or protein expression, refers to a reduction in the amount of transcription, gene or protein expression from a normal state or below the wild-type state in a particular cell subpopulation. The reduction may be significant, such that gene expression does not occur, or only a negligible amount of expression occurs.

[0345] In an embodiment, "overexpression" as used herein refers to an increase in the amount of transcription, gene or protein expression from a normal state or exceeding the wild-type state in a specific cell subpopulation.

[0346] Equivalent solutions

[0347] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments specifically described herein. Such equivalents are intended to be encompassed within the scope of the appended claims.

[0348] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may apply equally to all sections.

[0349] Incorporated by Reference

[0350] All patents and publications cited herein are hereby incorporated by reference in their entirety, including published PCT application WO 2020 / 227369, filed on May 6, 2020, entitled “Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors,” and published U.S. non-provisional application US 20220040106A1, filed on August 3, 2021, entitled “Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases.”

[0351] Throughout this application, various publications (including U.S. patents) are cited by author and year, and by patent number. The complete citations for the publications are listed below. The disclosures of these publications and patents are hereby incorporated by reference in their entirety into this application in order to more fully describe the prior art to which this disclosure belongs.

[0352] The present disclosure has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

[0353] Obviously, many modifications and variations of the present disclosure are possible in light of the above teachings.It is therefore to be understood that within the scope of the appended claims, the present disclosure may be practiced otherwise than as specifically described.

Claims

1. A method for producing low immunogenic cells, the method comprising: (a) reducing or eliminating the expression and / or activity of one or more immunogenic proteins in a cell; as well as (b) expressing one or more immunoprotective proteins in said cells or increasing the expression and / or activity thereof, Thereby generating the low immunogenic cells.

2. The method of claim 1, wherein the cell is a stem cell, an induced pluripotent stem cell (iPSC), a reprogrammed pluripotent or multipotent cell, an embryonic stem cell, a mesenchymal stem cell, or a differentiated cell derived from any stem cell thereof.

3. The method of claim 2, wherein the differentiated cell is a T cell, a helper T cell, a T memory cell, or a NK cell.

4. The method of claim 2, wherein the differentiated cells are macrophages.

5. The method of claim 2, wherein the differentiated cells are monocytes.

6. The method of claim 2, wherein the differentiated cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, or retinal pigment epithelial (RPE) cells.

7. The method of any of the preceding claims, wherein the low immunogenic cells substantially lack one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins.

8. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises disruption of the β2-macroglobulin (B2M) gene and / or disruption that reduces or eliminates the expression and / or activity of MHC class I proteins.

9. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises disruption of the CIITA gene and / or disruption that reduces or eliminates the expression and / or activity of an MHC class II protein.

10. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises disruption of an HLA-A gene and / or disruption that reduces or eliminates the expression and / or activity of an HLA-A protein.

11. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of an HLA-B gene and / or disruption that reduces or eliminates expression and / or activity of an HLA-B protein.

12. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises disruption of an HLA-C gene and / or disruption that reduces or eliminates the expression and / or activity of an HLA-C protein.

13. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of an HLA-E gene or disruption of an HLA-G gene and / or disruption that reduces or eliminates expression and / or activity of an HLA-E or HLA-G protein.

14. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of an HLA-F gene and / or disruption that reduces or eliminates expression and / or activity of an HLA-F protein.

15. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of a T cell alpha constant (TRAC) gene and / or disruption that reduces or eliminates TRAC protein expression and / or activity.

16. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of a T cell beta constant (TRBC) gene and / or disruption that reduces or eliminates TRBC protein expression and / or activity.

17. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises disruption of the PD-1 gene and / or disruption that reduces or eliminates PD-1 protein expression and / or activity.

18. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of an IL-4 gene and / or disruption that reduces or eliminates IL-4 protein expression and / or activity.

19. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises disruption of an IL-6 gene and / or disruption that reduces or eliminates IL-6 protein expression and / or activity.

20. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of an IL-10 gene and / or disruption that reduces or eliminates IL-10 protein expression and / or activity.

21. The method of any of the preceding claims, wherein reducing or eliminating expression and / or activity of the one or more immunogenic proteins comprises disruption of an IL-16 gene and / or disruption that reduces or eliminates expression and / or activity of an IL-16 protein.

22. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins comprises a SerpinB9 gene disruption and / or a disruption that reduces or eliminates SerpinB9 protein expression activity.

23. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins is achieved at the DNA level by one or more of the following: a transposase-based method, a Cre / Lox-based method, an endonuclease-based method, a homologous recombination (HR)-based method, a non-homologous end joining (NEHJ)-based method, a microhomology-mediated end joining (MMEJ)-based method, a homology-mediated end joining (HMEJ)-based method, small RNA, or a combination thereof.

24. The method of claim 23, wherein the small RNA is or includes one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease and small non-coding RNA.

25. The method of any of the preceding claims, wherein reducing or eliminating the expression and / or activity of the one or more immunogenic proteins is achieved at the RNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA.

26. The method of any one of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CD34 gene and / or gene product.

27. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CCL2 gene and / or gene product.

28. The method of any one of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a PD-L1 gene and / or gene product, and wherein the cell is not activated.

29. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of H2-M3 genes and / or gene products.

30. The method of any one of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CD47 gene and / or gene product.

31. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CD24 gene and / or gene product.

32. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of chimeric CD24 / CD47 genes and / or gene products.

33. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CTLA-4 gene and / or gene product.

34. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a CD200 gene and / or gene product.

35. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products.

36. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of a MFG-E8 gene and / or gene product.

37. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of an NCAM gene and / or gene product.

38. The method of any of the preceding claims, wherein expressing or increasing the expression and / or activity of one or more immunoprotective proteins comprises expressing or increasing the expression of an alpha-phagocytic integrin gene and / or gene product.

39. The method of any of the preceding claims, wherein expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expressing or increasing the expression of an antibody or antibody-like molecule that targets an IL-6 surface receptor (anti-IL-6R).

40. The method of any of the preceding claims, wherein expressing one or more immunoprotective proteins or increasing their expression and / or activity comprises expression of a FasL gene and / or gene product.

41. The method of any of the preceding claims, wherein expressing one or more immunoprotective proteins or increasing their expression and / or activity does not comprise overexpression of the FasL gene and / or gene product.

42. methods as described in any one of the preceding claims, expression and / or the activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins or 12 or more immunogenic proteins of wherein said low immunogenic cells are reduced or eliminated.

43. methods as described in any one in claim 1 to 41, wherein said low immunogenicity cell has the expression of 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins or 10 or more immunoprotective proteins, or increases their expression.

44. The method of any one of claims 1 to 41, wherein the expression and / or activity of genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G of the low immunogenic cells is reduced or eliminated.

45. The method of any one of claims 1 to 41, wherein the expression and / or activity of genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G of the low immunogenic cells is reduced or eliminated.

46. ​​The method of any one of claims 1 to 41, wherein the expression and / or activity of genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G and one or more of IL-4, IL-10 and IL-16 of the low immunogenic cells is reduced or eliminated.

47. The method of any one of claims 1 to 41, wherein the low immunogenic cells express or increase the expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL; and wherein the low immunogenic cells are not activated by expression of PD-L1; and One of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200 is expressed, or the expression of these two is increased.

48. The method of any one of claims 1 to 41, wherein the low immunogenic cells express or increase the expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL; and wherein the low immunogenic cells are not activated by the expression of PD-L1; and One of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200 is expressed, or the expression of these two is increased.

49. The method of any one of claims 1 to 41, wherein the low immunogenic cells express or increase the expression of the CD200 gene and / or gene product and do not express and / or substantially lack the CD24 or CD47 genes and / or gene products.

50. The method of any one of claims 1 to 41, wherein the low immunogenicity cells do not have expression and / or activity of SerpinB9 gene and / or gene product and CD200 gene and / or gene product.

51. The method of any one of claims 26 to 40, wherein expressing one or more immunoprotective proteins or increasing their expression is achieved by introducing exogenous genetic elements.

52. The method of claim 51, wherein the introduction of the exogenous genetic element is achieved by stable integration into the cell genome.

53. The method of claim 52, wherein the stable integration is achieved by one or more of the following: a transposase-based method, a Cre / Lox-based method, an endonuclease-based method, a homologous recombination (HR)-based method, a non-homologous end joining (NEHJ)-based method, a microhomology-mediated end joining (MMEJ)-based method, a homology-mediated end joining (HMEJ)-based method, or a combination thereof.

54. The method of claim 53, wherein the stable integration is achieved via a viral vector.

55. The method of claim 51, wherein the introduction of the exogenous genetic elements is achieved by transient transfection.

56. A method as described in any one of claims 26 to 40, wherein expressing the one or more immunoprotective proteins or increasing their expression is achieved by an exogenous promoter and / or enhancer, and / or an endogenous promoter and / or enhancer, or a combination thereof.

57. The method of any one of claims 26 to 40, wherein expressing or increasing the expression of the one or more immunoprotective proteins is under the control of a constitutively active promoter.

58. The method of any one of claims 26 to 40, wherein expressing or increasing the expression of the one or more immunoprotective proteins is achieved at the DNA level by one or more of: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA.

59. The method of any of the preceding claims, wherein expressing or increasing the expression of the one or more immunoprotective proteins is achieved at the RNA level by one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotides (ASO), locked nucleic acid (LNA), splice switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonucleases, and small non-coding RNA.

60. The method of any one of claims 26 to 40, wherein expressing or overexpressing the one or more immunoprotective proteins is achieved by one or more of a small regulatory RNA, a miRNA, an IRES element, a transcription factor, or a combination thereof.

61. The method of any of the preceding claims, wherein the low immunogenic cells are allogeneic.

62. The method of any of the preceding claims, wherein the low immunogenic cells do not elicit an immune response in a patient to whom the cells or a biological composition derived therefrom is administered.

63. The method of any one of the preceding claims, wherein the low immunogenic cells comprise one or more targeting agents.

64. The method of claim 63, wherein the one or more targeting agents comprise a chimeric antigen receptor (CAR), an immunocompetent HLA complex and / or a T cell receptor (TCR).

65. The method of claim 64, wherein the CAR is bispecific.

66. The method of claim 64, wherein the CAR lacks an intracellular portion.

67. A method as claimed in claim 64, wherein the CAR comprises a targeting agent, a transmembrane domain and an intracellular domain, which comprises a co-stimulatory domain and / or a signaling domain.

68. The method of claim 67, wherein the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α or ICOS, or a fragment thereof.

69. A method as claimed in claim 67, wherein the intracellular domain comprises the intracellular signaling domain of the CD3ζ chain and / or one or more co-stimulatory molecules, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40.

70. The method of claim 63, wherein the one or more targeting agents comprise an antibody or an antibody form.

71. The method of claim 70, wherein the antibody or antibody form is selected from one or more of the following: monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), VERR, V NAR 、V H H, afilin, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody.

72. The method of claim 71, wherein the antibody format is a scFv.

73. The method of claim 63, wherein the one or more targeting agents comprise a viral epitope recognition receptor (VERR) or a viral ligand.

74. The method of claim 63, wherein the one or more targeting agents comprise a ligand for a receptor.

75. The method of claim 63, wherein the one or more targeting agents comprises a receptor for a ligand.

76. The method of claims 63 to 75, wherein the one or more targeting agents are operably linked to a regulatable expression element.

77. A low immunogenic cell produced by the method of any one of claims 1 to 76.

78. A pharmaceutical composition comprising the low immunogenicity cells of claim 77 and one or more excipients.

79. A low immunogenicity cell, comprising: (a) one or more membrane-embedded targeting agents that target one or more cellular biomarkers; (b) expression or increased expression of one or more immunoprotective proteins selected from the group consisting of: (i) α-phagocytic integrin, CCL2, H2-M3, MFG-E8, and FasL, among which FasL was not overexpressed; (ii) PD-L1 and / or CTLA-4, wherein the low immunogenic cells are not activated by expression of PD-L1; and (iii) one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200; and (c) substantially lacks the expression and / or activity of one or more immunogenic proteins selected from: (i) HLA-A, HLA-B, HLA-C, HLA-F, CIITA, PD-1, IL-6, T cell alpha chain (TRAC), and T cell beta chain (TRBC); and (ii) HLA-E or HLA-G.

80. A low immunogenicity cell, comprising: (a) one or more membrane-embedded targeting agents that target one or more cellular biomarkers; (b) expression or increased expression of one or more immunoprotective proteins selected from the group consisting of: (i) α-phagocytic integrin, CCL2, H2-M3, MFG-E8, and FasL, among which FasL was not overexpressed; (ii) PD-L1 and / or CTLA-4, wherein the low immunogenic cells are not activated by expression of PD-L1; and (iii) CD24 and CD47, or chimeric CD24 / CD47; and (c) substantially lacks the expression and / or activity of one or more immunogenic proteins selected from: (i) HLA-A, HLA-B, HLA-C, HLA-F, CIITA, PD-1, SerpinB9, IL-6, T cell alpha chain (TRAC) and / or T cell beta chain (TRBC); and (ii) HLA-E or HLA-G.

81. The low immunogenicity cell of claim 79 or 80, wherein the low immunogenicity cell substantially lacks expression and / or activity of one or more of IL-4, IL-10 and / or IL-16.

82. The low immunogenic cell of any one of claims 79 to 81, further comprising expression or increased expression of an antibody or antibody-like molecule targeting an IL-6 surface receptor (anti-IL-6R).

83. The low immunogenic cell of any one of claims 79 to 82, further comprising expression or increased expression of NCAM.

84. A low immunogenic cell as described in any one of claims 79 to 83, wherein the one or more targeting agents include a chimeric antigen receptor (CAR).

85. The low immunogenicity cell of any one of claims 79 to 84, wherein the one or more targeting agents are antibodies or antibody forms.

86. The low immunogenic cell of claim 85, wherein the antibody or antibody form is selected from one or more of the following: monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), V NAR 、V H H, afilin, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody.

87. The low immunogenicity cell of any one of claims 79 to 83, wherein the one or more targeting agents are viral epitope recognition receptors (VERRs) or viral ligands.

88. The low immunogenic cell of any one of claims 79 to 83, wherein the one or more targeting agents are ligands of a receptor or receptors of a ligand.

89. The low immunogenic cell of any one of claims 79 to 88, wherein the low immunogenic cell is allogeneic.

90. The low immunogenic cells of any one of claims 79 to 89, wherein the low immunogenic cells do not induce an immune response in a patient to whom the cells or a biological composition derived therefrom is administered.

91. A pharmaceutical composition comprising the low immunogenic cells of any one of claims 79 to 90 and one or more excipients.

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