A low immunogenic biomimetic nanovesicle delivery system for hemophagocytic lymphohistiocytosis (HLH)-associated diseases

Abstract

Disclosed herein are compositions comprising allogeneic, hypoimmunogenic, cell-targetable biomimetic nanovesicles (BioNVs) and methods of using same for the treatment of hemophagocytic lymphohistiocytosis (HLH) and HLH-related disorders.

Description

[Technical Field]

[0001] The present disclosure provides, in part, compositions and methods comprising allogeneic, hypoimmunogenic, biomimetic nanovesicles and methods of using same for the treatment of hemophagocytic lymphohistiocytosis (HLH)-associated diseases in mammalian subjects, e.g., humans.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 425,175, filed November 14, 2022, and U.S. Provisional Patent Application No. 63 / 425,214, filed November 14, 2022, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Hemophagocytic lymphohistiocytosis (HLH) is a disease defined by the hyperactivation of immune cells (i.e., histiocytes), including T cells, NK cells, B cells, and macrophages. Although several clinical manifestations are associated with HLH, approximately 60% of cases are due to mutations in the PRF1 and / or UNC13D genes. HLH is a rare syndrome first characterized in the mid-20th century, primarily resulting in systemic hyperinflammation defined by recurrent fever, erythropenia, neutropenia, bone marrow, and hepatosplenomegaly (Asher R, "Histiocytic medullary reticulosis; a case without lymphdenopathy," Lancet, 1946 Vol. 1, No. 6401, 1946: pp. 650-1).

[0004] The diagnosis of HLH was later defined by persistent fever, splenomegaly, erythropenia, hypertriglyceridemia / hyperfibrinogenemia, and hemophagocytosis. New indicators of HLH, such as elevated circulating ferritin levels and high levels of soluble CD25, have recently been reported, the latter being a predictor of poor outcome (ESUMI N, et al., "Hyperferritinemia in malignant histiocytosis and virus-associated hemophagocytic syndrome," N Engl J Me, Vol. 316, No. 6, 1987: pp. 346-7) and (IMASHUKU S, et al., "Soluble interleukin-2 receptor: a useful prognostic factor for patients with hemophagocytic lymphohistiocytosis," Blood, 1995 Vol. 86, No. 12, 1995: pp. 4706-7).

[0005] Currently, few options exist for treating HLH and HLH-related disorders. Options include treating patients at the onset of HLH (early symptoms based on elevated levels of IL-2 and soluble CD25, among other physical characteristics) with cytokine replacement therapy, immunosuppressants, or monoclonal antibodies to suppress cytokine release syndrome (CRS). However, mortality remains high, with approximately 50% of HLH cases resulting in death, and HLH associated with malignancy has a poor prognosis. Most HLH patients die within the first 30–50 days and are young, under the age of 1 year. There remains a need for 1) effective correction of genetic mutations in genes that directly or indirectly affect perforin activity, and 2) delivery of corrective gene editing payloads to cells harboring the mutations in a highly targeted, allogeneic, stable, CRS-preventing, safe, and effective manner. Summary of the Invention

[0006]

[0003] Thus, in one aspect, the present invention relates to a biomimetic nanovesicle (BioNV) comprising a targeting agent targeted to at least a first cell surface marker associated with hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease and a gene editing payload targeted to one or more mutations associated with HLH or an HLH-associated disease, wherein the BioNV is configured to encapsulate and deliver the gene editing payload to a target cell expressing the first cell surface marker. Alternatively, in one embodiment, the BioNV comprises a targeting agent targeted to at least a first cell surface marker associated with hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease and one or more of perforin, granzyme, p53, and one or more nucleic acids encoding these proteins, wherein the BioNV is configured to encapsulate and deliver one or more of perforin, granzyme, p53, and one or more nucleic acids encoding these proteins to a target cell expressing the first cell surface marker.

[0007] In various embodiments, the HLH or HLH-associated disease is HLH, FLH4, FLH2, FLH3, FLH5, Griscelli syndrome type 2, Chedik-Higashi syndrome, PID, EBV, PID and EBV, XLP-1, XLP-5 and EBV, immunodeficiency 24 (IMD24), immunodeficiency 27A / B (IMD27A / B), immunodeficiency 18 (IMD18), Wiskott-Aldrich syndrome, immunodeficiency 9 (IMD9), X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (CGDX), Wolman disease, or galactosemia. One or more of the following disorders are present: I, Gaucher disease type 1, galactosialidosis, methylmalonic aciduria, Shaheen syndrome, mevalonic aciduria, biotin deficiency, LCHAD deficiency, lysinuric protein intolerance and orotic aciduria, multisulfatase deficiency and metachromatic leukodystrophy, propionic acidemia and multicarboxylase deficiency, lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, Takeuchi-Kosaki syndrome and neonatal-onset severe multisystem autoinflammatory disease, familial cold autoinflammatory syndrome 4, and autoinflammatory disorders with infantile enteritis.

[0008] In embodiments, the HLH or HLH-associated disease is selected from Table 1, and / or the BioNV targeting moiety and target cells are matched to the disease and selected from Table 1.

[0009] In embodiments, the first cell surface marker is or comprises CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, or TIM-3. In embodiments, the first cell surface marker is or comprises CD34. In various embodiments, the first cell surface marker is or includes CD56 (e.g., for NK cell targeting), or CD16 (also known as FcγRIII), which is found on the surface of NK cells, neutrophils, monocytes, macrophages, and T cells.

[0010] In embodiments, the gene editing payload comprises one or more gene editors and / or nucleic acids encoding one or more gene editors, in embodiments, the one or more gene editors are a site-specific endonuclease, a CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, a transposase, and / or an ortholog or homolog thereof.

[0011] In some embodiments, the gene editor is a CRISPR / Cas nuclease. In some embodiments, the gene editor is a site-specific endonuclease. In some embodiments, the gene editing payload comprises one or more gRNAs and / or nucleic acids encoding the one or more gRNAs. In some embodiments, the one or more gRNAs are targeted to one or more mutations in one or more genes in Table 2 and / or one or more mutations in Table 3, Table 4, or Table 5.

[0012] In embodiments, the one or more genes are STX11, PRF1, UNC13D, STXBP2, RAB27A, RHOG, LYST, CD27, SH2D1A, CTPS1, ITK, MAGT1, RASGRP1, STAT1, STAT2, IFNGR1 / 2, IL7RA, RAG1 / 2, CD3E, WAS, ORAI1, BTK, CYBB / CYBA / NCF1, LIPA, GALT, GBA, CTSA, MUT, COG6, MVK, BTD, HADHA, SLC7A7, SUMF1, PCCA / PCCB, XIAP, CDC42, or NLRC4.

[0013] In embodiments, the gene editing payload comprises one or more nucleic acids encoding functional versions of one or more genes in Table 2. In embodiments, the gene editing payload comprises at least two gRNAs that target regions adjacent to one or more genes in Table 2 and / or one or more mutations in Table 3, Table 4, or Table 5.

[0014] In various embodiments, the HLH or HLH-associated disease is HLH and the gene editing payload targets a mutant ROG gene; the HLH or HLH-associated disease is FLH4 and the gene editing payload targets a mutant STX11 gene; the HLH or HLH-associated disease is FLH2 and the gene editing payload targets a mutant PRF1 gene; the HLH or HLH-associated disease is FLH3 and the gene editing payload targets a mutant UNC13D gene; or the HLH or HLH-associated disease is FLH5 and the gene editing payload targets a mutant UNC13D gene. The gene editing payload targets a mutant STXBP2 gene; the HLH or HLH-associated disease is Griscelli syndrome type 2; the gene editing payload targets a mutant RAB27A gene; the HLH or HLH-associated disease is Chedik-Higashi syndrome; the gene editing payload targets a mutant LYST gene; the HLH or HLH-associated disease is PID and / or EBV; and the gene editing payload targets a mutant CD27 gene, ITK gene, MAGT1 gene, RASGRP1 gene, STAT1 gene, STA The gene editing payload targets the T2 gene, the IL7RA gene, and the RAG1 / 2 gene; the HLH or HLH-associated disease is XLP-1 / EBV, and the gene editing payload targets a mutant SH2D1A gene; the HLH or HLH-associated disease is immunodeficiency 24 (IMD24), and the gene editing payload targets a mutant CTPS1 gene; the HLH or HLH-associated disease is immunodeficiency 27A / B (IMD27A / B), and the gene editing payload targets a mutant IFNGR1 / 2 gene; the HLH or HLH-associated disease is immunodeficiency the HLH or HLH-associated disease is Immunodeficiency 9 (IMD9) and the gene editing payload targets a mutant ORAI1 gene; the HLH or HLH-associated disease is X-linked agammaglobulinemia (XLA) and the gene editing payload targets a mutant BTK gene;The HLH or HLH-associated disease is X-linked chronic granulomatous disease (CGDX), and the gene editing payload targets a mutant CYBB / CYBA / NCF1 gene; the HLH or HLH-associated disease is Wolman disease, and the gene editing payload targets a mutant LIPA gene; the HLH or HLH-associated disease is galactosemia I, and the gene editing payload targets a mutant GALT gene; the HLH or HLH-associated disease is Gaucher disease type 1, and the gene editing payload targets a mutant GBA gene; the HLH or HLH-associated disease is galactosemia I. the HLH or HLH-associated disease is methylmalonic aciduria, and the gene editing payload targets a mutant MUT gene; the HLH or HLH-associated disease is Shaheen syndrome, and the gene editing payload targets a mutant COG6 gene; the HLH or HLH-associated disease is mevalonic aciduria, and the gene editing payload targets a mutant MVK gene; the HLH or HLH-associated disease is biotin deficiency, and the gene editing payload targets a mutant BTD gene; The HLH or HLH-associated disease is LCHAD deficiency, and the gene editing payload targets a mutant HADHA gene; the HLH or HLH-associated disease is lysinuric protein intolerance and orotic aciduria, and the gene editing payload targets a mutant SLC7A7 gene; the HLH or HLH-associated disease is multisulfatase deficiency and metachromatic leukodystrophy, and the gene editing payload targets a mutant SUMF1 gene; the HLH or HLH-associated disease is propionic acidemia and multicarboxylase deficiency, and the gene editing payload targets a mutant SUMF1 gene. The gene editing payload targets a mutant PCCA / PCCB gene; the HLH or HLH-associated disease is lymphoproliferative syndrome or X-linked lymphoproliferative syndrome 2, and the gene editing payload targets a mutant XIAP gene; the HLH or HLH-associated disease is Takeuchi-Kosaki syndrome or neonatal-onset severe multisystem autoinflammatory disease, and the gene editing payload targets a mutant CDC42 gene; or the HLH or HLH-associated disease is familial cold autoinflammatory syndrome 4 or autoinflammation with infantile enterocolitis, and the gene editing payload targets a mutant NLRC4 gene.

[0015] In various embodiments, BioNV harbors one or more different types of granzyme proteins selected from granzymes A, B, H, K, and M.

[0016] In embodiments, one or more of perforin, granzyme, and p53 comprise a modification, hi embodiments, the modification comprises one or more of an amino acid mutation, a fusion, the addition of a fluorescent label, PEGylation, and a post-translational modification (PTM).

[0017] In some embodiments, BioNV encapsulates one or more nucleic acids, including DNA or RNA. In some embodiments, the RNA is mRNA, optionally modified mRNA, or circular RNA. In some embodiments, the DNA is a plasmid or vector.

[0018] In embodiments, BioNV further encapsulates and delivers one or more caspase proteins and / or one or more nucleic acids encoding one or more caspase proteins, in various embodiments, the one or more caspase proteins are caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10.

[0019] In embodiments, the BioNV is derived from a hypoimmunogenic modified cell. In embodiments, the hypoimmunogenic modified cell is a differentiated cell derived from a stem cell, an induced pluripotent stem cell (iPSC), a reprogrammed pluripotent or multipotent cell, an embryonic stem cell, a mesenchymal stem cell, or any modified cell thereof. In embodiments, the hypoimmunogenic modified cell is an iPSC. In embodiments, the differentiated cell is a T cell, a helper T cell, a T memory cell, a γδ T cell, a NK cell, a monocyte, or a macrophage.

[0020] In embodiments, BioNV substantially lacks one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins. In embodiments, BioNV has reduced or eliminated β2-macroglobulin (B2M) protein expression and / or activity and / or MHC class I protein expression and / or activity. In embodiments, BioNV has reduced or eliminated CIITA protein expression and / or activity and / or MHC class II protein expression and / or activity.

[0021] In embodiments, BioNV has reduced or eliminated HLA-A protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-B protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-C protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-E or HLA-G protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-F protein expression and / or activity.

[0022] In embodiments, the BioNV has reduced or eliminated T cell alpha constant (TRAC) protein expression and / or activity. In embodiments, the BioNV has reduced or eliminated T cell beta constant (TRBC) protein expression and / or activity. In embodiments, the BioNV has reduced or eliminated PD-1 protein expression and / or activity, or the BioNV has PD-1 protein expression and / or activity.

[0023] In some embodiments, BioNV has reduced or eliminated IL-4 protein expression and / or activity. In some embodiments, BioNV has reduced or eliminated IL-6 protein expression and / or activity. In some embodiments, BioNV has reduced or eliminated IL-10 protein expression and / or activity. In some embodiments, BioNV has reduced or eliminated IL-16 protein expression and / or activity.

[0024] In embodiments, BioNV has SerpinB9 protein expression and / or activity. In embodiments, BioNV has reduced or absent SerpinB9 protein expression and / or activity. In embodiments, BioNV has CD34 protein expression and / or activity. In embodiments, BioNV has CCL2 protein expression and / or activity. In embodiments, BioNV has PD-L1 protein expression and / or activity. In embodiments, BioNV has H2-M3 protein expression and / or activity.

[0025] In some embodiments, BioNV has expression and / or activity of a CD47 protein. In some embodiments, BioNV has expression and / or activity of a CD24 protein. In some embodiments, BioNV has expression and / or activity of a chimeric CD24 / CD47 protein. In some embodiments, BioNV has expression and / or activity of a CD200 protein. In some embodiments, BioNV has expression and / or activity of a chimeric CD24 / CD200 protein, or a chimeric CD47 / CD200 protein and / or activity.

[0026] In various embodiments, BioNV has expression and / or activity of CTLA-4 protein. In various embodiments, BioNV has expression and / or activity of MFG-E8 protein. In various embodiments, BioNV has expression and / or activity of NCAM protein. In various embodiments, BioNV has expression and / or activity of alpha phagocytic (anti-phagocytic) integrin protein. In various embodiments, BioNV has expression and / or activity of FasL protein.

[0027] In embodiments, BioNV has reduced or eliminated protein expression 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.

[0028] In various embodiments, BioNV has expression and / or activity of three or more immune defense proteins, four or more immune defense proteins, five or more immune defense proteins, six or more immune defense proteins, seven or more immune defense proteins, eight or more immune defense proteins, nine or more immune defense proteins, or ten or more immune defense proteins.

[0029] In embodiments, the BioNV is allogeneic. In embodiments, the BioNV does not provoke a harmful immune response in a subject to which it is administered.

[0030] In various embodiments, BioNV substantially lacks the proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and any one of HLA-E or HLA-G.

[0031] In various embodiments, BioNV substantially lacks the proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, PD-1, and any one of HLA-E or HLA-G.

[0032] In embodiments, BioNV substantially lacks the protein and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, PD-1, HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16.

[0033] In various embodiments, the BioNV comprises membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0034] In various embodiments, the BioNV comprises membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0035] In various embodiments, BioNV has a membrane-embedded CD200 protein and is substantially devoid of either CD24 or CD47 protein.

[0036] In embodiments, the targeting agent is one or more of a chimeric antigen receptor (CAR), a viral epitope-recognizing receptor (VERR), a viral ligand, a viral receptor, or an antibody or antibody format selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab'), Fv, a single-chain Fv (scFv), a diabody, a nanobody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody. In embodiments, the VERR or viral ligand is a gp120 / gp41 complex. In embodiments, the targeting agent is an scFv.

[0037] In some embodiments, the targeting agent is a CAR (e.g., optionally with an scFv antigen-binding portion). In some embodiments, the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or fragments and / or combinations thereof. In some embodiments, the CAR comprises an intracellular signaling domain of the CD3ζ chain, and / or an intracellular domain optionally further comprising one or more costimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40. In some embodiments, the CAR is activated, and optionally, the CAR is activated via its target, through another receptor and / or a virus.

[0038] In various embodiments, BioNVs are about 10 nm to about 1200 nm in size. In various embodiments, BioNVs are about 10 nm to about 100 nm in size. In various embodiments, BioNVs are about 100 nm to about 200 nm in size. In various embodiments, BioNVs are about 200 nm to about 500 nm in size. In various embodiments, BioNVs are about 500 nm to about 1200 nm in size.

[0039] In embodiments, BioNV is stored at or suitable for storage at about −80° C. In embodiments, BioNV is lyophilized or suitable for lyophilization and / or formulated as a pharmaceutical composition.

[0040] In aspects, described herein are pharmaceutical compositions comprising biomimetic nanovesicles (BioNVs) described herein and one or more pharmaceutically acceptable excipients.

[0041] In aspects, the present disclosure relates to a method of treating hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease, the method comprising administering BioNV, or a pharmaceutical composition comprising BioNV, as described herein, to a subject in need of treatment.

[0042] In embodiments, the methods herein are directed to the treatment of HLH, FLH4, FLH2, FLH3, FLH5, Griscelli syndrome type 2, Chedic-Higashi syndrome, PID, EBV, PID and EBV, XLP-1, XLP-5 and EBV, immunodeficiency 24 (IMD24), immunodeficiency 27A / B (IMD27A / B), immunodeficiency 18 (IMD18), Wiskott-Aldrich syndrome, immunodeficiency 9 (IMD9), X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (CGDX), Wolman disease, galactosemia I, Gaucher disease type 1, galactosemia and treating HLH or HLH-associated diseases including one or more of: lysinuric protein intolerance and orotic aciduria, methylmalonic aciduria, Shaheen syndrome, mevalonic aciduria, biotin deficiency, LCHAD deficiency, lysinuric protein intolerance and orotic aciduria, multisulfatase deficiency and metachromatic leukodystrophy, propionic acidemia and multicarboxylase deficiency, lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, Takeuchi-Kosaki syndrome and severe multisystem autoinflammatory disease of neonatal onset, or familial cold autoinflammatory syndrome 4, and autoinflammatory disease with infantile enteritis.

[0043] In embodiments, the method involves an HLH or HLH-associated disease selected from Table 1, and / or the BioNV targeting moiety and target cells are matched to the disease and selected from Table 1.

[0044] In embodiments, the methods herein involve administering a BioNV that targets one or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, or TIM-3. In embodiments, the BioNV targets CD34, CD56, or CD16.

[0045] In embodiments, the methods herein include variant-adapted disease treatments, wherein the HLH or HLH-associated disease is HLH and the gene editing payload targets a mutant ROG gene; the HLH or HLH-associated disease is FLH4 and the gene editing payload targets a mutant STX11 gene; the HLH or HLH-associated disease is FLH2 and the gene editing payload targets a mutant PRF1 gene; the HLH or HLH-associated disease is FLH3 and the gene editing payload targets a mutant UNC13D gene; The associated disease is FLH5, and the gene editing payload targets a mutant STXBP2 gene; the HLH or HLH-associated disease is Griscelli syndrome type 2, and the gene editing payload targets a mutant RAB27A gene; the HLH or HLH-associated disease is Chedik-Higashi syndrome, and the gene editing payload targets a mutant LYST gene; the HLH or HLH-associated disease is PID and / or EBV, and the gene editing payload targets a mutant CD27 gene, ITK gene, MAGT1 gene, RASGRP1 gene, S The gene editing payload targets the TAT1 gene, the STAT2 gene, the IL7RA gene, and the RAG1 / 2 gene; the HLH or HLH-associated disease is XLP-1 / EBV, and the gene editing payload targets a mutant SH2D1A gene; the HLH or HLH-associated disease is immunodeficiency 24 (IMD24), and the gene editing payload targets a mutant CTPS1 gene; the HLH or HLH-associated disease is immunodeficiency 27A / B (IMD27A / B), and the gene editing payload targets a mutant IFNGR1 / 2 gene; the HLH or HLH-associated disease. is immunodeficiency 18 (IMD18) and / or EBV, and the gene editing payload targets a mutant CD3E gene; the HLH or HLH-associated disease is Wiskott-Aldrich syndrome, and the gene editing payload targets a mutant WAS gene; the HLH or HLH-associated disease is immunodeficiency 9 (IMD9), and the gene editing payload targets a mutant ORAI1 gene; the HLH or HLH-associated disease is X-linked agammaglobulinemia (XLA), and the gene editing payload targets a mutant BTK gene;The HLH or HLH-associated disease is X-linked chronic granulomatous disease (CGDX), and the gene editing payload targets a mutant CYBB / CYBA / NCF1 gene; the HLH or HLH-associated disease is Wolman disease, and the gene editing payload targets a mutant LIPA gene; the HLH or HLH-associated disease is galactosemia I, and the gene editing payload targets a mutant GALT gene; the HLH or HLH-associated disease is Gaucher disease type 1, and the gene editing payload targets a mutant GBA gene; the HLH or HLH-associated disease is galactosemia I. the HLH or HLH-associated disease is methylmalonic aciduria, and the gene editing payload targets a mutant MUT gene; the HLH or HLH-associated disease is Shaheen syndrome, and the gene editing payload targets a mutant COG6 gene; the HLH or HLH-associated disease is mevalonic aciduria, and the gene editing payload targets a mutant MVK gene; the HLH or HLH-associated disease is biotin deficiency, and the gene editing payload targets a mutant BTD gene; The HLH or HLH-associated disease is LCHAD deficiency, and the gene editing payload targets a mutant HADHA gene; the HLH or HLH-associated disease is lysinuric protein intolerance and orotic aciduria, and the gene editing payload targets a mutant SLC7A7 gene; the HLH or HLH-associated disease is multisulfatase deficiency and metachromatic leukodystrophy, and the gene editing payload targets a mutant SUMF1 gene; the HLH or HLH-associated disease is propionic acidemia and multicarboxylase deficiency, and the gene editing payload targets a mutant SUMF1 gene. The gene editing payload targets a mutant PCCA / PCCB gene; the HLH or HLH-associated disease is lymphoproliferative syndrome or X-linked lymphoproliferative syndrome 2, and the gene editing payload targets a mutant XIAP gene; the HLH or HLH-associated disease is Takeuchi-Kosaki syndrome or neonatal-onset severe multisystem autoinflammatory disease, and the gene editing payload targets a mutant CDC42 gene; or the HLH or HLH-associated disease is familial cold autoinflammatory syndrome 4 or autoinflammation with infantile enterocolitis, and the gene editing payload targets a mutant NLRC4 gene.

[0046] In embodiments, administration of BioNV delivers a gene editing payload comprising one or more gene editors and / or nucleic acids encoding the one or more gene editors. In embodiments, the one or more gene editors are a site-specific endonuclease, a CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, a transposase, and / or an ortholog or homolog thereof. In some embodiments, the gene editor is a CRISPR / Cas nuclease. In some embodiments, the gene editor is a site-specific endonuclease.

[0047] In embodiments, administration of BioNV results in delivery of one or more gRNAs and / or nucleic acids encoding one or more gRNAs, in embodiments, the one or more gRNAs are targeted to one or more mutations in one or more genes in Table 2 and / or one or more mutations in Table 3, Table 4, or Table 5.

[0048] In some embodiments, administration of BioNV results in nucleotide repair of one or more mutations. In some embodiments, the one or more mutations are in one or more of the following genes: STX11, PRF1, UNC13D, STXBP2, RAB27A, RHOG, LYST, CD27, SH2D1A, CTPS1, ITK, MAGT1, RASGRP1, STAT1, STAT2, IFNGR1 / 2, IL7RA, RAG1 / 2, CD3E, WAS, ORAI1, BTK, CYBB / CYBA / NCF1, LIPA, GALT, GBA, CTSA, MUT, COG6, MVK, BTD, HADHA, SLC7A7, SUMF1, PCCA / PCCB, XIAP, CDC42, or NLRC4.

[0049] In some embodiments, administration of BioNV results in excision of the genomic sequence by a gene editing payload. In some embodiments, administration of BioNV results in excision of the genomic sequence by a gene editing payload, replacing the genomic sequence with a functional gene. In some embodiments, the functional gene is one or more of STX11, PRF1, UNC13D, STXBP2, RAB27A, RHOG, LYST, CD27, SH2D1A, CTPS1, ITK, MAGT1, RASGRP1, STAT1, STAT2, IFNGR1 / 2, IL7RA, RAG1 / 2, CD3E, WAS, ORAI1, BTK, CYBB / CYBA / NCF1, LIPA, GALT, GBA, CTSA, MUT, COG6, MVK, BTD, HADHA, SLC7A7, SUMF1, PCCA / PCCB, XIAP, CDC42, or NLRC4.

[0050] In embodiments, administration of BioNV results in the expression of functional perforin protein. In embodiments, administration of BioNV results in the exocytosis of functional perforin protein.

[0051] In some embodiments, administration of BioNV results in delivery of one or more of perforin, granzymes, p53, caspases, and one or more nucleic acids encoding these proteins to one or more diseased cells. In some embodiments, the granzymes are selected from granzymes A, B, H, K, and M.

[0052] In some embodiments, the administered BioNV comprises one or more of perforin, granzyme, p53, and caspase proteins having modifications, including one or more of amino acid mutations, fusions, fluorescent labels, PEGylation, and post-translational modifications (PTMs).

[0053] In some embodiments, BioNV is administered to deliver one or more nucleic acids, such as DNA or RNA. In some embodiments, the RNA is mRNA, optionally modified mRNA, or circular RNA. In some embodiments, the DNA is a plasmid or vector.

[0054] In embodiments, administration of BioNV results in delivery of one or more caspase proteins and / or one or more nucleic acids encoding one or more caspase proteins. In embodiments, the one or more caspase proteins are caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10. In embodiments, administration of BioNV results in activation of one or more of caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10 in the target cell.

[0055] In embodiments, administration of BioNV results in targeted apoptosis.

[0056] In various embodiments, the methods of treatment herein include administering one or more additional therapeutic agents, including anti-inflammatory agents, cytokine therapy reagents, immunosuppressants, anti-infective agents, monoclonal antibodies, and analgesics.

[0057] In embodiments, administering comprises providing one or more doses of at least about 1 ng / kg to at least about 10 mg / kg of BioNV. In embodiments, administration is intravenous, intramuscular, or parenteral.

[0058] In various embodiments, disclosed herein are methods of treating HLH or FLH2 in a subject, the method comprising administering to the subject a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least one gRNA and one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the gene editing payload is capable of correcting at least one mutation in the PRF1 gene, and wherein correction of the at least one or more mutations results in expression of a functional perforin protein.

[0059] In various embodiments, disclosed herein are methods of treating HLH or FLH3 in a subject, the method comprising administering to the subject a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least one gRNA and one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the gene editing payload is capable of correcting at least one mutation in the UNC13D gene, and wherein correction of the at least one or more mutations results in exocytosis of functional perforin protein.

[0060] In various embodiments, disclosed herein are methods of treating HLH or FLH2 in a subject, the method comprising administering to the subject a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least two gRNAs, one or more gene editors, and / or one or more nucleic acids encoding the one or more gene editors, and a functional PRF1 nucleic acid sequence, wherein the gene editing payload is capable of excising a mutated PRF1 genomic sequence and replacing the mutated PRF1 genomic sequence with a functional PRF1 nucleic acid sequence, resulting in expression of a functional perforin protein.

[0061] In various embodiments, disclosed herein are methods of treating HLH or FLH3 in a subject, the method comprising administering to the subject a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least two gRNAs, one or more gene editors, and / or one or more nucleic acids encoding the one or more gene editors, and a functional UNC13D nucleic acid sequence, wherein the gene editing payload is capable of excising a mutated UNC13D genomic sequence and replacing the mutated UNC13D genomic sequence with a functional UNC13D nucleic acid sequence, resulting in exocytosis of functional perforin protein.

[0062] In various embodiments, the therapeutic methods herein comprise administering a BioNV that targets a first cell surface marker that is or includes one or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, or TIM-3.

[0063] In various embodiments, the therapeutic methods herein include a gene editing payload comprising one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the one or more gene editors are a site-specific endonuclease, a CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, a transposase, and / or an ortholog or homolog thereof.

[0064] In various embodiments, administering includes providing one or more doses of BioNV of at least about 1 ng / kg to at least about 10 mg / kg, and administration is intravenous, intramuscular, or parenteral.

[0065] In various embodiments, disclosed herein are methods for treating HLH or an HLH-associated disease in a subject, the method comprising administering biomimetic nanovesicles (BioNVs) to a subject in need of treatment, wherein the BioNVs comprise a targeting moiety that targets at least a first cell surface marker of a hematopoietic cell, and the BioNVs are configured to deliver perforin protein and / or a nucleic acid encoding a perforin protein, and wherein administration of the BioNVs results in targeted apoptosis of one or more cells.

[0066] In various embodiments, disclosed herein are methods for treating HLH or an HLH-associated disease in a subject, the method comprising administering biomimetic nanovesicles (BioNVs) to a subject in need of treatment, wherein the BioNVs comprise a targeting moiety that targets at least a first cell surface marker of a hematopoietic cell, and the BioNVs are configured to deliver one or more granzyme proteins and / or nucleic acids encoding one or more granzyme proteins, and wherein administration of the BioNVs results in targeted apoptosis of one or more cells.

[0067] In embodiments, disclosed herein are ex vivo methods of treating HLH or FLH2 in a subject, the methods comprising isolating cells from the subject, contacting the cells with biomimetic nanovesicles (BioNVs), and reintroducing the cells into the subject, wherein the BioNVs are configured to deliver a gene editing payload capable of correcting at least one mutation in the PRF1 gene.

[0068] In embodiments, disclosed herein is an ex vivo method of treating HLH or FLH2 in a subject, the method comprising isolating cells from the subject, contacting the cells with biomimetic nanovesicles (BioNVs), and reintroducing the cells into the subject, wherein the BioNVs are configured to deliver a gene editing payload capable of correcting at least one mutation in the UNC13D gene.

[0069] In embodiments, disclosed herein are ex vivo methods of treating HLH or FLH2 in a subject, the methods comprising isolating cells from the subject, contacting the cells with biomimetic nanovesicles (BioNVs), and reintroducing the cells into the subject, wherein the BioNVs are configured to deliver a gene editing payload capable of excising a mutant PRF1 genomic sequence and replacing the mutant PRF1 genomic sequence with a functional PRF1 nucleic acid sequence.

[0070] In various embodiments, disclosed herein is an ex vivo method of treating HLH or FLH2 in a subject, the method comprising isolating cells from the subject, contacting the cells with biomimetic nanovesicles (BioNVs), and reintroducing the cells into the subject, wherein the BioNVs are configured to deliver a gene editing payload capable of excising a mutant UNC13D genomic sequence and replacing the mutant UNC13D genomic sequence with a functional UNC13D nucleic acid sequence.

[0071] In embodiments, the therapeutic methods herein include administering one or more additional therapeutic agents, which in embodiments include one or more cytokine therapy reagents, immunosuppressants, anti-infective agents, monoclonal antibodies, and / or analgesics.

[0072] In aspects, described herein are host cells for producing biomimetic nanovesicles (BioNVs). In various embodiments, host cells (e.g., human pluripotent cells) are edited to comprise one or more membrane-embedded targeting agents, to substantially lack the protein and / or activity of one or more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, CIITA, IL-6, TRAC, TRBC, IL-4, IL-6, IL-10, IL-16, and PD-1, and to express the protein and / or activity of one or more membrane-embedded proteins including one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 CTLA-4, CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200. In embodiments, the host cells are mammalian host cells, optionally human host cells. In embodiments, the human host cells are human female fibroblast reprogrammed induced pluripotent stem cells (iPSCs). In embodiments, the host cells are gene-edited to be B2M- / - (e.g., B2M negative) and CIITA- / - (e.g., CIITA negative). In embodiments, the host cells do not elicit, or elicit a significantly reduced, adverse immune response in a subject to which they are administered. [Brief explanation of the drawings]

[0073] [Figure 1] FIG. 1 shows an exemplary, non-limiting diagrammatic representation of hypoimmunogenic iPSC-derived BioNVs. [Figure 2] 1 shows exemplary, non-limiting diagrammatic representations of chimeric antigen receptor (CAR) constructs for expression in host cells (primary and secondary generations) that can be expressed in host cells to generate BioNV. For example, a targeting moiety is suitable for knocking in for stable expression within the antibody locus (Ab locus) while simultaneously knocking out the immunogenic protein(s) into which the targeting moiety is knocked in. [Figure 3]1 shows a non-limiting diagrammatic representation of the production of BioNV from a host cell. [Figure 4] This shows a graphical representation of the size distribution of BioNVs generated from gene-edited iPSCs as measured by dynamic light scattering (DLS). BioNVs were detected at sizes between approximately 100 nm and approximately 1000 nm, with size-based resolution excluding cellular debris, organelles, and protein aggregates (e.g., smaller than 100 nm and larger than 1200 nm). [Figure 5]

[0023] Figure 1 shows an exemplary, non-limiting diagrammatic representation of continuous extrusion for producing BioNVs from gene-edited iPSCs. The iPSCs are engineered to express a targeting agent (e.g., a CAR), which are then optionally differentiated into an appropriate cell type and / or activated. BioNVs are then produced by continuous extrusion, disrupting the cells and removing cellular debris and contaminants, followed by HPLC purification. [Figure 6] 1 illustrates an expression vector for use in host cells for knock-in of human CD47 (hCD47) isoform 2 expression. [Figure 7A] Figure 7A illustrates the expression of hCD47 isoform 2 in human female fibroblast-reprogrammed iPSCs, as determined by quantitative PCR (QT-PCR). Human female fibroblast-reprogrammed iPSCs were gene-edited to knock out B2M (B2M- / -) and CIITA (CIITA- / -), eliminating the expression of human leukocyte antigen (HLA) molecules, MHC class I, and MHC class II. Clonal populations were selected based on expected expression profiles, and hCD47 expression (e.g., expression from the plasmid in Figure 6) was analyzed by measuring mRNA levels using quantitative PCR (QT-PCR). Fold changes are relative to baseline expression in gene-edited human female fibroblast-reprogrammed iPSCs. Figure 7A shows the clonal increase in expression at the exon 1-2 junction. [Figure 7B]Figure 7B illustrates the expression of hCD47 isoform 2 in human female fibroblast-reprogrammed iPSCs, as determined by quantitative PCR (QT-PCR). Human female fibroblast-reprogrammed iPSCs were gene-edited to knock out B2M (B2M- / -) and CIITA (CIITA- / -), eliminating the expression of human leukocyte antigen (HLA) molecules, MHC class I, and MHC class II. Clonal populations were selected based on expected expression profiles, and hCD47 expression (e.g., expression from the plasmid in Figure 6) was analyzed by measuring mRNA levels using quantitative PCR (QT-PCR). Fold changes are relative to baseline expression in gene-edited human female fibroblast-reprogrammed iPSCs. Figure 7B shows the clonal increase in expression at the exon 3-4 junction. DETAILED DESCRIPTION OF THE INVENTION

[0074] Hemophagocytic lymphohistiocytosis (HLH) and HLH-related disorders There are several types of familial hemophagocytic lymphohistiocytosis (HLH) and HLH-related disorders. HLH, in various embodiments, is defined as a spectrum of hypo- or hyper-inflammatory responses.

[0075] Familial HLH2 (FHL2) is caused by mutations in PRF1, which expresses perforin. Perforin destroys infected or diseased cells (e.g., cancer cells). Therefore, loss-of-function mutations in perforin result in an inability to kill infected / disease cells. This leads to increased recruitment of T cells, macrophages, and other cells to the site of infection / disease, resulting in increased cytokine release, which contributes to hyperinflammation. Autosomal recessive variants in the perforin gene (PRF1) have been observed to be directly associated with perforin deficiency in FLH2. Perforin not only functions to kill abnormal cells but also to downregulate cellular immune activation, thereby linking low perforin activity to reduced cell killing and hyperimmune activation (StepP SE, et al., "Perforin gene defects in familial hemophagocytic lymphohistiocytosis," Science, Vol. 286, No. 5446, 1999: pp. 1957-9).

[0076] FHL3 is caused by mutations in UNC13D. For example, biallelic nonsense variants in UNC13D result in a defective protein called Munc13-4, which is involved in the exocytosis of perforin-containing lytic granules from T cells to infected / diseased cells. Therefore, mutations in UNC13D result in the inability to release perforin (FELDMANN J, et al., "Munc13-4 is essential for cytolytic granule fusion and is present in a form of familial hemophagocytic lymphhisiocytosis (FHL3)," Cell, Vol. 115, No. 4, 2003: pp. 461-73).

[0077] Biallelic nonsense variants in the STX11 and STXBP2 genes, which encode the cytosolic proteins syntaxin-11 and Munc18-2, respectively, are associated with familial HLH4 and 5 (FHL4 and FHL5) disorders (Zur Stadt U, et al., "Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11," Hum Mol Gene, Vol. 14, No. 6, 2005, pp. 827-34) (Cote M, et al., "Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells," J Clin Invest. Vol. 119, No. 12, 2009: pp. 3765-73), and (Zur Stadt U, et al., "Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is caused by mutations in Munc18-2 and impaired binding to syntaxin 11," Am J Hum Genet, Vol. 85, No. 4, 2009: pp. 482-92). These genes are expressed in lymphocytes and multiple hematopoietic lineages and are required for the exocytosis of lymphocyte cytotoxic granules, which is indirectly related to perforin deficiency.

[0078] Biallelic variants of PRF1 and UNC13D have been observed to cause HLH in early infancy (SEPULVEDA FE, et al., "Distinct severity of HLH in both human and murine mutants with complete loss of cytotoxic effector PRF1, RAB27A, and STX11," Blood, Vol. 121, No. 4, 2013: pp. 595-603), (HORNE A, et al., "Characterization of PRF1, STX11, and UNC13D genotype-phenotype correlations in familial hemophagocytic lymphohistiocytosis," Br J Haematol. Vol. 143, No. 1, 2008: pp. 75-83), and (SIENI E, et al., "Genotype-phenotype study of familial heemophagocytic lymphohistiocytosis type 3." J Med Genet. Vol. 48, No. 5 2011: pp. 343-52). On the other hand, nonsense variants in STXBP2, RAB27A, and STX11 result in HLH lesions that develop several years later. However, in some cases, exocytosis can be restored with cytokine therapy, particularly in STX11 variants (BRYCESON, et al., "Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients," Blood, Vol. 110, No. 6, 2007: pp. 1906-1915). In embodiments, the therapeutic methods disclosed herein include co-administration of one or more additional therapeutic agents, including cytokine therapy reagents.

[0079] Several other mutations that result in HLH or HLH-adjacent disorders have been identified, including mutations in the SHSD1A gene (which may present with EBV infection) and mutations in the RAB27A gene (which causes a rare disorder called Griscelli syndrome type 2 (GS2)). Mutations in the RAB27A gene, which encodes a small GTP-binding protein preferentially expressed by melanocytes and hematopoietic cells, are required for cytotoxic granule exocytosis and have been observed to cause GS2 (MENASCHE, et al., "Mutations in RAB27A cause Griscelli syndrome associated with heemophagocytic syndrome," Nat Genet, Vol. 25, No. 2, 2000: pp. 173-6). RAB27A promoter deletions have also been associated with GS2 (TESI, et al. "A RAB27A 5' untranslated region structural variant associated with late-onset hemophagocytic lymphohistiocytosis and normal pigmentation," J Allergy Clin Immunol, Vol. 142, No. 1, 2018: pp. 317-321) and (GRANDIN, et al. "A RAB27A duplication in several cases of Griscelli syndrome type 2: An explanation for cases lacking a genetic diagnosis," Hum Mutat, Vol. 38, No. 10, 2017: pp. 1355-1359).

[0080] Deleterious RHOG gene variants, encoding RhoG, a small GTPase involved in vesicle transport by interacting with anillin, a scaffold protein that interacts with actin, RhoA, and myosin to coordinate cytoskeletal rearrangements at immune synapses (KALINICHENKO et al., "RhoG Deficiency Abrgates Cytotoxicity of Human Lymphocytes and Enables Hemophagocytic Lymphatic Cytosis," Blood, Vol. 137, No. 15, 2021: pp. 2033-2045).

[0081] Several activating mutations have been observed to cause HLH and HLH-associated disorders. For example, a novel activating NLRC4 gene variant causes excessive / severe HLH (CANNA et al., "An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome," Nat Genet, Vol. 46, No. 10, 2014, pp. 1140-1146). Furthermore, a novel variant in the cdc42 gene causes HLH in the absence of infection. CDC42 is a small Rho GTPase that regulates actin dynamics. However, IL-18 is upregulated with cdc42 variants, suggesting IFN-γ costimulation leading to HLH, a mechanism distinct from antigen-driven activation of HLH (CANNA et al., 2014).

[0082] Non-coding gene variants are associated with HLH in more than 50% of cases, often differing by geographic location, gender, and ethnicity (MEETHS et al., "Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D," Blood, Vol. 118, No. 22, 2011: pp. 5783-93), (SEO et al., "Founder effects in two predominant intronic mutations of UNC13D, c. 118-308C>T and c. 754-1G>C underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (FHL3) in Korea," Ann Hematol, Vol. 92, No. 3, 2013: pp. 357-64), (ENTESARIAN et al., "Novel deep intronic and missense UNC13D mutations in familial haemophagocytic lymphohistiocytosis type 3,”Br J Haematol,Vol.162,No.3,2013:pp.415-8), and (QIAN et al.“The 253-kb inversion and deep intronic mutations in UNC13D are present in North American patients with familial hemophagocytic lymphohistiocytosis 3,”Pediatr Blood Cancer,Vol.61,No.6,2014:pp.1034-40).Studies have revealed that lymphocyte-specific intronic enhancers and alternative transcription start sites in UNC13D, which are regulated by ETS family transcription factors, play a role in HLH (MEETHS et al. 2011, SEO et al. 2013, ENTESARIAN et al. 2013, and QIAN et al. 2014). Non-coding mutations can occur simultaneously and in parallel with mutations in the open reading frame to enhance the effect in HLH.

[0083] HLH can also be caused or exacerbated by several infectious diseases and can be defined by either a primary mutation or a secondary etiology. For example, viral infections can cause HLH in individuals with certain mutations, leading to uncontrolled immune cell proliferation and / or increased cytokine release, resulting in hyperinflammation. Hyperactivated T cells and macrophages are associated with intracellular infections caused by herpesviruses, influenza, HIV, CMV, Epstein-Barr virus, dengue virus, and visceral leishmaniasis. During infection, HLH triggers an exaggerated immune response, which can be treated with immunosuppressive approaches to limit its effects, while secondary treatment against the infectious agent is also administered simultaneously. However, this strategy does not address the cause of HLH. In various embodiments, the treatment methods described herein include the concomitant administration of one or more additional immunosuppressive agents.

[0084] Variants in the SHSD1A gene are responsible for X-linked lymphoproliferative disorder type 1 (XLP1) in HLH caused by EBV. (COFFEY et al., “Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene,” Nat Genet, Vol. 20, No. 2, 1998: pp. 129-35), (NICHOLS et al., “Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome,” Proc Natl Acad Sci USA, Vol. 95, No. 23, 1998: pp. 13765-70), and (SAYOS et al., “The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM,” Nature. Vol. 395, No. 6701, 1998: pp. 462-9). Genetic susceptibility to EBV and HLH has been observed in patients with SHSD1A variants (Coffey et al. 1998, and Nichols et al. 1998). Variants in the XIAP gene, which encodes the XIAP protein, function to inhibit apoptosis but also regulate inflammasome activity caused by X-linked lymphoproliferative syndrome type 2 (XLP2) in EBV-infected patients with HLH symptoms (Schmidt et al., "Clinical similarities and differences of patients with X-linked lymphoproliferative syndrome type 1 (XLP-1 / SAP deficiency) versus type 2 (XLP-2 / XIAP deficiency)," Blood, Vol. 117, No. 5, 2011, pp. 1522-9).

[0085] HLH, in various embodiments, is also defined as a range of hypoinflammatory responses. It results from the interplay between severely impaired NK cell responses and hyperactive CD8+ T cell and macrophage responses, depending on the underlying causative agent. For example, NK cell cytotoxicity is severely impaired in patients with Chediak-Higashi syndrome (CHS), which is characterized by abnormal leukocyte granulation and increased susceptibility to infection (RODER et al., "A new immunodeficiency disorder in humans involving NK cells," Nature, Vol. 284, No. 5756, 1980: pp. 553-5). Deleterious LYST gene variants have been observed in patients with CHS, which is defined by a secretory defect that prevents exocytosis of perforin-containing granules in NK and T cells.

[0086] HLH has also been associated with malignancies, immunodeficiency disorders, and autoinflammatory syndromes. Reported malignancies include natural killer cell leukemia, multiple myeloma, SCLC, and gastric cancer. Documented autoinflammatory disorders include histiocytic glomerulopathy and subacute thrombotic microangiopathy. Latrogenic HLH, or acquired HLH, has also been associated with CAR-T cell therapy, gene therapy, antibody therapy, immune checkpoint inhibitors, and allogeneic stem cell transplantation.

[0087] Several genetic determinants have been identified for primary immunodeficiency diseases (PIDs) and / or metabolic disorders (EIMs) associated with the development of HLH. For example, genetic variants associated with PIDs and caused by EBV include CD27 (CD27), ITK (ITK), RASGRP1 (RASGRP1), TNFSFR9 (TNFSFR9), MAGT1 (MAGT1), CD70 (CD70), CORO1A (CORO1A), and CTPS1 (CTPS1). HLH is associated with a number of EIMs with common variants such as COG6 (COG6), including Wolman disease, Niemann-Pick disease, Gaucher disease, lysinuric protein intolerance, multisulfatase deficiency, galactosemia, Pearson syndrome, galactosialidosis, propionic acidemia, methylmalonic acidemia, biotinidase deficiency, cobalamin C deficiency, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, and glycosylation disorders.

[0088] Non-coding mutations may occur simultaneously / in parallel with ORF mutations and enhance any deleterious effects. Thus, in embodiments, double, triple (or more) targets for gRNA or gene replacement therapy are used in some patients, especially when the mutation burden within a gene or set of genes is high. In embodiments, relevant CRISPR methods / strategies for repair are listed in Tables 2-5 herein.

[0089] Thus, the present disclosure relates, in part, to allogeneic, hypoimmunogenic, modified cell-derived biomimetic nanovesicles (BioNVs) for use in the treatment of HLH or HLH-related diseases (e.g., diseases depicted in FIG. 1). In various embodiments, BioNVs are targeted to hematopoietic cells (e.g., hematopoietic stem cells (HSCs) with mutations and / or dysfunctional or defective genes, which can differentiate into all types of blood cells (including myeloid and lymphoid cells)) using a surface-directed targeting agent (e.g., viral epitope-recognizing receptors (VERRs), viral ligand / receptor complexes, CARs, etc.), where the targeting agent can recognize a single target or multiple targets via binding moieties for desired / specific biomarkers. In various embodiments, the binding moieties can be, for example, Fab, Fab', Fab'-SH, F(ab'), scFv, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and antibodies, V, among other antibody formats. H H nanobody, V NARS The antibody constructs may include one or more variants of antibody constructs (targeting any type of cell surface biomarker) that allow BioNV to target any cell of interest, including fusion proteins containing the antigen-binding portion of (i.e., a fusion protein ...

[0090] In various embodiments, BioNVs are generated from hypoimmunogenic modified cells and are approximately 20-1200 nm in size, much smaller than conventional cell-based T / NK cell therapies (e.g., as shown in Figures 3-4). In various embodiments, the hypoimmunogenic modified cells have differentiated into activatable lymphocytes (e.g., cytotoxic T cells, macrophages, NK cells, etc.). In various embodiments, cell membrane-derived BioNVs retain the hypoimmunogenic properties of the modified cells (e.g., iPSCs), which are conferred by genetic engineering focused on knockout of specific immunogenic cell surface markers (e.g., MHC class I / II proteins, T cell receptor (TCR) proteins, cytokine release syndrome (CRS) proteins, etc.) and / or expression or overexpression of immunoprotective cell surface markers (e.g., CD47, CD34, CD24, CD200, α-phagocyte integrin, etc.). In embodiments, BioNV can deliver one or more gene editing payloads, for example, by encapsulating one or more guide RNAs (gRNAs), CRISPR / Cas nucleases, or site-specific endonucleases. Alternatively, in embodiments, BioNV can deliver one or more perforin and / or granzyme proteins, including one or more nucleic acids encoding perforin and / or granzymes.

[0091] Furthermore, the present disclosure relates, in part, to methods for treating HLH or HLH-associated disorders using the BioNV compositions. For example, in various embodiments, the methods involve administering a BioNV targeted to the hematopoietic cell surface receptor CD34. In various embodiments, the treatment method involves a BioNV configured to deliver a encapsulated gene editing payload containing at least one gRNA targeted to, for example, a mutated, non-functional, or otherwise defective gene involved in encoding perforin (e.g., PRF1) or a mutated, non-functional, or otherwise defective gene in perforin exocytosis (e.g., UNC13D). Such a BioNV, in various embodiments, functions to correct one or more mutations and improve perforin function and / or exocytosis. In various embodiments, the BioNV-delivered gene editing payload delivers two or more gRNAs that target sequences adjacent to a mutated, non-functional, or otherwise defective genomic sequence, excising the genomic sequence and replacing the excised portion with a functional sequence, thereby enabling gene replacement therapy.

[0092] The therapeutic methods herein are configured to be in vivo methods, in which BioNV is administered to the subject being treated. Alternatively, in various embodiments, the therapeutic methods herein are configured to be ex vivo methods, in which cells are isolated from the subject being treated, BioNV contacts and edits the cells, and the edited cells are then reintroduced into the subject.

[0093] Upon reviewing this disclosure in its entirety, those skilled in the art will understand that therapeutic methods can be performed in subjects by correcting genomic mutations and / or replacing genomic sequences to generate healthy clonal populations or by delivering perforin and / or granzymes to select cell populations for targeted apoptosis. In embodiments, the method involves administering BioNV, which delivers a payload that allows for selective genomic integration by selective excision, to control the integration sequence and, therefore, the integration site(s). Mutation correction, in embodiments, includes monogenic (e.g., occurring within an open reading frame (ORF) or upstream of a promoter / enhancer region) or bigenic (e.g., both spatial mutations occur simultaneously) strategies. In embodiments, the genome replacement strategy is monoallelic or biallelic (e.g., correction of one or both alleles).

[0094] In embodiments, BioNVs and their methods of use described herein provide a delivery mechanism that overcomes the shortcomings of cell-based therapies, AAV, LNP, and exosome-based delivery systems in treating HLH and its related diseases.

[0095] Methods for treating HLH and HLH-related disorders In some embodiments, the present disclosure includes methods of treating HLH or an HLH-associated disorder in a subject. In some embodiments, the methods involve treating HLH or an HLH-associated disorder by administering a BioNV described herein to a subject in need of treatment. In some embodiments, the treatment method involves correcting mutations / variants in the genomic sequence of one or more coding and / or non-coding regions. Alternatively, in some embodiments, the treatment method involves gene replacement therapy to replace one or more mutated / non-functional sequences with a cognate functional sequence.

[0096] In embodiments, the present disclosure includes a method of treating HLH or FLH2 by administering a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least one gRNA and one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the gene editing payload is capable of correcting at least one mutation in the PRF1 gene, and wherein correction of the at least one or more mutations results in expression of a functional perforin protein.

[0097] In various embodiments, the present disclosure includes a method of treating HLH or FLH3 by administering a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least one gRNA and one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the gene editing payload is capable of correcting at least one mutation in the UNC13D gene, and wherein correction of the at least one or more mutations results in exocytosis of functional perforin protein.

[0098] In embodiments, the disclosure includes a method of treating HLH or FLH2 by administering a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least one gRNA, one or more gene editors, and / or one or more nucleic acids encoding the one or more gene editors, and a functional PRF1 nucleic acid sequence, wherein the gene editing payload is capable of excising a mutated PRF1 genomic sequence and replacing the mutated PRF1 genomic sequence with a functional PRF1 nucleic acid sequence, resulting in expression of a functional perforin protein.

[0099] In various embodiments, the present disclosure includes a method of treating HLH or FLH2 by administering a BioNV comprising a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, wherein the BioNV is configured to deliver a gene editing payload comprising at least one gRNA, one or more gene editors, and / or one or more nucleic acids encoding the one or more gene editors, and a functional UNC13D nucleic acid sequence, wherein the gene editing payload is capable of excising the mutated UNC13D genomic sequence and replacing the mutated UNC13D genomic sequence with a functional UNC13D nucleic acid sequence, resulting in expression of a functional perforin protein.

[0100] The therapeutic methods described herein, in embodiments, utilize disease-matched BioNVs, with the gene editing payload and targeting moiety selected as a function of the disease. In embodiments, the HLH or HLH-associated disease includes HLH, FLH4, FLH2, FLH3, FLH5, Griscelli syndrome type 2, Chedik-Higashi syndrome, PID, EBV, PID and EBV, XLP-1, XLP-5 and EBV, immunodeficiency 24 (IMD24), immunodeficiency 27A / B (IMD27A / B), immunodeficiency 18 (IMD18), Wiskott-Aldrich syndrome, immunodeficiency 9 (IMD9), X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (CGDX), Wolman disease, galactoglobulinemia, and leukemia. These include: rhesusemia type 1, Gaucher disease type 1, galactosialidosis, methylmalonic aciduria, Shaheen syndrome, mevalonic aciduria, biotin deficiency, LCHAD deficiency, lysinuric protein intolerance and orotic aciduria, multisulfatase deficiency and metachromatic leukodystrophy, propionic acidemia and multicarboxylase deficiency, lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, Takeuchi-Kosaki syndrome and neonatal onset severe multisystem autoinflammatory disease, familial cold autoinflammatory syndrome 4, or autoinflammatory disease with infantile enteritis.

[0101] The therapeutic methods herein, in embodiments, involve administering a disease-matched BioNV payload (e.g., perforin, granzyme, caspase, and / or p53) and a targeting moiety, as described in Table 1. In embodiments, the BioNV is targeted to one or more cell surface markers listed in Table 1. Table 1: Exemplary HLH or HLH-related disease triggers and associated exemplary biomarkers. [Table 1] TIFF2025538384000003.tif252159JPEG2025538384000004.jpg249159TIFF2025538384000005.tif168159

[0102] In various embodiments, BioNV is compatible with one or more disease triggers of B-cell lymphoma, which in non-limiting embodiments cause one or more of the following diseases: peripheral T-cell lymphoma, aggressive NK leukemia, gastric T-cell lymphoma, T-lymphoma, ALK+ anaplastic large cell lymphoma, ALK+ anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, panniculitis T-cell lymphoma, gamma delta T-cell lymphoma, hepatosplenic T-cell lymphoma, lymphoblastic lymphoma, and lymphoblastic leukemia. In various embodiments, the BioNV delivery target may be any of the following: (TEMBHARE et al., "Critical Role of Flow Cytometer Immunophenotyping in the Diagnosis, Subtyping, and Staging of T-Cell / NK-Cell Non-Hodgkin's Lymphoma in Real-world Practice: A Study of 232 CASes from a Tertiary Cancer Center in India," Front Oncol, Vol. 12, No. 779230, 2022: pp. 1-22) and (NIRMAL, "Diagnosis of malignant lymphoma—An Overview," J Oral Maxilofac As described in Pathol. Vol. 24, No. 2, 2020: pp. 195-199), the antigens are one or more of CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD16, CD25, CD26, CD30, CD38, CD43, CD56, CD57, CD68, TCRα / β, TCRγ / δ, CD94, CD163, CD185 (CXCR5), CD279 (PD-1), CD278 (ICOS), TCL1, ALK1, TCR-Vβ, and TRBC1.

[0103] In various embodiments, BioNV is compatible with one or more disease triggers of T-cell / NK lymphoma, which in non-limiting embodiments cause one or more of the following: diffuse large B-cell lymphoma, histiocytic-rich large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, intravascular large B-cell lymphoma, and Burkitt's lymphoma. In various embodiments, the BioNV delivery target is one or more of LCA (CD45), AE1 / AE3, S100 proteins, vimentin, CD30, CD43, EMA, CD20, bcl-2, CD3, CD10, CD5, CD19, Pax-5, TdT, CD79a, Oct-2, BOB.1, Bcl-1, Bcl-2, Bcl-6, MUM1, CD138, ALK, CD21, or CD35, for example, but not limited to, as described in (NIRMAL, 2020).

[0104] In various embodiments, BioNV is compatible with one or more disease triggers of Hodgkin's lymphoma, which in non-limiting embodiments causes one or more of lymphocyte-depleted Hodgkin's lymphoma, classical Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, T-cell-enriched B-cell lymphoma, and leukemic non-Hodgkin's lymphoma (NHL). In various embodiments, BioNV delivery targets are one or more of CD5, CD10, CD15, CD20, CD23, CD30, CD43, CD44, bcl-2, bcl-6, CD19, CD20, CD40, CD79a, CD138, bcl-6, PAX5, and MUM1 CD15, CD30, CD57, EBV-LMP, CD21, CD35, as described, for example, but not limited to, in (NIRMAL, 2020) and (TZANKOV, et al. "Expression of B-Cell Markers in Classical Hodgkin Lymphoma: A Tissue Microarray Analysis of 330 Cases," Nat Mod Pathol, Vol. 16, 2003: pp. 1141-1147).

[0105] In various embodiments, BioNV is adapted to one or more disease triggers of non-specific lymphoma. In non-limiting embodiments, such disease causes lymphocyte depletion lymphoma. In various embodiments, the BioNV delivery target is one or more of TdT, CD163, or CD68, for example, but not limited to, as described in (NIRMAL, 2020).

[0106] In various embodiments, BioNV is compatible with one or more disease triggers of leukemia, which in non-limiting embodiments cause one or more of acute lymphocytic leukemia, acute myeloid leukemia, acute monoblastic leukemia, acute erythroblastic leukemia, acute megakaryocytic leukemia, chronic lymphocytic leukemia, or leukemic non-Hodgkin's lymphoma (NHL). In various embodiments, the BioNV delivery target is one or more of CD117, CD33, CD123, CLL1, TIM3, CD244, CD47, CD96, CD157, CD7, CD34, CD38, CD45, CD13, CD15, CD11b, c-kit, HLA-DR, CD116, and CD16, as described, for example and without limitation, in (HAUBNER et al., "Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML," Leukemia Vol. 33, 2019: pp. 64-74) and (INOUE, et al., "Multicolor Analysis of Cell Surface Marker of Human Leukemia Cell Lines Using Flow Cytometry," Anticancer Research, Vol. 34, No. 8, 2014: pp. 4539-4550).

[0107] In various embodiments, BioNV is matched to one or more disease triggers of hematopoietic tumors, such as, in non-limiting embodiments, one or more of Castleman's disease, myelodysplastic syndrome, or multiple myeloma. In various embodiments, the BioNV delivery target is, for example, but not limited to, one or more of CD20, VEGF-R, IL-6R, MUM1, CD138, CD20, or EBV-LMP, as described in (CARBONE, et al., "Castleman disease," Nat Rev Dis Primers, Vol. 7, No. 84, 2021: pp. 1-18).

[0108] In various embodiments, BioNV is compatible with one or more disease triggers of solid tumors or localized malignancies, which in non-limiting embodiments cause one or more of Werms' tumor, germ cell tumors, lung cancer, prostate cancer, hepatocellular carcinoma, colon malignancies, or cervical squamous cell carcinoma. In embodiments, BioNV delivery targets include CTLA-4, PD-L1, PD-L2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, AFP, mesothelin, ER, PR, HER-2 / neu, EGFR, KRAS, UGT1A1, c-kit, CD30, PDGFR, TEM8, EIIIB, CA-125, CD2, CD9, CD11b, CD18, CD19, CD20, CD21, CD22, CD24, CD30, CD31, CD32a, CD38, CD41, CD4 2b, CD43, CD44, CD48, CD51, CD54, CD61, CD62P, CD63, CD66a, CD66b, CD66c, CD66e, CD74, CD87, CD96, CD98, CD105, CD106, CD107a, CD111, CD112, CD117, CD126, CD130, CD138, CD140b, CD143, CD144, CD146, CD147, CD166, CD171, CD202b, CD208, CD221, CD227, CD239, CD309, CD321, CD324, CD326, CD333, CD340, CLEC-2, CD147, or GPVI.

[0109] The therapeutic methods herein, in embodiments, utilize disease-adapted BioNVs to target one or more diseased cells associated with the virus that causes HLH or an HLH-associated condition.

[0110] In various embodiments, BioNV is compatible with one or more disease triggers of Epstein-Barr virus (EBV), which, for example, in a non-limiting embodiment, is the causative agent of T / NK cell lymphoproliferative disorder. In various embodiments, the BioNV delivery target is one or more of CD19, CD21, CD27, CD38, IRF4, EBNA3A, EBNA3B, EBNA3C, LMP2-A, EBNA-1, LMP-1, BZLF1 (RAK), or BMLF1 (GLC), as described, for example and without limitation, in (FOURNIER et al., "Rapid identification and characterization of infected cells in blood during chronic active Epstein-Barr virus infection," J Exp Med, Vol. 217, No. 11, 2020: pp. 1-20) and (HATTON et al., "The interplay between Epstein-Barr virus and B lymphocytes: implications for infection, immunity, and disease," Immunol Res, Vol. 58 No. 2-3, 2014: pp. 268-76).

[0111] In various embodiments, BioNV is compatible with one or more disease triggers of HIV (e.g., HIV-1 or HIV-2). HIV is, for example, in non-limiting embodiments, the causative agent of Kaposi's sarcoma, AIDS-related lymphoma, and cervical cancer. In various embodiments, BioNV delivery targets are one or more of Siglec-1, CD2, CD3, CD4, CCR5, CD20, CD25, CD30, CD32a, CD69, CD91, CD160, CD257, LAG-3, CD147, CD231, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, BTK, cyclophilin B, Sec62, Rab10, or SPCS.

[0112] In various embodiments, BioNV is compatible with one or more disease triggers of cytomegalovirus (CMV), which, in non-limiting embodiments, is the causative agent of various cancers, including breast cancer, colon cancer, prostate cancer, rhabdomyosarcoma, hepatocellular carcinoma, salivary gland tumors, and neuroblastoma. In various embodiments, the BioNV delivery target is one or more of, for example, but not limited to, KLRG1, CD44, CD69, CD103, CD62L, CD127, CD27, CD28, IL-2, TNF-α, IL-6, CRP, CD-57, PD-1, CD95, CXCR3, CD45, or CCR7, as described in (VAN DEN BERG et al., "Latent CMV Infection Is Associated With Lower Influenza Virus-Specific Memory T-Cell Frequencies, but Not With an Impaired T-Cell Response to Acute Influenza Virus Infection," Front. Immunol., Vol. 12, No. 663664, 2021: pp. 1-15).

[0113] In various embodiments, BioNV is compatible with one or more disease triggers of human T-cell lymphotropic virus 1 (HTLV) (e.g., HTLV-1), which, for example, in non-limiting embodiments, is a causative agent of various cancers, including T-cell lymphoma, chronic myeloid leukemia, bladder cancer, biliary tract cancer, esophageal cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, glioblastoma, and pancreatic cancer. In various embodiments, the BioNV delivery target is one or more of IL-2, PTPRC, CD45R, CD69, CD80, B7-1, TNFRSF18, GITR, TNFRSF7, CD27, TNFRSF, 4-1BB, TNFSF9, 4-1BBL, CCR7, EBI1, IL2RAc, CD25, IL7R, or CD127, as described, for example but not limited to, (KRESS, et al., "Cell surface markers in HTLV-1 pathogenesis," Viruses, Vol. 3, No. 8, 2011: pp. 1439-59).

[0114] In various embodiments, BioNV is compatible with one or more disease triggers of the dengue virus (DENV). DENV is a causative agent of various cancers, such as, for example, in non-limiting embodiments, B-cell lymphoma and myeloid lymphoma. In various embodiments, BioNV delivery targets are, for example, but not limited to, one or more of CD69, HLA-DR, CD38, TIA-1, CD44, CD11a, VEGFRI, VEGFRII, CD244, ICAM, or VCAM, as described in (JOHN et al., "Biomarkers of severe dengue disease—a review," J. Biomed. Sci., Vol. 22, No. 83, 2015: pp. 1-7).

[0115] In various embodiments, BioNV is compatible with one or more disease triggers of the Japanese encephalitis virus (JEV), which, for example, in non-limiting embodiments, is a causative agent of various cancers, including B-cell lymphoma and myeloid lymphoma. In various embodiments, the BioNV delivery target is one or more of CD274, NS1, CX3CR1, PLVAP, or GKN3, as described, for example, but not limited to, (MURKHERJEE et al., "PLVAP and GKN3 Are Two Critical Host Cell Receptors Which Facilitate Japanese Encephalitis Virus Entry Into Neurons," Nat Sci Rep, Vol. 8, No. 11784, 2018: pp. 1-16) and (LANNES et al., "CX3CR1-CX3CL1-dependent cell-to-cell Japanese encephalitis virus transmission by human microglial cells," Nat Sci Rep, Vol. 9, No. 4833, 2019: pp. 1-13).

[0116] In various embodiments, the BioNV is compatible with one or more disease triggers of West Nile Virus (WNV). WNV is a causative agent of various cancers, including, for example, in non-limiting embodiments, B-cell lymphoma and myeloid lymphoma. In various embodiments, the BioNV delivery target is one or more of NS4B, RLR, CD11c, CD80, CD86, CD40, HLA-DR, or CD38, as described, for example, but not limited to, in (Zimmerman et al., "West Nile Virus Infection Blocks Inflammatory Response and T Cell Costimulatory Capacity of Human Monocyte-Derived Dendritic Cells," J. Virol., Vol. 93, No. 23, 2019: pp. e00664-19).

[0117] In various embodiments, BioNV is compatible with one or more disease triggers of human papillomavirus (HPV). HPV-Head and Neck is, for example, in non-limiting embodiments, a causative agent of various cancers, such as cervical cancer, as well as cancers of the vulva, vagina, penis, anus, and oropharynx. In various embodiments, BioNV delivery targets are, for example, but not limited to, one or more of CD3, CD4, CD8, FoxP3, CD25, CD95, MICA / B, CD39, CD73, NKp30, NKp46, CD44, CD24, NKG2A, CTLA-4, CD68, CD45, or CD57, as described in (LITWIN et al., "Infiltrating T-cell markers in cervical carcinogenesis: a systematic review and meta-analysis," Br J Cancer, Vol. 124, 2021: pp. 831-841).

[0118] In various embodiments, BioNV is compatible with one or more disease triggers of metabolic diseases linked to HLH and HLH-related diseases, including Fallmann's disease, galactosemia, Gaucher disease type 1, galactosialidosis, methylmalonic aciduria, Shaheen syndrome, mevalonic aciduria, biotin deficiency, LCHAD deficiency, lysinuric protein intolerance and / or orotic aciduria, multisulfatase deficiency and / or metachromatic leukodystrophy, propionic acidemia and / or multicarboxylase deficiency.

[0119] In various embodiments, BioNV is tailored to one or more disease triggers of autoinflammatory diseases associated with HLH and HLH-related diseases, including histiocytic glomerulopathy and subacute thrombotic microangiopathy.

[0120] The difference in BioNV marker-disease pairings is, in embodiments, based on age and / or disease severity.

[0121] Due to the promiscuous nature of the presence of biomarkers in the diagnosis of HLH and HLH-related diseases (e.g., viral infection, cancer, autoimmune, autoinflammatory, etc.), the BioNVs used in the therapeutic methods herein, in embodiments, are targeted to one or more cell surface markers listed in Table 1 (e.g., to target mutations in Tables 2-5) for the treatment of any of the diseases described herein. For example, in embodiments, the BioNVs have a bispecific CAR that targets two or more cell surface markers in Table 1, e.g., from one or more disease subsets.

[0122] Administration of BioNV, in embodiments, provides highly specific and targeted delivery of perforin, granzymes, p53, and / or caspases to mutant, diseased, and / or transformed cells, replacing and / or complementing endogenous dysfunctional perforin / granzyme-mediated cell apoptosis. In embodiments, administration of BioNV prevents one or more toxicities and / or side effects associated with current therapies (e.g., excessive inflammation, off-target effects, nonspecific activation of T cells, etc.).

[0123] The granzyme, in embodiments, is selected from granzymes A, B, H, K, and M. In embodiments, the delivered protein / nucleic acid (e.g., perforin, one or more granzyme proteins, and / or caspases) comprises one or more modifications. In embodiments, exemplary, non-limiting modifications include one or more of an amino acid variant, a fusion, the addition of a fluorescent label, PEGylation, and / or a post-translational modification (PTM). Such modifications, in embodiments, are used to modulate the biochemical function of perforin and / or a granzyme, e.g., altering half-life, serum stability, catalytic activity, etc. In embodiments, an "amino acid mutation," as used herein, refers to one or more amino acid substitutions, additions, deletions, or variants (e.g., resulting from SNPs, etc.). For example, in a non-limiting embodiment, BioNV herein can encapsulate perforin and / or granzyme, or a nucleic acid encoding perforin and / or granzyme, which contains one or more amino acid substitutions, additions, deletions, or mutations intended to reduce protease susceptibility, increase or decrease half-life, improve kinetics (e.g., granzyme B cleavage of procaspase-3), or improve Ca2+ retention (e.g., in the case of perforin).

[0124] The perforin and / or granzyme variants used in the methods herein are, in various embodiments, engineered to have reduced activity levels to prevent off-target effects. In various embodiments, the variants have activity levels of about or at least about 10% of the wild-type perforin and / or granzyme activity level, about or at least about 20% of the wild-type perforin and / or granzyme activity level, about or at least about 30% of the wild-type perforin and / or granzyme activity level, about or at least about 40% of the wild-type perforin and / or granzyme activity level, or about or at least about 50% of the wild-type perforin and / or granzyme activity level. 0%, about or at least about 60% of the activity level of wild-type perforin and / or granzyme, about or at least about 70% of the activity level of wild-type perforin and / or granzyme, about or at least about 70% of the activity level of wild-type perforin and / or granzyme, about or at least about 80% of the activity level of wild-type perforin and / or granzyme, about or at least about 90% of the activity level of wild-type perforin and / or granzyme, or about or at least about 100% of the activity level of wild-type perforin and / or granzyme.

[0125] Perforin generally requires calcium (Ca2+) binding to be activated, which typically occurs during endosomal acidification during uptake and release. BioNVs for perforin delivery are, in embodiments, engineered so that the encapsulated perforin is Ca2+ bound (e.g., prepared with a Ca2+ concentration similar to that of acidified endosomes).

[0126] In some embodiments, BioNV encapsulates and / or delivers the tumor suppressor p53 to target cells. p53, in some embodiments, includes higher-order forms of p53 (e.g., homotetrameric forms) in addition to monomeric forms. In some embodiments, the p53 protein comprises one or more structure-function features of a protein, including, from N- to C-terminus, a transactivation domain, a proline-rich domain, a DNA-binding domain, a nuclear localization sequence (NLS), a tetramerization domain, a nuclear export sequence (NES), and a basic domain. In some embodiments, the p53 is a wild-type form or a variant p53 having one or more substitutions relative to the wild-type sequence.

[0127] In some embodiments, BioNV encapsulates and delivers to target cells perforin, one or more granzymes (e.g., A, B, H, K, or M), and p53. In some embodiments, BioNV encapsulates and delivers to target cells perforin, one or more granzymes (e.g., A, B, H, K, or M), p53, and p53 caspase. In some embodiments, the combination of two or more of these proteins results in efficient, targeted apoptosis of target cell subsets in diseased subjects.

[0128] Nucleic acid delivery, in embodiments, includes delivery of one or more nucleic acids comprised of DNA and / or RNA. RNA, in embodiments, includes mRNA (e.g., transcripts for expression of perforin and / or granzymes) and / or circular RNA (e.g., for increased cytosolic half-life). mRNA, in embodiments, is modified mRNA. DNA, in embodiments, includes a plasmid or vector suitable for delivery to cells for expression of perforin and / or granzymes.

[0129] In various embodiments, the methods herein include delivering one or more caspase proteins and / or one or more nucleic acids encoding one or more caspase proteins. In various embodiments, BioNV is administered for highly targeted apoptosis of cells contributing to a disease state, and caspases are delivered along with perforin and / or granzymes to help enhance the intrinsic perforin / granzyme apoptotic pathway. In various embodiments, delivery of one or more caspase proteins increases the rate of the caspase cascade in one or more target cells; for example, without limitation, BioNV is configured to deliver granzyme B (GzmB) along with caspase-3, since GzmB cleaves caspase-3 to activate the caspase apoptotic pathway. In such embodiments, GzmB and / or caspase-3 can be provided as mRNA to prevent substrate cleavage prior to delivery.

[0130] In various embodiments, the one or more caspase proteins include caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, and / or any pro-caspases related thereto.

[0131] In various embodiments, the methods of treatment herein include administering one or more additional therapeutic agents (such as second-line therapeutic agents).

[0132] In various embodiments, the one or more additional therapeutic agents are one or more cytokine therapy reagents, immunosuppressants, anti-infective agents, monoclonal antibodies, and / or analgesics.

[0133] In some embodiments, the therapeutic method utilizes BioNVs targeted to at least a portion of one or more lymphoid biomarkers. In some embodiments, administering the BioNVs targets the entrapped payload to target cells expressing a target that is or includes one or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3. In some embodiments, the cell surface target is CD34. In some embodiments, the cell surface target is CD56 (e.g., NK cells or cell subsets). In embodiments, BioNVs are targeted to portions of any cell surface marker described herein. In embodiments, BioNVs are targeted to two or more biomarkers (e.g., in the case of bispecific CARs, or multiple targeting agents). In embodiments, BioNVs are targeted to one or more cell surface markers of NK cells, T cells, macrophages, monocytes, dendritic cells, granulocytes / polymorphonuclear leukocytes (i.e., neutrophils, basophils, eosinophils), among other immune cell types and subsets.

[0134] In various embodiments, the treatment method involves BioNV containing a payload that is targeted to target cells using a disease-matched gene editing payload, for example according to the mutations listed in Tables 2-5. Table 2: Exemplary BioNV gene editing targets and / or gene replacement candidates for treating HLH and HLH-associated diseases. [Table 2] JPEG2025538384000007.jpg246159JPEG2025538384000008.jpg137159

[0135] In some embodiments, the therapeutic methods herein utilize a gene editing payload adapted to the disease. In some embodiments, the disease is HLH, and the gene editing payload targets a mutant RHOG gene. In some embodiments, the disease is FLH4, and the gene editing payload targets a mutant STX11 gene. In some embodiments, the disease is FLH2, and the gene editing payload targets a mutant PRF1 gene. In some embodiments, the disease is FLH3, and the gene editing payload targets a mutant UNC13D gene. In some embodiments, the disease is FLH5, and the gene editing payload targets a mutant STXBP2 gene.

[0136] In embodiments, the disease is Grischelli syndrome type 2 and the gene editing payload targets a mutant RAB27A gene. In embodiments, the disease is Chediak-Higashi syndrome and the gene editing payload targets a mutant LYST gene.

[0137] In embodiments, the disease is PID and / or EBV, and the gene editing payload targets one or more of a mutant CD27 gene, an ITK gene, a MAGT1 gene, a RASGRP1 gene, a STAT1 gene, a STAT2 gene, an IL7RA gene, or a RAG1 / 2 gene.

[0138] In embodiments, the disease is XLP-1 / EBV and the gene editing payload targets a mutant SH2D1A gene. In embodiments, the disease is immunodeficiency 24 (IMD24) and the gene editing payload targets a mutant CTPS1 gene. In embodiments, the disease is immunodeficiency 27A / B (IMD27A / B) and the gene editing payload targets a mutant IFNGR1 / 2 gene. In embodiments, the disease is immunodeficiency 18 (IMD18) and / or EBV and the gene editing payload targets a mutant CD3E gene. In embodiments, the disease is Wiskott-Aldrich syndrome and the gene editing payload targets a mutant WAS gene. In embodiments, the disease is immunodeficiency 9 (IMD9) and the gene editing payload targets a mutant ORAI1 gene. In embodiments, the disease is X-linked agammaglobulinemia (XLA) and the gene editing payload targets a mutant BTK gene. In embodiments, the disease is X-linked chronic granulomatous disease (CGDX) and the gene editing payload targets a mutant CYBB / CYBA / NCF1 gene.

[0139] In embodiments, the disease is Wolman disease and the gene editing payload targets a mutant LIPA gene; the disease is galactosemia I and the gene editing payload targets a mutant GALT gene. In embodiments, the disease is Gaucher disease type 1 and the gene editing payload targets a mutant GBA gene. In embodiments, the disease is galactosialidosis and the gene editing payload targets a mutant CTSA gene. In embodiments, the disease is methylmalonic aciduria and the gene editing payload targets a mutant MUT gene. In embodiments, the disease is Shaheen syndrome and the gene editing payload targets a mutant COG6 gene. In embodiments, the disease is mevalonic aciduria and the gene editing payload targets a mutant MVK gene.

[0140] In embodiments, the disease is biotin deficiency and the gene editing payload targets a mutant BTD gene. In embodiments, the disease is LCHAD deficiency and the gene editing payload targets a mutant HADHA gene. In embodiments, the disease is multisulfatase deficiency and metachromatic leukodystrophy and the gene editing payload targets a mutant SUMF1 gene. In embodiments, the disease is propionic acidemia and multicarboxylase deficiency and the gene editing payload targets a mutant PCCA / PCCB gene.

[0141] In embodiments, the disease is lysinuric protein intolerance and orotic aciduria, and the gene editing payload targets a mutant SLC7A7 gene. In embodiments, the disease is lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, and the gene editing payload targets a mutant XIAP gene. In embodiments, the disease is Takeuchi-Kosaki syndrome and severe multisystem autoinflammatory disease of neonatal onset, and the gene editing payload targets a mutant CDC42 gene. In embodiments, the disease is familial cold autoinflammatory syndrome 4 and autoinflammation with infantile enterocolitis, and the gene editing payload targets a mutant NLRC4 gene.

[0142] In various embodiments, the gene editing strategy utilizes CRISPR-mediated repetitive single base pair mutation repair (CMSBpMutR), which is used in a variety of applications, including, but not limited to, (GAO, et al. "Optimized CRISPR / Cas9-mediated single nucleotide mutation in adherent cancer cell lines," STAR Protoc, Vol. 2, No. 2, 2021: 100419), (USHER, et al. "Optimizing CRISPR / Cas9 Editing of Repetitive Single Nucleotide Variants," Front Genome Ed. Vol. 4, No. 932434, 2022: pp. 1-11), (KOMOR et al. "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature. Vol. 533, No. 7603, 2016: pp. 420-4), and (KIM, et al. "Increasing the genome-targeting scope and precision of base “Cytidine deaminase fusions,” Nat Biotechnol, Vol. 35, No. 4, 2017, pp. 371-376.

[0143] In various embodiments, the gene editing strategy utilizes CRISPR-mediated codon or triple base pair mutation repair (CTrMR), as described, for example, but not by way of limitation, in (XU et al. "CRISPR / Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1 / ABCC1 and Restores Its Proper Folding and Function," Mol Ther Nucleic Acids, Vol. 7, 2017: pp. 429-438).

[0144] Gene editing strategies may, in embodiments, utilize CRISPR-mediated sticky-end non-homologous end joining (S-NHEJ), CRISPR-mediated blunt-end non-homologous end joining (B-NHEJ), CRISPR-mediated homology-directed repair (HDR), and / or homology-directed repair (HDR) coupled gene replacement (HDR / CGR).

[0145] In some embodiments, the strategy for correcting multiple mutations in a single gene involves excising the mutated region of the gene followed by a HDR-coupled gene replacement strategy. In some embodiments, the strategy for correcting multiple mutations in multiple genes associated with HLH (such as UNC13B) involves excising each mutated gene followed by a HDR-coupled gene replacement strategy for multiple genes. In some embodiments, the strategy for correcting multiple genes with a single mutation in each gene utilizes a multi-gRNA approach to correct mutations, which may include any one of the gene editing strategies described herein.

[0146] The methods herein, in embodiments, include a BioNV gene editing approach to target the PRF1 gene to treat FHL2 (e.g., as shown in Table 3). Table 3 shows an exemplary, non-exhaustive list of PRF1 mutations arranged in descending order of mutation frequency in FLH2 patients, as described in (GADOURY-LEVESQUE, et al. "Frequency and spectrum of disease-causing variants in 1892 patients with suspected genetic HLH disorders," Blood Adv. Vol. 4, No. 12, 2020: pp. 2578-2594). In embodiments, methods of targeting PRF1 include correcting mutations occurring in the PRF1 gene (e.g., located in exons, introns, and other non-coding regions, such as promoters or promoter enhancers) to correct perforin dysfunction. For example, in embodiments, the BioNV gene editing payload delivers a single gRNA that targets common mutations occurring in the PRF1 gene (e.g., in the case of single gene mutations / variants), or delivers multiple gRNAs that target several common mutations (e.g., in the case of bigenic mutants / variants), and / or delivers partial or total functional gene replacements of PRF1 that are compatible with HDR to target a wider range of common mutations. Table 3: Exemplary mutations in the PRF1 gene associated with FHL2 and corresponding exemplary gene editing strategies for each mutation (GADOURY-LEVESQUE, et al. 2020). CRISPR-mediated iterative single base pair mutation repair (CMSBpMutR); CRISPR-mediated codon or triple base pair mutation repair (CTrMR); CRISPR-mediated sticky-end non-homologous end joining (S-NHEJ); CRISPR-mediated blunt-end non-homologous end joining (B-NHEJ); CRISPR-mediated homology-directed repair (HDR); HDR-coupled gene replacement (HDR / CGR); and membrane attack complex / perforin family (MACPF). [Table 3] TIFF2025538384000010.tif231159TIFF2025538384000011.tif238159TIFF2025538384000012.tif92159

[0147] The methods herein, in embodiments, include a BioNV gene editing approach to target the UNC13D gene to treat FHL3 (e.g., as shown in Table 4). Table 4 shows an exemplary, non-exhaustive list of UNC13D mutations, sorted by descending mutation frequency in FLH3 patients, as described in (GADOURY-LEVESQUE et al., 2020). In embodiments, methods of targeting UNC13D include correcting mutations occurring in the UNC13D gene (e.g., located in exons, introns, and other non-coding regions, such as promoters or promoter enhancers) to modify Munc13-4 protein activity in perforin release. For example, in embodiments, the BioNV gene editing payload delivers a single gRNA that targets common mutations occurring in the UNC13D gene (e.g., in the case of single gene mutations / variants), or delivers multiple gRNAs that target several common mutations (e.g., in the case of bigenic mutants / variants), and / or delivers partial or total functional gene replacements of UNC13D that are compatible with HDR to target a wider range of common mutations. Table 4: Exemplary mutations in the UNC13D gene associated with FHL3 and corresponding exemplary gene editing strategies for each mutation (GADOURY-LEVESQUE, et al. 2020). CRISPR-mediated iterative single base pair mutation repair (CMSBpMutR); CRISPR-mediated codon or triple base pair mutation repair (CTrMR); CRISPR-mediated sticky-end non-homologous end joining (S-NHEJ); CRISPR-mediated blunt-end non-homologous end joining (B-NHEJ); CRISPR-mediated homology-directed repair (HDR); HDR-coupled gene replacement (HDR / CGR). [Table 4] TIFF2025538384000014.tif238159TIFF2025538384000015.tif244159TIFF2025538384000016.tif224159

[0148] The methods herein, in embodiments, include a BioNV gene editing approach to target the STXBP2 gene to treat FHL5 (e.g., as shown in Table 5). Table 5 shows an exemplary, non-exhaustive list of STXBP2 mutations sorted in descending order of mutation frequency in FLH5 patients, as described in (GADOURY-LEVESQUE et al., 2020). In embodiments, methods of targeting STXBP2 include correcting mutations occurring in the STXBP2 gene (e.g., located in exons, introns, and other non-coding regions, such as promoters or promoter enhancers) to modify perforin release. For example, in embodiments, the BioNV gene editing payload delivers a single gRNA that targets common mutations occurring in the STXBP2 gene (e.g., in the case of single gene mutations / variants), or delivers multiple gRNAs that target several common mutations (e.g., in the case of bigenic mutants / variants), and / or delivers partial or total functional gene replacements of STXBP2 that are compatible with HDR to target a wider range of common mutations. Table 5: Exemplary mutations in the STXBP2 gene associated with FHL5 and corresponding exemplary gene editing strategies for each mutation (GADOURY-LEVESQUE, et al. 2020). CRISPR-mediated iterative single base pair mutation repair (CMSBpMutR); CRISPR-mediated codon or triple base pair mutation repair (CTrMR); CRISPR-mediated sticky-end non-homologous end joining (S-NHEJ); CRISPR-mediated blunt-end non-homologous end joining (B-NHEJ); CRISPR-mediated homology-directed repair (HDR); HDR-coupled gene replacement (HDR / CGR). [Table 5] TIFF2025538384000018.tif119159

[0149] The methods herein, in some embodiments, include a BioNV gene editing approach to target the SHSD1A gene to treat XLP1. In some embodiments, the SHSD1A targeting method involves correcting mutations occurring in the SHSD1A gene (e.g., located in exons, introns, and other non-coding regions, such as promoters or promoter enhancers) to correct dysfunctional SH2 domain-containing protein 1A associated with T cell and B cell proliferation. For example, in some embodiments, the BioNV gene editing payload delivers a single gRNA targeting a common mutation occurring in the SHSD1A gene (e.g., in the case of a single gene mutation / variant), or delivers multiple gRNAs targeting several common mutations (e.g., in the case of a bigenic mutant / variant), and / or delivers a partial or full functional gene replacement of SHSD1A compatible with HDR to target a wider range of common mutations.

[0150] In embodiments, methods using a BioNV gene editing approach to target the SHSD1A gene include treating HLH and HLH-related diseases associated with EBV infection. In embodiments, the BioNV carrying a gene editing payload targeting SHSD1A comprises one or more targeting moieties for EBV-infected cells. The BioNV used in the methods described herein, in embodiments, comprises an activated surface-directed CAR.

[0151] The methods herein, in embodiments, include a BioNV gene editing approach to target the RAB27A gene to treat GS2. In embodiments, the method of targeting RAB27A includes correcting a mutation occurring in the RAB27A gene (e.g., located in an exon, intron, or other non-coding region, such as a promoter or promoter enhancer) to correct a dysfunctional small GTP-binding protein that is preferentially expressed in melanocytes and hematopoietic cells and is required for the exocytosis of cytotoxic granules (e.g., as described in MENASCHE et al., 2000). For example, in embodiments, the BioNV gene editing payload delivers a single gRNA that targets common mutations occurring in the RAB27A gene (e.g., in the case of single gene mutations / variants), or delivers multiple gRNAs that target several common mutations (e.g., in the case of bigenic mutants / variants), and / or delivers partial or full functional gene replacements of RAB27A that are compatible with HDR to target a wider range of common mutations.

[0152] The methods herein, in embodiments, include administering a BioNV containing a corrective gene-editing payload to an adult, for example, but not limited to, where prospective parents are screened for the presence of a mutation / variant in HLH and HLH-associated disease and the mutation / variant is corrected before conception. The therapeutic methods herein, in embodiments, include administering a BioNV containing a corrective gene-editing payload to a newborn or young child at the time of or prior to the onset of HLH and HLH-associated disease. The therapeutic methods herein, in embodiments, include administering a BioNV containing a corrective gene-editing payload to an adult who has accumulated a genetic abnormality that leads to HLH and HLH-associated disease, or treating an adult who is likely to be infected with HLH prior to the onset of symptoms. In embodiments, the therapeutic methods described herein include administering a BioNV prior to the clinical manifestation of symptoms.

[0153] In various embodiments, administration of BioNV in the therapeutic methods herein restores functional perforin protein expression and / or exocytosis. In various embodiments, functional perforin protein expression is at or above about 10% expression level, at or above about 20% expression level, at or above about 20% expression level, at or above about 30% expression level, at or above about 40% expression level, at or above about 50% expression level, at or above about 60% expression level, at or above about 70% expression level, at or above about 80% expression level, at or above about 90% expression level, at or above about 100% expression level, or at or above about 100% expression level, compared to wild-type expression levels in healthy subjects. An expression level of at least about 100%, an expression level of about 110% or at least about 110%, an expression level of about 120% or at least about 120%, an expression level of about 130% or at least about 130%, an expression level of about 140% or at least about 140%, an expression level of about 150% or at least about 150%, an expression level of about 160% or at least about 160%, an expression level of about 170% or at least about 170%, an expression level of about 180% or at least about 180%, an expression level of about 190% or at least about 190%, and an expression level of about 200% or at least about 200%.

[0154] In various embodiments, the exocytosis of functional perforin protein is at or above about 10% exocytosis level, at or above about 20% exocytosis level, at or above about 20% exocytosis level, at or above about 30% exocytosis level, at or above about 40% exocytosis level, at or above about 40% exocytosis level, at or above about 50% exocytosis level, at or above about 60% exocytosis level, at or above about 60% exocytosis level, at or above about 70% exocytosis level, at or above about 80% exocytosis level, at or above about 90% exocytosis level, at or above about 100% exocytosis level, or the like, as compared to the exocytosis level of wild-type perforin in a healthy subject. includes an exocytosis level of at least about 100%, about 110% or at least about 110%, about 120% or at least about 120%, about 130% or at least about 130%, about 140% or at least about 140%, about 150% or at least about 150%, about 160% or at least about 160%, about 170% or at least about 170%, about 180% or at least about 180%, about 190% or at least about 190%, and about 200% or at least about 200% exocytosis level.

[0155] In various embodiments, restored perforin function may be about 10% or at least about 10% of the activity level, about 20% or at least about 20% of the activity level, about 30% or at least about 30% of the activity level, about 40% or at least about 40% of the activity level, about 50% or at least about 50% of the activity level, about 60% or at least about 60% of the activity level, about 70% or at least about 70% of the activity level, about 80% or at least about 80% of the activity level, about 90% or at least about 90% of the activity level, about 100% or at least about 100% of the activity level, or at least about 100% of the activity level, compared to the level of wild-type perforin activity and / or function in a healthy subject. or at least about 100% activity level, about 110% or at least about 110% activity level, about 120% or at least about 120% activity level, about 130% or at least about 130% activity level, about 140% or at least about 140% activity level, about 150% or at least about 150% activity level, about 160% or at least about 160% activity level, about 170% or at least about 170% activity level, about 180% or at least about 180% activity level, about 190% or at least about 190% activity level, and about 200% or at least about 200% activity level.

[0156] The therapeutic methods herein, in various embodiments, include administering one or more additional therapeutic agents, in various embodiments, the one or more additional therapeutic agents are one or more cytokine therapy reagents, immunosuppressants, anti-infective agents, monoclonal antibodies, and / or analgesics.

[0157] The methods herein, in embodiments, utilize allogeneic BioNV gene editing payloads (or therapeutic protein payloads) that are tunable as a function of population-based genetic determinants to precisely treat HLH and HLH-associated diseases, for example, but not limited to, in relation to gender, race, geographic location, etc., including, but not limited to, (CHEN, et al. "Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: Molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP," Clin Genet, Vol. 94, No. 2, 2018: pp. 200-212), (CETICA, et al. "Genetic predisposition to hemophagocytic lymphohistiocytosis: Report on 500 patients from the Italian registry," J Allergy Clin Immunol,Vol.137,No.1,2016:pp.188-196), (CHINN,et al.“Genetic and mechanistic diversity in pediatric hemophagocytic lymphohistiocytosis,”Blood,Vol.132,No.1,2018:pp.89-100),(ZUR STADT,et al.“Mutation spectrum in children with primary hemophagocytic lymphohistiocytosis: molecular and functional analyzes of PRF1,UNC13D,STX11,and RAB27A,”Hum Mutat,Vol.27,No.1,2006:pp.62-8), (AMMANN,et al.“HLH study of the GPOH.“遗传分析的有效免疫指导,包括对噬血细胞性淋巴组织细胞增生症患者进行外显子组测序”,《临床免疫学杂志》,第37卷,第8期,2017年:第770 - 780页)、(卢等人,“噬血细胞性淋巴组织细胞增生症患儿穿孔素基因突变”,《中华医学杂志》,第122卷,第23期,2009年:第2851 - 2855页)、(李等人,“非洲裔噬血细胞性淋巴组织细胞增生症患者与具有50delT突变的PRF1共同单倍型”,《儿科学杂志》,第149卷,第1期,2006年:第134 - 137页)、(施等人,“家族性噬血细胞性淋巴组织细胞增生症2型中的PRF1基因突变:一例家族报告及文献综述”,《药物基因组学与个人医学》,第14卷,2021年:第1637 - 1645页)、(哈齐克等人,“婴儿急性肝衰竭中噬血细胞性淋巴组织细胞增生症的高患病率”,《儿科学杂志》,S0022 - 3476(22),2022:00625 - 4)以及(毕等人,“一名中国女性新生儿的家族性噬血细胞性淋巴组织细胞增生症2型:一例病例报告及文献综述”,《世界临床病例杂志》,第9卷,第21期,2021年:第6056-6066).

[0158] In various embodiments, patient choice and / or medical history will determine the gene editing payload used, the cell surface markers targeted, the BioNV surface expression profile, the BioNV dosage, and / or the concomitant therapeutic agents (if any).

[0159] Biomimetic nanovesicles (BioNVs) for treating HLH and HLH-related diseases In aspects, the disclosure includes a BioNV comprising a targeting agent targeted to at least a first cell surface marker associated with hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease, wherein the BioNV is configured to deliver either a gene editing payload targeted to one or more mutations associated with HLH as described herein, or one or more of perforin, granzyme, caspase, and / or p53 proteins, and / or nucleic acids encoding one or more thereof.

[0160] In various embodiments, BioNVs used in the methods herein are derived from hypoimmunogenic modified cells, such as stem cells, induced pluripotent stem cells (iPSCs), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any of these modified cells. In various embodiments, the modified cells are iPSCs. In various embodiments, the iPSCs are differentiated into cells that produce perforin and / or granzymes. In various embodiments, the differentiated cells are myeloid cells (e.g., macrophages, monocytes, neutrophils, etc.). In various embodiments, the differentiated cells are lymphoid cells (e.g., T cells, helper T cells, T memory cells, NK cells, etc.), such as, for example, but not limited to, those described in (Wang, et al., "3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR T cells," Cell Stem Cell. Vol. 29, 2022: pp. 515-527). In some embodiments, BioNVs are produced from γδ T cells. In some embodiments, BioNVs inherit the barrier-crossing functionality of the cells from which they are derived, for example, but not limited to, the blood-brain barrier-crossing functionality of macrophage or monocyte origin. This, in some embodiments, enables BioNVs to penetrate tissues and target cells that may contribute to HLH or HLH-associated diseases, such as immune cells that require perforin expression and / or exocytosis.

[0161] In embodiments, BioNVs are generated from hypoimmunogenic modified cells created by knocking out, silencing, inactivating, blocking, or otherwise abolishing the expression, transcriptional efficiency, and / or activity of one or more immunogenic molecules. In embodiments, the hypoimmunogenic modified cells, and BioNVs derived therefrom, substantially lack one or more MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, and / or CRS proteins.

[0162] In various embodiments, BioNVs are generated from hypoimmunogenic modified cells with reduced or absent expression and / or activity of beta2-macroglobulin (B2M) protein and / or expression and / or activity of MHC class I proteins, as in the case of CD8+ T cell lineages. In various embodiments, BioNVs are generated from hypoimmunogenic modified cells with reduced or absent expression and / or activity of CIITA protein and / or expression and / or activity of MHC class II proteins, as in the case of CD4+ T cell lineages. Without wishing to be bound by theory, these proteins contribute to the immunogenicity of human leukocyte antigens (HLA), necessitating donor-recipient HLA allele matching for cell-based therapy treatment. In embodiments, allogeneic and / or low immunogenic properties are achieved by reducing or eliminating the expression and / or activity of genes encoding T cell receptor (TCR) proteins, including, for example, the α and β chains (in the case of αβ T cells) or the γ and δ chains (in the case of γδ T cells) that form the ligand binding site, and the signaling modules CD3δ, CD3γ, CD3ε, and CD3ζ. In embodiments, this is done to reduce foreign T cell receptor types other than those of the targeting agent cassette, further improving the homogeneity of the targeting agent of interest and reducing off-target effects due to BioNV administration.

[0163] In some embodiments, the presence of B2M risks preventing long-term acceptance of the BioNV by the recipient, as observed with the whole cell-based therapeutics described above, thereby reducing the number of doses that can be administered. To overcome this problem, in some embodiments, the HLA-E or HLA-G genes are left intact, allowing the immune system to adapt to the resulting BioNV. In some embodiments, HLA-A, HLA-B, HLA-C, HLA-F, and either HLA-E or HLA-G (but not both) are knocked out sequentially.

[0164] In embodiments, BioNV has reduced or eliminated HLA-A protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-B protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-C protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-E or HLA-G protein expression and / or activity. In embodiments, BioNV has reduced or eliminated HLA-F protein expression and / or activity.

[0165] In embodiments, BioNV lacks MHC class I and MHC class II complexes by knocking out (in the modified cells) key proteins involved in the expression of MHC class I and MHC class II complexes, such as β2-macroglobulin (B2M), a serum protein found in association with MHC class I heavy chains on the surface of nearly all nucleated cells and involved in peptide antigen presentation to the immune system. In embodiments, the allogeneic iPSCs have disrupted the CIITA gene, a master control transcription factor that controls the expression of all MHC II genes. In embodiments, the allogeneic iPSCs have disrupted the CIITA gene, a master control transcription factor that controls the expression of all MHC II genes, such that the resulting differentiated cell lines (e.g., DCs, mononuclear phagocytes, endothelial cells, thymic epithelial cells, B cells, etc.) do not express or have reduced expression of MHC class II proteins.

[0166] In embodiments, the BioNV has reduced or eliminated T cell alpha constant (TRAC) protein expression and / or activity. In embodiments, the BioNV has reduced or eliminated T cell beta constant (TRBC) protein expression and / or activity. In embodiments, the BioNV has reduced or eliminated PD-1 protein expression and / or activity, or the BioNV has PD-1 protein expression and / or activity.

[0167] CRS is a major concern in whole cell therapies, and despite being engineered to be less immunogenic, effector functions and other consequences of cell interactions after infusion can result in the release of biomolecules, which can lead to systemic inflammatory syndromes characterized by fever, multiple organ dysfunction, etc. In embodiments, BioNVs lack one or more proteins that contribute to CRS. In embodiments, BioNVs have reduced or abolished protein expression and / or activity of CRS-associated cytokines.

[0168] In some embodiments, the BioNV has reduced or eliminated IL-4 protein expression and / or activity. In some embodiments, the BioNV has reduced or eliminated IL-6 protein expression and / or activity. In some embodiments, the inclusion of unwanted IL-6 in the BioNV may result in the BioNV contributing to localized (and localized by biomarker targeting) and / or potentially systemic CRS events. In some embodiments, the BioNV has reduced or eliminated IL-10 protein expression and / or activity. In some embodiments, the BioNV has reduced or eliminated IL-16 protein expression and / or activity. In some embodiments, the reduction or elimination of interleukins reduces the likelihood of CRS.

[0169] In embodiments, the BioNV is derived from cells that have been modified to be less immunogenic by the expression and / or activity of one or more immunoprotective proteins that, in some embodiments, prevent or reduce an immune response in a subject, prevent or reduce early clearance of the BioNV in a subject, prevent or reduce phagocytosis, or confer barrier penetration function, such as, but not limited to, CD47, CD24, CD200, CD34, CCL2, H2-M3, MFG-E8, PD-L1 (from a non-activated cell source), CTLA-4, and the like.

[0170] Serine protease inhibitor B9 (SerpinB9) is a member of the serine protease inhibitor superfamily. In embodiments, SerpinB9 protects cells from the immune-killing effects of granzyme B. In embodiments, BioNV is not designed to deliver granzymes, and BioNV has SerpinB9 expression and / or activity. In embodiments, expression of SerpinB9 suppresses granzyme B functions associated with immunostimulatory responses, such as apoptosis of target cells and / or infected cells. Alternatively, in embodiments, BioNV is designed to deliver granzymes, and BioNV substantially lacks SerpinB9 expression and / or activity.

[0171] In some embodiments, BioNV has CD34 protein expression and / or activity. In some embodiments, BioNV has CCL2 protein expression and / or activity. In some embodiments, BioNV has PD-L1 protein expression and / or activity. In some embodiments, BioNV has H2-M3 protein expression and / or activity.

[0172] In some embodiments, BioNVs express and / or have CD47 protein activity. Exosomes and cell-derived vesicles (CDVs) are easily removed from the body by macrophages through phagocytosis. Phagocytosis significantly impacts the therapeutic benefits and efficacy of CDVs. Without wishing to be bound by theory, in some embodiments, BioNVs are CD47-tagged on their surface to prevent macrophage depletion of BioNVs. The CD47tg tag provides a "don't eat me" signal, which in some embodiments increases the half-life and serum stability of BioNVs in subjects. In some embodiments, the molecule CD47 isoform 2 (an isoform that interacts with the SIRPα receptor on macrophages) is incorporated into modified cells (e.g., iPSC cell lines). Without CD47tg, the half-life of BioNVs is shortened due to phagocytosis inhibition, resulting in the need for higher and / or more frequent administration. In various embodiments, the potential inhibitory phenotype of CD47 expression throughout the cell is prevented by interfering with the inhibitory mechanism of a series of microRNAs on the 3'UTR of the CD47 gene. This is done by deleting this region in a stable construct or eliminating / inhibiting the expression of the microRNA. In various embodiments, this can solve the problem of inhibition caused by microRNAs throughout differentiated cell subsets.

[0173] In some embodiments, BioNV has expression and / or activity of CD24 protein. CD24 is a sialoglycoprotein and anti-phagocytic protein expressed on mature granulocytes and B cells. CD24 prevents phagocytosis through interaction with Siglec-G / 10 on macrophages. In some embodiments, BioNV has expression and / or activity of a chimeric CD24 / CD47 protein. In some embodiments, BioNV expresses a chimeric CD24 / CD47 with a tethered transmembrane domain. In some embodiments, the CD47 isoform 2 and CD24 domains can be expressed separately or linked to form a bi-lobed chimeric protein. In some embodiments, BioNV is derived from iPSCs derived from fibroblasts rather than ABO cells.

[0174] In some embodiments, BioNV has expression and / or activity of CD200 protein. In some embodiments, the CD200 tag minimizes phagocytosis by macrophages and also prevents granulocyte activation. In some embodiments, when a CD47 or CD24 tag is used, or when a CD24 / CD47 chimeric bilobe protein tag (each of which prevents phagocytosis) is used in combination with overexpressed H2-M3 (which attenuates NK responses), stability is achieved without CD200 while allowing adequate BioNV clearance. In some embodiments, CD200 can be expressed to prevent granulocyte activation, while the CD47 or CD24 tag (but not both tags) can be removed. In some embodiments, BioNV has expression and / or activity of a chimeric CD24 / CD200 protein, or a chimeric CD47 / CD200 protein and / or activity.

[0175] In some embodiments, BioNV does not express and / or has the activity of all three of CD47, CD24, and CD200. In some embodiments, BioNV is engineered to be stable in a subject's body but not prevent the BioNV from being cleared from the body. BioNV that is too stable may ultimately provoke a humoral response, thereby limiting the number of doses or treatments.

[0176] In various embodiments, the BioNV has expression and / or activity of the CTLA-4 protein. In various embodiments, the BioNV has expression and / or activity of the MFG-E8 protein. In various embodiments, the BioNV has expression and / or activity of the NCAM protein. In various embodiments, the BioNV has expression and / or activity of the alpha phagocytic integrin protein. In various embodiments, the BioNV has expression and / or activity of the FasL protein.

[0177] In embodiments, the BioNV has reduced or eliminated expression and / or activity of one or more immunogenic proteins, such as proteins that result in an immune response in a subject, donor-recipient mismatch, HLA alloimmunity, inflammation, CRS, etc., including, but not limited to, MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In embodiments, the BioNV has reduced or eliminated expression and / or activity of three or more immunoprotective proteins, four or more immunoprotective proteins, five or more immunoprotective proteins, six or more immunoprotective proteins, seven or more immunoprotective proteins, eight or more immunoprotective proteins, nine or more immunoprotective proteins, ten or more immunoprotective proteins, eleven or more immunoprotective proteins, or twelve or more immunoprotective proteins.

[0178] In various embodiments, BioNV has expression and / or activity of three or more immune defense proteins, four or more immune defense proteins, five or more immune defense proteins, six or more immune defense proteins, seven or more immune defense proteins, eight or more immune defense proteins, nine or more immune defense proteins, or ten or more immune defense proteins.

[0179] In embodiments, the BioNV is allogeneic. In embodiments, the BioNV does not elicit an immune response in a subject to which it is administered.

[0180] In various embodiments, BioNV substantially lacks the proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and any one of HLA-E or HLA-G.

[0181] In various embodiments, BioNV substantially lacks the proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, PD-1, and any one of HLA-E or HLA-G.

[0182] In embodiments, BioNV substantially lacks the protein and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, PD-1, HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16.

[0183] In embodiments, a BioNV that is "substantially devoid" of protein expression and / or activity refers to a significantly reduced level compared to native or wild-type cells, as determined, for example, using a protein quantification assay or protein activity assay. In embodiments, such assays include, but are not limited to, immunoassays, immunohistochemical staining, and antibody-based detection (e.g., Western blotting, dot blotting, ELISA, multiplex assays, etc.), quantitative PCR, reverse transcriptase (RT) PCR, genomic sequencing, RNA profiling, mass spectrometry, fluorescence-based in situ detection, fluorescence-activated cell sorting (FACS), flow cytometry of cell surface markers, cell Western blotting, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), fluorogenic and colorimetric enzyme kinetic analysis, titration, calorimetry, etc. In various embodiments, a BioNV that is "substantially devoid" of protein expression and / or activity includes having protein expression and / or activity that is about 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, or below undetectable levels of protein expression and / or activity compared to a native or wild-type cell of the same species (e.g., a non-edited cell).

[0184] In various embodiments, the BioNV comprises membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0185] In various embodiments, the BioNV comprises membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0186] In various embodiments, BioNV has a membrane-embedded CD200 protein and is substantially devoid of either CD24 or CD47 protein.

[0187] In some embodiments, BioNV comprises one or more targeting agents directed against one or more surface markers of diseased cells associated with HLH and / or HLH-associated disorders. In some embodiments, the targeting agent is one or more of a CAR, a VERR, a viral ligand, a viral receptor, or an antibody or antibody format selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv (scFv), a diabody, a nanobody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody. In some embodiments, the VERR or viral ligand is a gp120 / gp41 complex. In some embodiments, the targeting agent is an scFv. In some embodiments, the targeting agent is a CAR.

[0188] In embodiments, the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or fragments and / or combinations thereof. In embodiments, the CAR comprises an intracellular signaling domain of the CD3ζ chain, and / or an intracellular domain optionally further comprising one or more costimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40. In embodiments, the CAR is activated, optionally via its target, another receptor, and / or a virus.

[0189] In various embodiments, BioNV has a VERR ligand-binding ectodomain or viral ligand (cellular receptor ligand), a hinge derived from CD8 or IgG4, a transmembrane domain, costimulatory molecule(s) (e.g., CD27, CD28, ICOS, 4-1BB, and / or OX40), and / or a stimulatory molecule (e.g., CD3 zeta chain or FcR gamma chain).

[0190] In various embodiments, BioNV has a VERR and / or viral ligand fused to the Vp1 AAV protein ectodomain, transmembrane domain, and PLA2 domain from AAV (priming). The PLA2 domain may reside on the external, solvent-exposed surface of BioNV in an inactive form. Alternatively, or in addition, the PLA2 domain may be engineered to be active from within BioNV.

[0191] In embodiments, BioNV has a surface-exposed transmembrane-anchored membrane fusion protein(s) fused to an internally directed inactive (or activated) lipid fusion domain (e.g., PLA2).

[0192] In embodiments, BioNV has an externally oriented transmembrane-anchored membrane lipid fusion protein and / or lipid-protein complex. In embodiments, BioNV can fuse with the plasma membrane of a target cell, resulting in direct injection or deposition of the payload into the cell's cytoplasm. In embodiments, VERR or the viral ligand can have a common internal coiled trimer that is linked to the PLA2 protein of the lipid-protein complex. Upon binding of the VERR / viral ligand to the target, the coiled trimer can undergo a conformational change that activates the fusion protein (or protein complex) and initiates the target cell's internalization mechanism(s).

[0193] In embodiments, BioNV has surface-expressed HIV gp120 / gp41. In embodiments, BioNV expressing the gp120 / gp41 complex can also promote fusion with the plasma membrane of target cells. The WT gp120 / gp41 complex on BioNV can target the CD4 / CXCR5 receptor on hematopoietic cells and deliver therapeutic payloads, such as CRISPR / Cas-based gene editing machinery, gRNA(s), functional copies of genes, perforin, granzymes, caspases, p53, etc., to the cytoplasm. Without wishing to be bound by theory, this delivery mechanism has the advantage of bypassing the standard endosomal processing pathway. In embodiments, the surface epitope of the gp120 receptor ligand may be mutated to target cellular markers other than CD4 / CXCR5, expanding the therapeutic repertoire.

[0194] In embodiments, BioNV comprises a gp120 / gp41 complex for delivering a gene editing payload. In embodiments, the complex may be expressed on the surface of a cell (transmembrane) and surface-exposed in the resulting BioNV (after processing). In embodiments, the gp120 / gp41 complex may be used to recognize CD4 / CCR5 receptors on target cells and deliver gene editors or therapeutic proteins to cells expressing these receptors. In embodiments, the gp120 / gp41 receptor complex is used to treat virus-infected immune cells because it combines a mechanism for target cell recognition (via gp120 interacting with the CD4 receptor) and a mechanism for injecting the gene editor into the cytoplasm, bypassing the less efficient endosomal pathway (via gp41 interacting with the CCR5 receptor). In embodiments, gp120 is the targeting portion of the complex, and gp41 is the harping / fusion portion of the complex. In embodiments, the gp120 / gp41 complex has high precision and reduced off-target delivery to unintended cells.

[0195] In some embodiments, the BioNV targets cell surface markers of immune cells, including cell surface markers of stem cells and multipotent cells that differentiate into immune cells. In some embodiments, the cell surface markers are or include CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3, and combinations thereof. In some embodiments, the BioNV comprises a ligand and / or receptor transmembrane construct that targets cells that contribute to HLH and / or HLH-associated disorders.

[0196] In embodiments, the fusion of BioNV into the cell is mediated by a protein described herein.

[0197] In some embodiments, the BioNV has a targeting agent targeted to at least a first cell surface marker of cells with a deficiency in perforin and / or perforin release. In some embodiments, the BioNV may have more than one targeting agent, such as a bispecific CAR that can target a first and a second cell surface marker of an immune cell. In some embodiments, the marker is expressed on one or more of T cells, dendritic cells, macrophages, or any cell type that harbors a defective perforin. The T cells may be CD4+ T cells or CD8+ cytotoxic T cells (CTLs). The marker may be expressed by immune cells, including any lymphoid precursor lineage (e.g., T cell subsets, e.g., Tregs, Th17, Th2, Th1, Th0, and Th22), or any myeloid precursor lineage (e.g., mast cells, myoblasts, monocytes, eosinophils, basophils, neutrophils, DCs, and macrophages).

[0198] In various embodiments, the first cell surface marker is or comprises a sialic acid-binding immunoglobulin-like lectin (Siglec) molecule. In various embodiments, the Siglec is or comprises Siglec-1. In various embodiments, Siglecs, such as Siglec-1, are valid targets for the therapeutic methods described herein. Siglec targets can be divided into subsets based on sequence and structural similarities, such as CD33-related Siglecs (e.g., Siglec-H, Siglec-5, and Siglec-14) and CD22-related Siglecs.

[0199] In some embodiments, the first cell surface marker is or includes CD32a (also known as FcγRIIa). In some embodiments, targeting the CD32a cell surface expression of the low-affinity Fc receptor CD32a may enable targeting of CD4+ T cells. In some embodiments, targeting the CD32a biomarker delivers a payload, e.g., a gene editor, to cells that contribute to HLH or HLH-associated diseases. In some embodiments, BioNV (e.g., BioNV targeting CD32a) carries a gene editing payload that is not active or functional in cells that do not contribute to HLH or HLH-associated diseases.

[0200] In embodiments, the first cell surface marker is or includes Siglec, PD-1, CD4, CCR5, CD32a, CD91, CD257, LAG-3, CD147, CD231, cell adhesion molecule 1 (CEACAM1), Plexin B2 (PLXNB2), or a combination thereof.

[0201] In some embodiments, BioNV comprises a bispecific chimeric receptor targeted to two or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3, including, but not limited to, one or more antibodies or antibody formats described herein. In some embodiments, BioNV comprises a bispecific chimeric receptor targeting a Siglec and CD32a. In some embodiments, BioNV comprises a bispecific chimeric receptor targeting CD4 and a co-receptor.

[0202] In embodiments, BioNV encapsulates a gene editing payload, e.g., a "lumenally loaded" (i.e., BioNV can load the gene editing payload into the lumen (the space within the biomimetic nanovesicle)). In embodiments, the payload is a gene editing payload that includes one or more gene editors. In embodiments, the one or more gene editors are complexed with at least one of the two gRNAs.

[0203] In embodiments, the one or more gene editors are gene-editing nucleic acids and / or proteins, such as, for example, a site-specific endonuclease, a CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, a transposase, and / or an ortholog or homolog thereof. In embodiments, the gene editor is a CRISPR / Cas nuclease. In embodiments, the gene editor is a site-specific endonuclease.

[0204] In embodiments, the gene editing payload also includes a gRNA, which as used herein is referred to as a guide RNA. In embodiments, the size of the guide RNA is from about 16 nucleotides (or mer) to about 100 nucleotides. In embodiments, the small RNA has a length of at least about 16 mer, at least about 17 mer, at least about 18 mer, at least about 19 mer, at least about 20 mer, at least about 21 mer, at least about 22 mer, at least about 23 mer, at least about 24 mer, at least about 25 mer, at least about 26 mer, at least about 27 mer, at least about 28 mer, at least about 29 mer, at least about 30 mer, at least about 35 mer, at least about 40 mer, at least about 45 mer, at least about 50 mer, at least about 55 mer, at least about 60 mer, at least about 65 mer, at least about 75 mer, at least about 80 mer, at least about 85 mer, at least about 90 mer, at least about 95 mer, or at least about 100 mer or more.

[0205] In various embodiments, the gRNA can be complementary to a coding or non-coding sequence and tailored to the specific sequence being targeted, including mutant / variant sequences. In various embodiments, the gRNA can be complementary to a coding or non-coding sequence of a gene, e.g., a sequence encoding one or more coding and / or non-coding regions, such as those listed in Tables 2-5. In various embodiments, the gRNA sequence can be a sense or antisense sequence. In various embodiments, the gene editor composition administered herein preferably includes, but is not limited to, two or more gRNAs. However, a single gRNA can also be used.

[0206] In embodiments, BioNV harbors one or more gene editing payloads that target and correct one or more mutations / variants in Tables 2-5. Mutations, in embodiments, include one or more of frameshifts, duplications, deletions, insertions, missense mutations, splicing mutations, silent mutations, base exchanges, etc.

[0207] In embodiments, the mutations are single or multiple point mutations (e.g., missense mutations, such as Gly149Ser to Gln446Pro in the PRF1 MACPF domain). In embodiments, the point mutations are targeted and corrected by a deaminase domain-based editor, for example, as described in (CHU et al., "Rational Designed Base Applications for Precise Editing of the SICKle Cell Disease Mutation," CRISPR J, Vol. 4, No. 2, 2021: pp. 169-177).

[0208] The mutation, in embodiments, is a frameshift (e.g., p.Leu17fs at the PRF1 N-terminus of the MACPF domain). In embodiments, the frameshift is targeted and corrected by a CRISPR enzyme, such as Cpf1, which is used to cleave the mutated sequence at a specific site and ligate via the cohesive ends to generate a corrected transcript.

[0209] In embodiments, the mutation(s) are located in exons, introns, and other non-coding regions, such as promoters or promoter enhancers, occurring in the PRF1 gene. Mutations, in embodiments, are either monogenic (e.g., within the PRF1 open reading frame (ORF) or upstream of the promoter) or bigenic, where multiple spatial mutations occur simultaneously. In embodiments, these types of mutations are targeted by simultaneously delivering multiple gRNAs or by delivering multiple separate BioNV treatments in a single treatment regimen.

[0210] In embodiments, BioNV harbors one or more gene editors configured to excise sequences of one or more genes listed in Table 2.

[0211] In embodiments, BioNV is useful for gene replacement therapy, and BioNV harbors one or more nucleic acids encoding functional versions of one or more genes listed in Table 2. The genes are, in embodiments, introduced into the genome at the same location as the mutated / non-functional gene so as to maintain sensitivity to the same controls (e.g., promoters / enhancers, transcriptional controls, etc.).

[0212] The mutation, in embodiments, is a deletion (e.g., an in-frame deletion (p.Lys285del) or a frameshift deletion (p.Thr51fs) in the PRF1 MACPF domain). In embodiments, the deletion is targeted and corrected using gene replacement therapy. For example, in a non-limiting embodiment, CRISPR / Cas nucleases can be coupled with homologous recombination to restore the deleted sequence.

[0213] The BioNV-encapsulated nucleic acid construct, in various embodiments, contains a nucleic acid sequence operably linked to one or more genetic control elements, including tissue-specific promoters, cell-specific promoters, suicide promoters, and the like, that control expression of the BioNV-encapsulated element restricted to target cells.

[0214] In various embodiments, the BioNVs are about 10-1200 nm in diameter. In various embodiments, the BioNVs are about 10 nm in size, about 20 nm in size, about 30 nm in size, about 40 nm in size, about 50 nm in size, about 60 nm in size, about 70 nm in size, about 80 nm in size, about 90 nm in size, about 100 nm in size, about 120 nm in size, about 140 nm in size, about 160 nm in size, about 180 nm in size, about 200 nm in size, about 300 nm in size, about 400 nm in size, about 500 nm in size, about 600 nm in size, about 700 nm in size, about 800 nm in size, about 900 nm in size, about 1000 nm in size, about 1100 nm in size, or about 1200 nm in size. In various embodiments, the size of the BioNVs ranges from 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, about 1000 nm to 1200 nm, or about 10 nm to 1200 nm.

[0215] In embodiments, BioNV is stored at or suitable for storage at about -80°C. In embodiments, BioNV is lyophilized (e.g., reconstituted in a buffer) or suitable for lyophilization. In embodiments, BioNV is stable at about ambient temperature, at about -20°C, at about 4°C, at about 25°C, or at 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 1 week, or at least about 1 month or longer. Medication and Administration

[0216] The dosage and administration schedule of the BioNVs disclosed herein may depend on various parameters and factors, including, but not limited to, the specific marker target BioNV, the severity of the condition, the age, weight, and overall health of the subject, and the discretion of the administering physician. Furthermore, pharmacogenomic information (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic drug) about a particular subject may influence the dosage used. Furthermore, the exact individual dosage may be adjusted somewhat depending on various factors, such as the specific combination of drugs administered, the duration of administration, the route of administration, the nature of the formulation, the rate of excretion, the disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage may be expected.

[0217] In various embodiments, delivery of BioNV is similar to that of vesicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0218] The therapeutic methods using BioNV described herein, in various embodiments, include a dose range in terms of the concentration of BioNV per kilogram (kg) of subject body weight. A suitable dose range for the therapeutic methods described herein is about 10 3 BioNV / kg ~ approx. 10 12 In various embodiments, the BioNV may comprise about 10 3 BioNV / mL ~ approx. 10 14 Alternatively, in various embodiments, the BioNV composition is present in the composition at a concentration of about 5 ng / mL to about 500 mg / mL weight / volume, or at least about 1 ng / kg to at least about 10 mg / kg weight / volume.

[0219] Therapeutic methods using BioNV described herein, in various embodiments, include intravenous, intramuscular, or parenteral administration, i.e., BioNV or edited cells are infused into a subject via infusion or injection into the subject's blood or tissues.

[0220] In various embodiments, the BioNVs disclosed herein are administered by controlled or sustained release means or delivery devices known to those skilled in the art, including, but not limited to, those described in U.S. Patent 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 can be useful for controlling or sustaining the release of one or more active ingredients, for example, by using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof, to provide desired release profiles at various rates.The controlled or sustained release of active ingredients can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by light of appropriate wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0221] In various embodiments, polymeric materials are 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).

[0222] In various embodiments, controlled-release systems are placed in proximity to the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in 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 also be used.

[0223] In various embodiments, methods of using BioNV include applying BioNV to the surface of a device (e.g., a catheter) or containing it within 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 essential ingredients for use in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary), references well known in the art.

[0224] In various embodiments, BioNVs can be administered at a dose tailored to the dosage of whole cells, e.g., based on CAR concentration. In various embodiments, while a typical concentration range of CAR protein per microgram of T cells is 0.20 ng to 0.70 ng, a single BioNV may have a total number of CARs that is 5 to 10,000 times lower than in whole cells. Consequently, when converting the mass of a BioNV to CAR concentration, its CAR concentration can be assumed to be equivalent (e.g., in the case of exosomes) or increased (e.g., in the case of BioNVs) to the cells from which it was derived (e.g., T cells). In various embodiments, the concentration and / or surface density of a targeting agent (e.g., CAR) is increased on the BioNV compared to the whole cells from which it was derived. In various embodiments, the concentration and / or surface density of a targeting agent (e.g., CAR) is enriched by sequential extrusion processing of whole cells. In various embodiments, the concentration and / or surface density of a cell surface molecule, such as a targeting agent (e.g., CAR), on the BioNV is increased by 2- to 100-fold compared to whole cells. In various embodiments, because exosomes are naturally excreted, their concentration and / or surface density of cell surface molecules, such as targeting agents (e.g., CARs), is substantially the same as that of whole cells.

[0225] The dosing regimen utilizing any of the BioNVs disclosed herein can be selected depending on various factors, such as the type, species, age, weight, sex, and medical condition of the subject; the severity of the condition being treated; the route of administration; the subject's renal or hepatic function; the individual's pharmacogenomic makeup; and the particular composition of the invention being used. Any of the BioNVs disclosed herein may be administered once daily, or the total daily dose may be administered in divided doses two, three, or four times daily. Furthermore, any of the BioNVs disclosed herein may be administered continuously rather than intermittently throughout the dosing regimen.

[0226] In various embodiments, BioNV is administered in successive doses about every hour, about every 2 hours, about every 6 hours, about every 12 hours, about every 24 hours, about every 2 days, about every 4 days, about every 7 days, about every 2 weeks, about every 4 weeks, about every month, about every 2 months, about every 6 months, or about every year.

[0227] In various embodiments, mixed remission or clinical remission of the disease state, cancer, infection, or undetectable viral DNA or RNA is achieved within about 1 year, within about 6 months, within about 24 weeks, within about 18 weeks, within about 12 weeks, within about 8 weeks, within about 6 weeks, within about 4 weeks, within about 2 weeks, within about 1 week of administration of the compositions and methods using such compositions.

[0228] composition In aspects, the present disclosure relates to compositions for the treatment of HLH and / or HLH-associated diseases, the compositions comprising: a) a polypeptide encoding a polypeptide that encodes ... These include allogeneic, low-immunogenic BioNVs with membrane-embedded targeting agents (e.g., CAR, VERR / viral ligand) targeted against markers of HIV-1, PD-1, CTLA-4, TIGIT, LAG-3, and / or TIM-3, which harbor gene editing payloads that specifically target one or more genomic sequences, or which harbor payloads of perforin, granzymes, caspases, p53, and / or one or more nucleic acids encoding these proteins.

[0229] In some embodiments, the composition comprises BioNV. In some embodiments, the composition comprises BioNV and at least one additional combination therapy, as described herein. In some embodiments, the composition comprises BioNV and at least one gene editing payload and / or at least one additional combination therapy, encapsulated within an aqueous core. In some embodiments, the composition comprises BioNV and at least one of perforin, granzyme, caspase, p53, and one or more nucleic acids encoding these proteins, and / or at least one additional combination therapy, encapsulated within an aqueous core. In some embodiments, the composition comprises a therapeutically effective amount of BioNV and / or a therapeutically effective amount of at least one additional combination therapy. In some embodiments, BioNV and one or more checkpoint inhibitors are combined in solution or are in separate solutions that are co-administered.

[0230] In various embodiments, the compositions are allogeneic and / or hypoimmunogenic, hi various embodiments, the compositions are derived from iPSCs (or other cell types) that have been modified to reduce expression of immunogenic molecules and / or increase expression of immunoprotective molecules.

[0231] In embodiments, the composition is hypoimmunogenic. For example, in embodiments, the composition does not elicit an inflammatory and / or immune response upon administration. In embodiments, BioNV is hypoimmunogenic. In embodiments, upon administration to a subject, the composition, optionally the BioNV therein, elicits a reduced level of, among other things, 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, TNF-α / β, IDO, HLA-G, HGF, PGE2, or any combination thereof, compared to its allogeneic whole cell therapy counterpart. Induce 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 the inflammatory or immune response as measured by cytokine, chemokine, or immunomodulatory enzyme concentrations, such as a combination of

[0232] In various embodiments, BioNV is about 10 3 NV / mL ~ approx. 10 14 Alternatively, in various embodiments, the BioNV composition is present in the composition at a weight / volume range of about 5 ng / mL to about 500 mg / mL.

[0233] In some embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasma, and pyrogens, and in more particular embodiments, substantially free of all of the foregoing. In some embodiments, the composition is substantially free of whole cells and intracellular components, including organelles such as nuclei, mitochondria, and Golgi, and / or substantially free of non-targeting agent ligand-expressing nanovesicles (NVs), and / or substantially free of ruptured, damaged NVs. In some embodiments, the composition is substantially free of cellular chromatin, nucleosomes, and other genetic material and non-therapeutic gene-editing nucleic acids. In some embodiments, BioNV and BioNV compositions are substantially free of cellular genomic DNA.

[0234] Pharmaceutical Compositions and Formulations In various aspects, the composition is a pharmaceutical composition. In various embodiments, the pharmaceutical composition of the present invention is formulated to provide a therapeutically effective amount of BioNV and / or a gene editing payload as an active ingredient. In various embodiments, the pharmaceutical composition of the present invention is formulated to provide a therapeutically effective amount of BioNV and / or one or more of perforin, granzyme, caspase, p53, and nucleic acids encoding these proteins. In various embodiments, the pharmaceutical composition of the present invention is formulated to provide a therapeutically effective amount of one or more additional combination therapies and / or gene editing as a payload within BioNV as an active ingredient. In various embodiments, the pharmaceutical composition of the present invention is formulated to provide a therapeutically effective amount of one or more additional combination therapies and / or therapeutic proteins as a payload within BioNV as an active ingredient. Typically, the pharmaceutical composition also includes one or more pharmaceutically acceptable excipients, carriers, such as inert solid diluents and fillers, diluents, such as sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers, and adjuvants.

[0235] Pharmaceutical excipients can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents may be used. Pharmaceutically acceptable excipients are generally sterile when administered to a subject. Water is a useful excipient when any of the agents disclosed herein are administered intravenously. Saline and aqueous solutions of dextrose and glycerol can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any composition disclosed herein can also be formulated with wetting or emulsifying agents, or pH buffering agents, as needed. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaroeds., 19th ed. 1995), which is incorporated herein by reference.

[0236] In embodiments, the composition comprises an excipient or carrier. In embodiments, the diluent is a pharmaceutically acceptable excipient or carrier.

[0237] In various embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof, as well as inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In various embodiments, the diluent comprises one or more of saline, phosphate-buffered saline, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified minimal essential medium (alphaMEM), Roswell Park Memorial Institute Medium 1640 (RPMI Media 1640), HBSS, human albumin, Ringer's solution, etc., or any combination thereof.

[0238] In various embodiments, the active ingredient is typically mixed with an excipient, diluted by an excipient, or enclosed within such a carrier, for example, in the form of a capsule, tablet, sachet, paper, or other container. When an excipient functions as a diluent, it can be a solid, semi-solid, or liquid material (e.g., physiological saline) and acts as a vehicle, carrier, or medium for the active ingredient. In various embodiments, the composition can 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 capsule, suppository, sterile injection solution, and sterile packaged powder. As is known in the art, the type of diluent can vary depending on the intended route of administration. In various embodiments, the resulting composition may include additional agents such as preservatives, cryopreservatives (e.g., DMSO), and / or lyoprotectants (e.g., polyols, salts). In various embodiments, the carrier may be or include a lipid-based or polymer-based colloid. In various embodiments, the carrier material may be a colloid formulated as a liposome, hydrogel, microparticle, nanoparticle, or block copolymer micelle. In various embodiments, the carrier material may form a capsule, which may be a polymer-based colloid.

[0239] In various embodiments, the pharmaceutical composition comprising BioNV comprises a solubilizing agent. In various embodiments, the pharmaceutical composition comprising BioNV comprises a cryoprotectant, such as DMSO or glycerol, or an agent that improves thermal stability. In various embodiments, the pharmaceutical composition may be delivered using a suitable vehicle or delivery device known in the art.

[0240] In various embodiments, the composition comprises a scaffold. In various embodiments, the scaffold comprises a biomaterial. In a non-limiting example, the three-dimensional biomaterial comprises BioNVs embedded in an extracellular matrix attached to, dispersed within, or entrapped within the scaffold. In various embodiments, the biomaterial is biodegradable and / or synthetic.

[0241] In various embodiments, the scaffold comprises a biodegradable biomaterial, non-limiting examples of which include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstituted basement membrane matrix, starch, dextran, alginate, hyaluron, chitin, chitosan, agarose, sugars, 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 species include, but are not limited to, polydioxanones, polycarbonates, polyoxalates, poly(α-esters), polyanhydrides, polyacetates, polycaprolactones, poly(orthoesters), polyamino acids, polyamides, and mixtures and copolymers thereof, L-lactic acid and D-lactic acid stereopolymers, copolymers of bis(para-carboxyphenoxy)propanoic 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, α-benzyl glutamic acid and polyethylene glycol copolymers, succinate and poly(glycol) copolymers, polyphosphazenes, polyhydroxyalkanoates, and mixtures thereof. Binary and ternary systems are also contemplated. In various embodiments, the scaffold comprises one or more of collagen, various proteoglycans, alginate-based matrices, and chitosan. In various embodiments, the scaffold comprises one or more of hydrogel, silk, Matrigel, acellular and / or decellularized scaffold, poly-ε-caprolactone scaffold, resorbable scaffold, and nanofiber hydrogel composite.

[0242] In various embodiments, the scaffold comprises a synthetic biomaterial, non-limiting examples of which include lactone-based polyesters or copolyesters such as polylactic acid, polycaprolactone glycolide, 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., polyhydroxyethylmethylmethacrylate, polyvinylpyrrolidinone), fluorinated polymers such as polytetrafluoroethylene, and cellulose esters.

[0243] In various embodiments, the compositions may be prepared by any method known in the pharmaceutical art and administered by various routes (e.g., subcutaneous, intravenous, etc.) depending on whether local or systemic treatment is desired and the area to be treated. In various embodiments, administration may be topical (including ophthalmic and mucosal delivery, including intranasal, intravaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epithelial, and transdermal), ocular, oral, or parenteral. In various embodiments, methods may include intraocular delivery, topical administration (eye drops), subconjunctival, periocular, or intravitreal injection, or introduction by a balloon catheter or ophthalmic insert surgically placed in the conjunctival sac. In various embodiments, parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular, administration. In various embodiments, parenteral administration can be in the form of a single bolus dose, or can be by, for example, a continuous infusion pump.

[0244] In various embodiments, pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. In various embodiments, methods of treating HLH and HLH-related disorders include the use of pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like.

[0245] In various embodiments, pharmaceutical compositions contain, as an active ingredient, the nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. In various embodiments, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not elicit adverse, allergic, or other undesirable responses when administered to an animal or human, as appropriate. The methods and compositions disclosed herein are applicable to a wide range of species, including, for example, humans, non-human primates (e.g., monkeys), horses, or other livestock, dogs, cats, ferrets, or other mammals kept as pets, rats, mice, or other laboratory animals. In various embodiments, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, and the like that may be used as a vehicle for a pharmaceutically acceptable substance.

[0246] In various embodiments, the compositions may be applied to the surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. In various embodiments, the compositions may be administered alone or in admixture with a pharmaceutically acceptable excipient or carrier (e.g., saline). The excipient or carrier is selected based on the mode and route of administration. Suitable pharmaceutical carriers and pharmaceutical essential ingredients for use in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary), references well known in the art.

[0247] In various embodiments, the compositions disclosed herein, eg, pharmaceutical compositions, are resuspended in a saline buffer (including but not limited to TBS, PBS, etc.).

[0248] The present technology includes the disclosed BioNV in various pharmaceutical composition formulations. In various embodiments, the BioNV disclosed herein may be in the form of a liquid, suspension, emulsion, drops, tablets, pills, pellets, capsules, liquid-containing capsules, powders, sustained-release formulations, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0249] Pharmaceutical compositions containing BioNV described herein can be conveniently provided in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agent into association with the carrier, which constitutes one or more accessory ingredients. Typically, pharmaceutical compositions are prepared by uniformly and intimately bringing the therapeutic agent into association with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired dosage form (e.g., wet or dry granulation, powder blending, etc., followed by tableting using conventional methods known in the art).

[0250] In various embodiments, any BioNV disclosed herein is routinely formulated as a pharmaceutical composition adapted for the modes of administration disclosed herein.

[0251] Additional therapeutic agents In various embodiments, the compositions or methods described herein further comprise a therapeutically effective amount of one or more additional therapeutic agents (i.e., combination therapy). In various embodiments, the therapeutically effective amount of one or more additional therapeutic agents may be in a solution containing the BioNVs, adsorbed onto the surface of the NVs, or as a payload encapsulated within the BioNVs. In various embodiments, the additional therapeutic agent is one or more of an immunosuppressant, a cytokine therapy reagent, an analgesic, and / or an anti-infective agent.

[0252] In various embodiments, the compositions or methods contemplate other additional therapeutic agents, such as, for example, an analgesic to help treat inflammation or pain at the site of administration, or an anti-infective to prevent infection at the site treated by the composition.Non-limiting examples of additional therapeutic agents include analgesics, such as nonsteroidal anti-inflammatory drugs, opiate agonists, and salicylates; anti-infectives, such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other B-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterial agents, antituberculous mycobacterial agents, antiprotozoal agents, antimalarial agents, antiviral agents, antiretroviral agents, antiscabies agents, Anti-inflammatory agents, corticosteroid anti-inflammatory agents, antipruritic / local anesthetics, topical anti-infective agents, antifungal topical anti-infective agents, antiviral topical anti-infective agents; electrolyte and renal acting agents, such as acidifying agents, alkalizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium sparing diuretics, thiazide diuretics, electrolyte replenishing agents, and uricosurics; enzymes, such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylate gastrointestinal anti-inflammatory agents, antacid anti-ulcer agents, gastric acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2 blocker anti-ulcer agents , gallstone solubilizers, digestive agents, emetics, laxatives and stool softeners, and gastrointestinal motility enhancers; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics; hormone modulating agents, such as abortifacients, adrenocortical agonists, corticosteroid agonists, androgens, antiandrogens, immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics, such as amide-type local anesthetics and ester-type local anesthetics; musculoskeletal agents, For example, anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, nonsteroidal 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.

[0253] Additional non-limiting examples of useful therapeutic agents in the above categories include: (1) general analgesics, such as lidocaine or its derivatives, and nonsteroidal anti-inflammatory drug (NSAID) analgesics, such as diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opiate 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) mupirocin, etc. (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 B-lactam antibiotic anti-infectives such as aztreonam and imipenem; (10) penicillin antibiotic anti-infectives such as nafcillin, oxacillin, penicillin G, and penicillin V; (11) ciprofloxacin and norfloxacin (12) quinolone antibiotic anti-infectives such as floxacin; (13) antituberculous mycobacterial anti-infectives such as isoniazid (INH) and rifampin; (14) antiprotozoal anti-infectives such as atovaquone and dapsone; (15) antimalarial anti-protozoal anti-infectives such as chloroquine and pyrimethamine; (16) antiretroviral anti-infectives such as ritonavir and zidovudine; (17) acyclovir, ganciclovir, interferon alfa; (18) topical antifungal antiinfectives such as amphotericin B, clotrimazole, miconazole, and nystatin; (19) topical antiviral antiinfectives such as acyclovir; (20) electrolyte and renal agents such as lactulose; (21) loop diuretics such as furosemide; (22) potassium-sparing diuretics such as triamterene; (23) thiazide diuretics such as hydrochlorothiazide (HCTZ); (24) uricosurics such as probenecid; (25) enzymes such as RNase and DNase;(26) antiemetics such as prochlorperazine; (27) salicylate-based gastrointestinal anti-inflammatory agents such as sulfasalazine; (28) gastric acid pump inhibitor antiulcer agents such as omeprazole; (29) H2 blocker antiulcer agents such as cimetidine, famotidine, nizatidine, and ranitidine; (30) digestive agents such as pancrelipase; (31) gastrointestinal prokinetic agents such as erythromycin; (32) ester-type local anesthetics such as benzocaine and procaine; (33) musculoskeletal adrenal corticosteroids such as beclomethasone, betamethasone, cortisone, 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, ketorolac, 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.

[0254] BioNV In various aspects, the present invention includes BioNVs. In various embodiments, BioNVs are approximately 10-1200 nm in size and contain an externally facing, membrane-embedded targeting agent capable of binding to target molecules. In various embodiments, BioNVs are composed of plasma membrane material from the host cells from which they are derived. Without wishing to be bound by theory, their biomimetic qualities depend on the nanovesicle composition derived from the plasma membrane of allogeneic, hypoimmunogenic modified cells. In various embodiments, BioNVs comprise a plasma membrane-derived lipid bilayer, completely encapsulating an aqueous core that houses various therapeutic molecules, including gene editing payloads, small RNAs, nucleic acids encoding functional proteins, perforin, granzymes, caspases, and / or p53.

[0255] To ensure proper orientation of the targeting agent and eliminate BioNVs lacking the targeting agent, in various embodiments, HPLC-based affinity chromatography techniques are used to select and enrich only BioNVs with sufficient surface concentrations of solvent-exposed targeting agent. HPLC-based affinity chromatography techniques are used to reduce the concentration of contaminating cellular material and NVs with immunogenic cell surface markers by either positive or negative selection.

[0256] In various embodiments, BioNV targeting agent constructs include various structural molecules, such as fusion proteins typically used in chimeric antigen receptors (CARs). Prototype CAR structure-functions include fusion proteins containing an extracellular (or externally facing) binding portion (e.g., scFv) connected to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.) by a hinge peptide (e.g., CH2 / CH3 domains from the IgG Fc region, Gly-Gly-Ser peptide bond, CD28 peptide, CD8α peptide, etc.), followed by various intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.). In various embodiments, BioNV lacks the intracellular machinery of whole cells, and therefore does not require intracellular signaling molecules in CARs. In embodiments, the targeting agent construct comprises an extracellular binding portion fused to the transmembrane domain of CD28 with an IgG CH2 / CH3 linker, and substantially lacks an intracellular domain or functionality. In embodiments, the targeting agent construct may be a fusion protein with Vp1 AAV, replacing the prototypical intracellular domain, or may otherwise be fused to an anchor protein, e.g., a PLA2 domain from AAV, a fusion protein, a cytoskeletal element, a small molecule transport domain, etc., which may assist in integration into the target cell and / or in the encapsulation and release of the therapeutic payload.

[0257] In embodiments, BioNV targeting agent antigen binding molecules include various targeting agents containing antibody-based or antibody format binding domains. In embodiments, BioNV comprises an antibody or antibody format binding portion selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, a VERR, a viral ligand, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv (scFv), a diabody, a nanobody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody. In embodiments, the targeting agent construct is a bispecific T cell engager (BiTE), a variable heavy chain IgG fragment V, or a fusion protein comprising an antigen-binding portion of an antibody. H H, V NAR In embodiments, the targeting agent is a fusion of a heavy chain (HC) or light chain (LC) variable portion with an scFv.

[0258] In embodiments, BioNVs are formed by disrupting the cell membrane of engineered iPSCs. In embodiments, hypoimmunogenic iPSCs are characterized by a B2M- / -, CIITA- / -, CD47+ / +, PD1- / - plasma membrane profile and can be used to generate the BioNVs. In embodiments, BioNVs can be generated from parent iPSC cell lines by methods such as sonication, adaptive focused acoustic technology, French press, extrusion, serial extrusion, detergent-mediated cell lysis, and electroporation. In embodiments, serial extrusion is the method used to generate BioNVs.

[0259] In embodiments, BioNVs can be selected for size uniformity by methods that determine particle size, such as dynamic light scattering (DLS), flow cytometry, or mass photometry. In embodiments, BioNVs can be filtered for a particle size or size range to optimize renal clearance and other clinically relevant NV properties. In embodiments, BioNVs can be in a size range of approximately 20 nm to 1200 nm. In various embodiments, the BioNVs are about 10 nm in size, about 20 nm in size, about 30 nm in size, about 40 nm in size, about 50 nm in size, about 60 nm in size, about 70 nm in size, about 80 nm in size, about 90 nm in size, about 100 nm in size, about 120 nm in size, about 140 nm in size, about 160 nm in size, about 180 nm in size, about 200 nm in size, about 300 nm in size, about 400 nm in size, about 500 nm in size, about 600 nm in size, about 700 nm in size, about 800 nm in size, about 900 nm in size, about 1000 nm in size, about 1100 nm in size, or about 1200 nm in size. The size of the BioNVs ranges from about 10 nm to 20 nm in size, about 20 nm to 30 nm in size, about 30 nm to 40 nm in size, about 40 nm to 50 nm in size, about 50 nm to 60 nm in size, about 60 nm to 70 nm in size, about 70 nm to 80 nm in size, about 80 nm to 90 nm in size, about 90 nm to 100 nm in size, about 10 nm to 100 nm in size, about 100 nm to 200 nm in size, about 200 nm to 400 nm in size, about 400 nm to 600 nm in size, about 600 nm to 800 nm in size, about 800 nm to 1000 nm in size, or about 1000 nm to 1200 nm in size.

[0260] In various embodiments, iPSC-derived BioNVs may contain only the outer plasma membrane leaflet, only the inner plasma membrane leaflet, and / or both leaflets of an intact plasma membrane lipid bilayer. In various embodiments, iPSC-derived NVs may contain additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, lecithin, etc.), non-ionic surfactants (e.g., sorbitan 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, etc.) to improve physicochemical properties such as thermal stability and encapsulation / release of therapeutic payloads. The amount of cholesterol and the length and saturation of the hydrocarbon chains of the phospholipids can affect the rigidity and stability of the bilayer, which in turn can affect the ability of the NV to accept and release drugs, biomolecules, and other therapeutic payloads. In embodiments, BioNV also incorporates zwitterionic lipids and methods of using zwitterionic lipids, such as those described in U.S. Patent Publication US20130216607, the contents of which are incorporated herein by reference in their entirety. Correspondingly, functionalization of the hydrophilic head groups of lipids with polymers or biomolecules can provide new features to the vesicle surface, allowing for interactions with blood components, tissues, and the immune system in vivo.

[0261] In embodiments, the targeting agent comprises a receptor and a fusion peptide combined in a single complex, such as a VERR / viral receptor / ligand complex. In embodiments, the VERR / viral ligand does not contain a combination of fusion and recognition functions. In embodiments, the VERR / viral ligand has the same cell and / or tissue tropism as the virus from which the VERR / viral ligand is derived. In embodiments, the VERR / viral ligand has altered cell and / or tissue tropism compared to the virus from which the VERR / viral ligand is derived. In embodiments, the VERR / viral ligand promotes endosomal uptake. In embodiments, the VERR / viral ligand promotes membrane fusion (fusogenicity).

[0262] In various embodiments, the VERR / viral ligand comprises a protein that targets a cell adhesion molecule (CAM). Most viral receptors identified to date are CAMs, which function in cell-cell and cell-extracellular matrix adhesion and are essential mediators of cellular processes such as development, maintenance of cellular structure, cell signaling, and tissue maintenance and repair. The CAM family includes selectins, cadherins, integrins, and IgSF members. The ubiquitous expression and multifactorial functions of CAMs in ligand binding, endocytosis, and signaling provide multiple cellular targeting mechanisms for BioNV to bind to CAMs. In various embodiments, the viral ligand comprises a fusion, variant, or portion of the viral ligands of HIV, paralytic virus, reovirus, rhinovirus, adenovirus, poliovirus, and coxsackievirus B (CVB) (IgSF receptors), which are known to bind to CAM receptors.

[0263] In embodiments, BioNV comprises one or more of the VERR and / or viral ligand and / or host cell receptor components in Table 6. Table 6: Exemplary viral ligand cellular receptors of BioNV targeted VERR / viral ligands [Table 6]

[0264] IgSF members have emerged as receptors for a wide range of viruses, including enveloped and non-enveloped viruses, including reovirus, adenovirus, coxsackievirus, rabies virus, paralytic virus, and HIV. In various embodiments, the VERR / viral ligand comprises a viral protein that targets the IgSF member CD4 as a primary receptor, as in the case of HIV, which also requires specific coreceptors CCR4 and CXCR5. In various embodiments, the VERR / viral ligand may comprise the HIV envelope (ENV) glycoprotein, which interacts with CD4 and mediates a specific ENV-CD4 interaction that promotes immune cell entry. ENV is a trimeric protein composed of gp120 and gp41, and gp120 mediates binding to CD4 via a conserved domain that results in a conformational change within gp120 and CD4. Following CD4 interaction, ENV viral ligands that bind to the chemokine coreceptors CXCR4 or CCR5 can be used to specifically target BioNV to macrophages and CD4+ T cells, respectively. However, the initial interaction between ENV and host cells occurs through nonspecific cellular receptors, including heparan sulfate proteoglycans, or specific receptors, such as α4β7 integrin or the innate immune receptor DC-SIGN. In embodiments, modified cells from which BioNV is derived express gp120 / 41 complexes that incorporate mutations to prevent these nonspecific interactions. In embodiments, without wishing to be bound by theory, the mutations generate gp120 / 41 complexes with reduced neurotoxicity (Jadhav and Nema. "HIV-Associated Neurotoxicity: The Interplay of Host and Viral Proteins." Mediators of Inflammation. Vol. 2021, No. 1267041, 2021:11 pages).

[0265] In various embodiments, VERR / viral ligands include viral proteins that target PtdSer receptors, including T-cell immunoglobulin and mucin domain (TIM) and TYRO3, AXL, and the MERTK family of receptor tyrosine kinases (TAMs), all of which have been shown to function as receptors for many enveloped viruses. PtdSer receptors have been reported to mediate viral entry for many enveloped viruses, including the filoviruses EBOV and Marburg virus (MARV); flaviviruses such as WNV, dengue virus (DENV), and Zika virus (ZIKV); arenaviruses such as Lassa virus; and poxviruses such as vaccinia virus. AXL is also an entry receptor for DENV. AXL-mediated entry for both ZIKV and DENV is mediated through Gas6, an AXL ligand that directly binds phosphatidylserine and binds to AXL.

[0266] In embodiments, BioNV targets T cells using sialyllactose derived from gangliosides, which act as viral attachment factors for sialylglycan receptor expression on T cell subsets, and targets immune cells via hemagglutinin proteins selected from, for example, influenza A H1, H7, H10, or H1N1, H3N2, H7N9, H10N8, among others. In embodiments, BioNV is targeted to cells via a viral fusogen or viral glycoprotein that promotes fusion of the NV to the cell's plasma membrane.

[0267] Those skilled in the art will appreciate that VERR / viral ligands can be designed to target cell surface markers of any cell subset of interest, and appropriate methods for optimization.

[0268] In various embodiments, BioNVs are CD34+ or are derived from CD34+ cells, such as human CD34+ umbilical cord blood. CD34+ cord blood-derived cell lines can serve as base cell lines for the development, production, and manufacturing of BioNVs for delivery of gene editing therapeutics.

[0269] In various embodiments, BioNV and / or compositions comprising BioNV are administered in combination with one or more additional compounds. In various embodiments, BioNV is pretreated with one or more additional compounds, e.g., prior to administration to a subject.

[0270] In embodiments, BioNVs are modular and allogeneic (off-the-shelf) due to the lack of immunogenicity derived from engineered iPSCs. In embodiments, the lack of all cell signaling components makes BioNVs easily tunable for target specificity and resistance to immunosuppression. In embodiments, BioNVs lack genetic elements that contribute to runaway cytokine storms, minimizing the risk of cytokine release syndrome (CRS) in patients. In embodiments, BioNVs are derived from cells that can cross biological barriers and / or have viral receptors that facilitate crossing.

[0271] Without wishing to be bound by theory, BioNVs generated from iPSC-engineered allogeneic-based cell lines represent immunoinvisible BioNVs with the potential for multiple administration. Antibody-mediated neutralization of BioNVs is minimized, and immune cell-mediated clearance is avoided (T cells and macrophages). In embodiments, BioNVs do not contain viable genetic material from cells that can cause CRS or teratomas. In embodiments, BioNVs are derived from different cell types, with or without barrier-permeable ligands, to further control activity after injection.

[0272] BioNV formation method In various embodiments, the modified cells are hypoimmunogenic cells derived from iPSCs engineered to reduce or eliminate the expression and / or activity of immunogenic proteins and / or to express or increase the expression of immune-protective proteins. In various embodiments, iPSCs are reverted from a somatic state using microRNA technology instead of small molecule transactivators. The use of microRNAs provides a more rigorous differentiation system, resulting in higher quality iPSCs. Without wishing to be bound by theory, such high-quality iPSCs are less susceptible to silencing (of proteins after genetic engineering, e.g., CD47) and genetic drift, and have superior quality / quantity of culture splits (cultures can be split more times than with other methods before cell integrity is compromised).

[0273] In non-limiting embodiments, an exemplary method for generating BioNVs is shown in Figure 5. In embodiments, continuous extrusion is used to disrupt gene-edited, hypoimmunogenic cells (e.g., iPSCs), where BioNVs form naturally from the reformation of the plasma membrane.

[0274] In embodiments, BioNVs derived from iPSC-derived hypoimmunogenic cells retain the function of the hypoimmunogenic cells, for example, but not limited to, the ability to cross the blood-brain barrier in the case of macrophages / monocytes, or tissue-specific factors in the case of cardiomyocytes, hepatocytes, etc.

[0275] In various embodiments, allogeneic iPSCs have disrupted MHC class I and MHC class II complexes by knocking out key proteins involved in their expression, such as B2M, a serum protein found in association with MHC class I heavy chains on the surface of nearly all nucleated cells and involved in peptide antigen presentation to the immune system.

[0276] In various embodiments, when B2M knockout (KO), CIITA KO, IL-6 KO, and CD47tg knockin (KI) are integrated into iPSCs, the TRAC and TRBC genes can be knocked out. In various embodiments, only one gene of each is knocked out, rather than both on separate alleles. In various embodiments, the TRAC and TRBC genes can be knocked out as described herein. The purpose of knocking out the TRAC and TRBC genes is to eliminate T cell receptors. In various embodiments, the modified cells are differentiated into a T cell subset lacking the T cell receptor for inducing BioNV. Genetically modifying cells to substantially lack TCRs can reduce the likelihood of competing ligands for the targeting agent construct that may nonspecifically target other tissues. Therefore, in various embodiments, TCR genes are knocked out as a strategy to reduce the off-target effects of BioNV. In various embodiments, TRAC / TRBC knockout generally reduces the likelihood of CRS and the toxicity of BioNV.

[0277] In various embodiments, the modified cells are expanded after genetic engineering. Any small-scale expansion method or large-scale feeder system expansion method known in the art can be used.

[0278] In various embodiments, after constructing B2M KO, CIITA KO, IL-6 KO, CD47tgKI, and IL-2 promoter-driven green fluorescent protein (GFP) (IL-2p GFP) reporters, a targeting agent construct can be incorporated / engineered into cells. In various embodiments, the targeting agent construct is knocked into the TRAC / TRBC gene while simultaneously knocking out the remaining TRAC / TRBC genes, resulting in targeting agent+ and TRAC / TRBC- / - cells. In various embodiments, the targeting agent construct is knocked into the TRAC / TRBC locus at both loci simultaneously, resulting in targeting agent+ / + and TRAC / TRBC- / - cells.

[0279] In embodiments, once B2M KO, CIITA KO, IL-6 KO, CD47tg KI, IL-2p GFP KI, and targeting agent-modified cells (e.g., iPSCs) are engineered, the immune synapse (IS) quality between the targeting agent recognition domain and the biomarker is measured. In embodiments, the quality of the IS of BioNVs can be directly correlated to the efficacy of whole cell therapy.

[0280] In some embodiments, BioNV, or its derived hypoimmunogenicity, includes a nucleic acid encoding GFP (among other fluorescent proteins). In some embodiments, when B2M KO, CIITA KO, CD47tgKI, IL-6 KO, or TRAC / TRBC single KO are integrated into iPSCs, GFP molecules are integrated into the modified cell line. In some embodiments, this serves as a control cell line. In some embodiments, the non-control cell line (therapeutic cell line) does not have GFP. In some embodiments, the nucleic acid encoding GFP is operably linked to a promoter derived from one or more of IL-2, perforin, granzyme, alarmin, TNF, INF, combinations thereof, and / or any other cell-specific or reporter gene. When lymphocytes are globally / globally activated from various stimuli, the IL-2 promoter is constitutively activated. In some embodiments, more focused activation / repression (modulation) is used. In some embodiments, the IL-2p GFP reporter gene serves as an indicator of the degree of global / global activation of the cells (part of the BioNV induction process). In embodiments, GFP signaling in combination with immunoblot analysis of cytokine levels (such as perforin, granzymes, alarmins, TNF, and INF) can effectively modulate the degree of global / global activation of lymphocytes upon exposure to an activating antigen. In embodiments, GFP is used to compare the degree of activation between manufacturing lots to ensure consistency in therapeutic development.

[0281] In various embodiments, the hypoimmunogenic cells are CD34+ or are derived from CD34+ cells, such as human CD34+ umbilical cord blood. In various embodiments, CD34+ cord blood-derived cell lines can serve as base cell lines for the development, production, and manufacturing of BioNV for delivery of gene editing therapeutics. In various embodiments, CD34+ cord blood-derived hypoimmunogenic cell lines have been experimentally confirmed for their low expression of HLA1 / 2 and overexpression of CD47 (Deuse, et al. "Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients." Nat Biotechnol. Vol. 37, 2019:252-258).

[0282] In various embodiments, hypoimmunogenic cells can be engineered using multiple hypoimmunogenic engineering techniques, such as those described by Deuse et al., Han et al., Xu et al., Harding et al., and in published U.S. Patent Applications Nos. 20190376045, 20190376045, 20210308183, and 20210292715 to Deuse, US 20210161971 to Nagy, US 20180141992 to Strominger, and published European Patent Application No. 3693384 to Poirot, each of which is incorporated by reference in its entirety. (Han, et al. "Generation of hypoimmunogenic human pluripotent stem cells." PNAS. Vol. 116, No. 212019: pp. 10441-10446.) (Xu, 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. and (Harding, et al., “Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts.”BioRxiv.716571[Preprint],July 30,2019.).

[0283] In various embodiments, BioNVs are derived from cells that have eliminated HLA genes encoding MHC membrane glycoproteins that mediate immune responses associated with GVHD rejection. HLA gene clusters can be classified into three categories: 1) the MHC class I pathway, 2) the MHC class II pathway, and 3) the MHC class III pathway. In GVHD, only the MHC class I and II pathways express protein complexes that trigger immune responses; the MHC class III complex is not involved in immune activity.

[0284] Removal of MHC class protein complexes can induce NK cells and macrophages into an active clearance mode, followed by cell destruction. To circumvent this killing mechanism, in various embodiments, the addition of a transmembrane molecule protein tag of CD47 isoform 2 can be incorporated into the cell membrane of modified cells, thereby avoiding natural killer and macrophage-mediated killing responses. 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: pp. 6662-7.). In various embodiments, cells can be engineered to prevent these responses using additional mechanisms, such as those described below: 1) CD24 transmembrane molecule protein tags, such as those described in Zhao et al. (Zhao W, et al. "Strategies for Genetically Engineering Hypoimmunogenic Universal Pluripotent Stem Cells." iScience. Vol. 23, No. 6, 2020: 101162.); 2) membrane-bound surfactant protein-D (SP-D), such as those described in Jiaravuthisan et al. (Jiaravuthisan P, et al. A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells." Transpl Immunol. Vol. 47, 2018: pp. 44-48.), and 3) a molecular PD-L1 tag to prevent T cell responses. In various embodiments, PD-L1 is overexpressed in BioNV derived from cells that are not activated and loaded with apoptotic cytokines.In various embodiments, PD-L1 is upregulated on non-activated, hypoimmunogenic cells, i.e., BioNV, used for gene editor delivery. In various embodiments, CD47 can be utilized in iPSCs engineered for immune tolerance to innate immune cells, as described, for example, in 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.) and (Jaiswal S, et al. "CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis," Cell. Vol. 138, No. 2, 2009:pp. 271-85.). In various embodiments, the cells may be modified as described in US Pat. No. 8,562,997 to Jaiswal et al., which is incorporated herein by reference in its entirety.

[0285] In various embodiments, rather than completely knocking out all HLA genes, as done, for example, in Xu et al. and Han et al., an approach can be used in which only HLA genes highly associated with immune responses are knocked out, leaving intact HLA genes that attenuate macrophage or NK responses (e.g., HLA-E, HLA-F, and HLA-G). In various embodiments, this approach does not require the addition of a CD47 tag, and modified cells can be engineered to produce BioNVs with or without CD47.

[0286] In embodiments, the methods improve upon the low immunogenicity approaches of Table 7. Table 7: Three cell modification methods using HLA knockout combined with CD47 isoform 2 tag and PD-L1 transmembrane tag (Zhao, et al.) (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.). [Table 7]

[0287] In embodiments, the development of allogeneic modified cells involves the ablation of the MHC class I and MHC class II protein complexes by disruption of certain HLA genes or knockout of B2M followed by knockout of the CIITA gene. In embodiments, due to the rapid mechanism of action, the knockout can be performed using a CRISPR gene editing approach. In embodiments, the knockout is performed using zinc finger nucleases (ZFNs) and / or TALENS. In embodiments, the Cre / Lox recombinase system is used to generate the modified cells. In embodiments, RNA silencing (such as RNAi, shRNA, microRNA, CRISPR Cas13a-d) is used to generate the modified cells.

[0288] In embodiments, the development of allogeneic modified cells includes the Harding et al. method of creating allogeneicity, which differs from the methods described above. In embodiments, instead of deleting MHC class I / II genes and risking long-term acceptance by the recipient, the Harding et al. method includes an alternative approach based on a naturally occurring immune escape mechanism. In embodiments, the method relies on Harding et al.'s biomimicry based on Devil Facial Tumor Disease (DFTD) type 2, a horizontally transmitted cancer common in Tasmanian devils. In embodiments, the development of allogeneic modified cells includes overexpression of the immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200, and / or MFG-E8 to protect the cell derivatives from long-term immune rejection in mice (and humans) without deleting MHC class I / II proteins. In embodiments, the modified cells express one or more of the proteins set forth in Table 4, including any splice variants and / or isoforms of any of the listed proteins (e.g., CD200 splice variants). In embodiments, this system can be used to interfere with the activity of antigen-presenting cells (APCs), macrophages, natural killer cells, and T lymphocytes. In embodiments, the modified cell lines can also include the safety cell system developed by Liang et al., in which a cell division gene is linked to a suicide gene to prevent runaway teratomas that lead to cancer (Liang, 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.).

[0289] In embodiments, the methods improve upon the low immunogenicity approaches of Table 8. Table 8: Expression or increased expression of exemplary proteins for generating allogeneic modified cells. [Table 8]

[0290] In embodiments, the modified cells from which the BioNVs are derived are engineered to have a knockout of one or more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, CIITA, IL-6, IL-4, IL-10, IL-16, TRAC, TRBC, and / or any combination thereof, and a knockin of one or more of CCL2, PD-L1, CTLA-4, H2-M3, CD24, CD47 (3'UTR region removed or alternative 3'UTR region not containing the binding site for an inhibitory microRNA), MFG-E8, CD200, and / or any combination thereof.

[0291] In embodiments, BioNV is produced from modified cells having one or more of the modifications in Table 9. Table 9: Exemplary engineered cell expression profiles for BioNV formation for human use (Fife BT and Bluestone JA. "Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways." Immunol Rev. Vol. 224, 2008: pp. 166-82.) and (Rong Z, et al. "An effective approach to prevent immune rejection of human ESC-derived allografts." Cell Stem Cell. Vol. 14, No. 1 2014: pp. 121-30.). [Table 9]

[0292] In various embodiments, gene inactivation / activation is controlled by an inducible promoter throughout the BioNV differentiation and production process. In various embodiments, disruption of MHC, TCR, and cytokine release syndrome (CRS) genes generates allogeneic iPSCs that are - / - CRS and - / - TCR, resulting in plasma membranes that are less immunogenic when injected into a subject. CRS genes involved in CRS pathogenesis include cytokines such as IL-6, IL-10, IFN-γ, monocyte chemoattractant protein 1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor-α, and IL-8. In various embodiments, one or more of these genes are inactivated, for example, in the cells from which the BioNV is derived.

[0293] In some embodiments, BioNVs are formed by disrupting the cell membrane of engineered iPSCs. In some embodiments, hypo-iPSCs are characterized by a B2M- / -, CIITA- / -, CD47+ / +, PD1- / - plasma membrane profile and can be used to generate BioNVs. Low-immunogenic BioNVs can be generated from parent iPSC cell lines by methods such as sonication, adaptive focused acoustics, French press, extrusion, serial extrusion, detergent-mediated cell lysis, and electroporation. In some embodiments, serial extrusion is a method used to generate low-immunogenic BioNVs. In some embodiments, serial extrusion of iPSCs can produce BioNVs that are tgCD47+ and HLA1 / HLA2-negative (low-immunogenic), which eliminates PD1 resistance.

[0294] In various embodiments, genetic manipulation of iPSCs includes gene editing techniques such as CRISPR-based gene editing systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, among other gene editing methods, to generate allogeneic / hypoimmunogenic iPSCs and / or for targeting agent cassette integration. In various embodiments, genetic manipulation of iPSCs can refer to the reduction or elimination of transcription of any genetic element. Similarly, genetic manipulation of iPSCs can refer to the increase or knock-in of expression of any genetic element, including both endogenous and exogenous genetic elements.

[0295] For example, in various embodiments, stable cellular integration (safe harbor gene locations) in iPSCs can be controlled by implementing a Tet-regulated CRISPR and 3x transcription factor-targeting gRNA system. The CRISPR activation system for three upstream transcription factors can trigger a signal cascade event that promotes the production of VERR / viral ligands, replacing the endogenous antibody ORFs at the specified locus(s). This system can be made "tunable" by including a Tet-regulated promoter, allowing for the alteration of the concentration of VERR / viral ligands on the cell surface. Next, stable cellular replacement with VERR / viral ligand cassettes for CDRs and heavy and light chain antibody regions can be achieved by Cpf-1-induced homologous recombination repair (HDR). Finally, the stably integrated targeting agent cassette can contain flanking gRNA binding sites, allowing for repeated exchange or alteration of the targeting agent binding moiety, resulting in rapid and consistent insertion of the desired sequence.

[0296] In various embodiments, the allogeneic and low immunogenic properties of iPSCs can be further improved by inducing overexpression of immune-protective molecules. For example, but not limited to, overexpression of CD47, among other cell surface integrins, can slow the response rate of macrophage depletion of BioNV products from the blood. In various embodiments, the allogeneic and low immunogenic properties of iPSCs can be further improved by expression of α and β phagocytic integrins. In various embodiments, overexpression of similar immunogenic and protective cell surface markers that signal leukocytes can be implemented as a strategy to increase the half-life of BioNVs after injection.

[0297] In various embodiments, iPSCs are genetically engineered to incorporate a targeting agent cassette. Targeting agent cassette integration can include both integrative and non-integrative transgenes. Non-limiting examples of non-integrative transgene insertion include mRNA, non-integrative lentivirus, and endonuclease targeting methods. Integrative targeting agent cassette insertion methods include stable retroviral vector insertion systems and transposase-based integration systems. Stable targeting agent cassette transduction can be achieved, for example, using retroviral vectors, which can enable iPSCs to maintain the genetic element encoding the targeting agent throughout differentiation, proliferation, and activation. In various embodiments, clinical-grade stable transduction of targeting agent cassettes into T cells has been achieved using GRV vectors with similar CAR cassettes, such as brexucabtagene autoleucel (Tecartus®, Kite Pharma Inc.) and axicabtagene ciloleucel (Yescarta®, Kite Pharma Inc.), and tisagenlecleucel (Kymriah®, Novartis International AG) has been delivered using lentiviral vectors (Labbe, RP, et al. "Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives." Viruses. Vol. 13, No. 8, 2021:1-22.).

[0298] In various embodiments, the concentration of targeting agents on the surface of iPSC-based cell lines or any downstream differentiated cells (and resulting BioNVs) can be regulated using various transcriptional control elements, such as a tetracycline on / off promoter (or similar drug-regulated promoter) to drive expression of the CRISPR activation / gRNA (CRISPRa) system. The CRISPRa system can then activate antibody regulatory transcription factors, e.g., Drm2, Fr5, and Bxp2, which regulate expression of engineered targeting agent cassettes integrated into the antibody locus at sites where the antibody genes have been replaced. Additionally, similar transcriptional control elements can be provided to control overexpression of genes (e.g., CD47), drive genes that control differentiation, and the like, during defined manufacturing stages.

[0299] In some embodiments, targeting agent expression can be initiated in modified cells regardless of whether they have been differentiated. In some embodiments, targeting agent expression can be performed in undifferentiated iPSCs to obtain BioNVs that lack cell surface markers from differentiated cell subsets. Alternatively, iPSCs can be differentiated into lymphoid or myeloid cells before initiating targeting agent expression of cell surface markers from selected immune cell types.

[0300] Subjects and / or Animals In various embodiments, the subject and / or animal is a mammal, such as a human, a primate, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, a rabbit, a sheep, or a non-human primate. In various embodiments, the subject and / or animal is a transgenic animal comprising fluorescent cells, such as RPE cells and / or GFP-bearing immune cells. In various embodiments, the subject and / or animal is a human. In various embodiments, the human is a pediatric human, an adult, an elderly person, an infant, or a child. In other embodiments, the human is referred to as a subject or a patient.

[0301] In various embodiments, the treatment methods include those for ages from about 0 months to about 6 months, from about 6 months to about 12 months, from about 12 months to about 18 months, from about 18 months to about 36 months, from about 1 year to about 5 years, from about 5 years to about 10 years, from about 10 years to about 15 years, from about 15 years to about 20 years, from about 20 years to about 25 years, from about 25 years to about 30 years, from about 30 years to about 35 years, from about 35 years to about 40 years, from about 40 years to about 50 years, from about 50 years to about 60 years, from about 60 years to about 70 years, from about 70 years to about 80 years, from about 80 years to about 90 years, from about 90 years to about 100 years, from about 100 years to about 150 years, from about 150 years to about 20 years, from about 200 years to about 25 years, from about 250 years to about 30 years, from about 300 years to about 35 years, from about 350 ...100 years to about 150 years, from about 150 This includes administering to a human of an age ranging from about 45 years old to about 45 years old, from about 45 years old to about 50 years old, from about 50 years old to about 55 years old, from about 55 years old to about 60 years old, from about 60 years old to about 65 years old, from about 65 years old to about 70 years old, from about 70 years old to about 75 years old, from about 75 years old to about 80 years old, from about 80 years old to about 85 years old, from about 85 years old to about 90 years old, from about 90 years old to about 95 years old, or from about 95 years old to about 100 years old.

[0302] In embodiments, the subject is a non-human animal, and thus the invention is for veterinary uses, hi embodiments, the non-human animal is a household pet, a livestock, or a laboratory animal.

[0303] In various embodiments, serum and / or immune cells are assessed and / or affected. In various embodiments, the immune cells include cells of the subject's and / or animal's innate immune system. In various 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 present invention provides for detecting the presence, absence, or amount of viral cDNA or RNA in a sample from a subject.

[0304] kit In various embodiments, the present disclosure provides a kit that can simplify the administration of any of the agents described herein. An exemplary kit of the present invention includes any of the agents described herein in a unit dosage form. In various embodiments, the unit dosage form is a container, such as a pre-filled syringe, which may be sterile, containing any of the agents described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In various embodiments, the kit further includes a label or printed instructions for use of any of the agents described herein. In various embodiments, the kit also includes an eyelid speculum, a local anesthetic, and an injection surface cleaner. In various embodiments, the kit further includes one or more additional agents described herein.

[0305] In various aspects, the present invention includes a syringe containing one or more compositions of the present invention. In various embodiments, the syringe is prefilled with a quantity of the composition. In various embodiments, the syringe is prefilled with a volume of about 1 mL to about 10 mL. In various embodiments, the syringe is prefilled 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.

[0306] In various embodiments, the syringe contains a composition that has a shelf stability ranging from about 1 hour to about 1 week. In various embodiments, the syringe contains a composition that has a shelf stability of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature ranging from about -85°C to about 25°C. In various embodiments, the syringe contains a composition that has a shelf stability of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature ranging from about 15°C to about 25°C.

[0307] 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.

[0308] In one embodiment, the kit comprises a container containing a composition comprising BioNV of the present invention, a therapeutically effective amount of an additional therapeutic agent as described herein, and instructions for use.

[0309] definition The following definitions are used in connection with the invention disclosed 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 invention belongs.

[0310] An "effective amount" or "therapeutically effective amount" is an amount effective to treat, prevent, or ameliorate HLH or an HLH-related disorder, such as those described herein, or an amount intended to reduce the number of cells harboring mutations / variants in one or more of the genes described herein.

[0311] As used herein, "a," "an," or "the" can mean one or more than one.

[0312] As used herein, the word "comprises" and variations thereof are intended to be open-ended, such that the recitation of items in a list does not exclude other similar items that may also be useful in the materials, compositions, devices, and methods of the present technology. Similarly, the terms "can" and "may" and variations thereof are intended to be open-ended, such that a recitation that certain embodiments can or may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0313] For purposes of describing and claiming the present invention, the open-ended term "comprising" is used herein as synonymous with terms such as including, containing, or having, although the invention, or embodiments thereof, may alternatively be described using alternative terms, for example, "consisting of" or "consisting essentially of."

[0314] In some embodiments, "BioNV," as referred to herein, refers to an allogeneic, low-immunogenic, biomimetic nanovesicle containing at least one surface-oriented, membrane-embedded targeting agent. In some embodiments, "nanovesicles (NV)" as referred to herein are lipid-bound vesicles approximately 10 nm to 1200 nm in size that encapsulate an aqueous core. In some embodiments, lipid-bound NVs can be formed using lipid monolayers, lipid bilayers, or can maintain a multilayered morphology. In some embodiments, BioNVs refer to biologically derived nanosized vesicles that can have engineered biological functionalization. In some embodiments, BioNVs are "biomimetic" in that they are derived from endogenous cellular material, and more specifically, they substantially replicate the plasma membrane material found within cells. In some embodiments, cells from which BioNVs are derived can include any type of stem cell, including cell types differentiated from such stem cells. In some embodiments, BioNVs are substantially free of encapsulated cellular debris, including nucleic acids, organelles, or organelle parts. In embodiments, BioNV is characterized by having one or more, two or more, three or more, four or more, five or more, or six or more of the following: a. A size of about 10 nm to about 1200 nm; b.About 500nm 3 ~approximately 5 μm 3 having a total volume of (assumed spherical); c. having a content of at least one of phospholipids and cholesterol; d. The surface membrane has one or more of CD34, CCL21, PD-L1 (in BioNV derived from a non-activated cell source), FasL, SerpinB9, H2-M3, CD47, CTLA-4, CD24, CD200, MFG-E8, NCAM, and / or α-phagocytic integrin, or chimeras of any one or more thereof; the surface membrane is substantially devoid of T cell receptor components (TRAC and / or TRBC), MHC class I components, and / or MHC class II components, and is devoid of one or more proteins 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, and is substantially devoid of one or more proteins of IL-4, IL-6, IL-10, and / or IL-16 within the vesicle; e. A CAR, VERR, viral ligand / receptor, antibody or antibody format selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab'), Fv, single-chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, fusion protein comprising the antigen-binding portion of an antibody, bispecific T cell engager (BiTE), or a variable heavy chain IgG fragment V H H or V NAR or having a membrane-embedded targeting agent that includes a target-binding moiety via the T cell receptor (TCR); f. Capable of adsorbing and / or encapsulating one or more gene editing payloads, gRNAs, small RNAs, small molecule inhibitors, therapeutic payloads such as perforin, granzymes, caspases, p53, cytokines, biologics, fluorescent proteins, fusion proteins, and / or any combination thereof; and g. Capable of not eliciting a harmful immune response in the subject.

[0315] In various embodiments, "induced pluripotent stem cells," "iPSCs," or "reprogrammed induced pluripotent stem cells" are stem cells that arise from differentiated cells and are reprogrammed back to an embryonic-like pluripotent state. iPSCs generally can proliferate indefinitely and become any cell type of the organism from which they originated.

[0316] In various embodiments, "allogeneic" as used herein refers to genetically dissimilar and inherently immunologically incompatible biological materials, tissues, or cells, despite being derived from the same species. For example, allogeneic BioNV is material derived from a first subject (iPSC donor) and can be provided to any number of different subjects who are not genetically identical.

[0317] In various embodiments, "low immunogenicity" or "low immunity," as used herein with respect to modified cells and / or BioNVs, refers to a reduced ability to generate an immune response. iPSCs and BioNVs may be low immunogenic due to reduced or absent expression of immunogenic cell surface proteins, such as T cell receptor (TCR) proteins, cytokine response syndrome proteins, and MHC class I or II proteins. iPSCs and BioNVs may be low immunogenic due to expression of one or more immune-protective cell surface proteins, such as CD47 and alpha-phagocytic integrin. BioNVs may be low immunogenic due to not inducing CRS in a subject and / or not inducing HLA mismatches.

[0318] In various embodiments, "knockout," "silencing," "inactivation," "disruption," or "blocking," and the like, with respect to transcription, gene, or protein expression, refers to a decrease in the amount of transcription, gene, or protein expression from the normal state, or to a level below the wild-type state, in a particular subset of cells. This decrease can be so significant that no gene expression occurs, or only a negligible amount of protein expression occurs.

[0319] In embodiments, "overexpression," as used herein, refers to an increase in the amount of transcription, gene, or protein expression from the normal state, or greater than the wild-type state, in a particular subset of cells.

[0320] In various embodiments, the term "site-specific endonuclease" as used herein refers to a targetable DNA cleavage reagent that can function to site-specifically incise (e.g., single-stranded DNA cleavage) or cleave (e.g., double-stranded DNA cleavage) within a genomic nucleic acid sequence. In various embodiments, site-specific endonucleases include zinc finger nucleases (ZFNs), restriction enzymes, and transcription activator-like effector nucleases (TALENs), among other site-specific endonucleases. [Example]

[0321] Example 1: Development of cell lines for human CD47 (hCD47) isoform 2 knock-in clone selection in low-immunogenic iPSCs Human female fibroblast-reprogrammed induced pluripotent stem cells (iPSCs) were isolated and genetically modified to knock out B2M (B2M- / -) and CIITA (CIITA- / -). The cells were shown to be "hypoimmunogenic" due to abolished expression of human leukocyte antigen (HLA) molecules, including MHC class I and MHC class II expression. Clonal populations were selected based on their predicted expression profiles and further modified for knock-in expression of hCD47, for example, using expression from an expression plasmid shown in Figure 6. A knock-in version of the CD47 cassette containing a deletion of the 3' UTR of CD47 was used for the expression method. Because the cells contain several microRNAs that bind to the 3' UTR of CD47 and naturally downregulate its expression, deletion of this portion allowed for stable clonal surface expression of CD47. As shown in Figure 6, a bGH poly(A) tail was used instead. Cells were transfected and maintained under neomycin selection, followed by total RNA extraction. Table 10: Total RNA extraction per CD47 stable clone population. [Table 10]

[0322] Cellular mRNA was extracted and analyzed by measuring specific hCD47 isoform 2 mRNA levels using quantitative PCR (QT-PCR). Fold changes were calculated relative to baseline expression in gene-edited human female fibroblast-reprogrammed iPSCs that had not undergone clonal knock-in. As shown in Figure 7A, select clonal populations achieved over 100% higher fold expression levels at the exon 1-2 junction than the baseline cells. As shown in Figure 7B, select clonal populations achieved nearly 100% higher fold expression levels at the exon 3-4 junction than the baseline cells.

[0323] Overall, the data indicate that, inter alia, hypoimmunogenic cell lines substantially free of MHC class I and MHC class II molecules and lacking expression of B2M and CIITA can be further modified to express human CD47, which prevents phagocytosis of the resulting BioNV. The cell lines are suitable for generating hypoimmunogenic BioNV, being substantially free of MHC class I and MHC class II molecules and expressing CD47, which prevents or evades phagocytosis.

[0324] equivalent 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 which equivalents are intended to be encompassed within the scope of the following claims.

[0325] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual section may be equally applicable to all sections.

[0326] Incorporation by Reference All patents and publications referenced herein are incorporated by reference in their entirety, including published PCT application WO2020 / 227369, filed May 6, 2020, entitled "Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors," published U.S. non-provisional application 20220040106A1, filed August 3, 2021, entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases," published PCT application WO2022 / 266399, filed June 17, 2022, entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases," published October 31, 2022, entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases," published PCT application WO2022 / 266399, filed October 31, 2022, entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases," published This includes published PCT application WO2023 / 081109 entitled "Infectious Diseases."

Claims

1. A biomimetic nanovesicle (BioNV), comprising: a targeting agent targeted to at least a first cell surface marker associated with hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease; a gene editing payload targeted to one or more mutations associated with HLH or an HLH-associated disorder; The BioNV, wherein the BioNV encapsulates the gene editing payload and is configured to deliver it to a target cell that expresses the first cell surface marker.

2. A biomimetic nanovesicle (BioNV), comprising: a targeting agent targeted to at least a first cell surface marker associated with hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease; and one or more of perforin, granzyme, p53, and one or more nucleic acids encoding these proteins; The BioNV encapsulates one or more of perforin, granzyme, p53, and one or more nucleic acids encoding these proteins, and is configured to be delivered to a target cell expressing the first cell surface marker.

3. The HLH or the HLH-related disease is HLH, FLH4, FLH2, FLH3, FLH5, Griscelli syndrome type 2, Chedik-Higashi syndrome, PID, EBV, PID and EBV, XLP-1, XLP-5 and EBV, immunodeficiency 24 (IMD24), immunodeficiency 27A / B (IMD27A / B), immunodeficiency 18 (IMD18), Wiskott-Aldrich syndrome, immunodeficiency 9 (IMD9), X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (CGDX), Wolman disease, galactosemia I, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, Gaucher disease type 4, Gaucher disease type 5, Gaucher disease type 6, Gaucher disease type 7, Gaucher disease type 8, Gaucher disease type 9, Gaucher disease type 1, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, Gaucher disease type 4, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 2, Gaucher disease type 3, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 2, Gaucher disease type 3, Gaucher disease type 2, Gaucher disease type 3, Gaucher disease type 4, Gaucher disease type 1, Gaucher disease type 2 ... The BioNV of claim 1 or 2, which is one or more of lactosialidosis, methylmalonic aciduria, Shaheen syndrome, mevalonic aciduria, biotin deficiency, LCHAD deficiency, lysinuric protein intolerance and orotic aciduria, multisulfatase deficiency and metachromatic leukodystrophy, propionic acidemia and multicarboxylase deficiency, lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, Takeuchi-Kosaki syndrome and severe multisystem autoinflammatory disease of neonatal onset, familial cold autoinflammatory syndrome 4, and autoinflammation with infantile enteritis.

4. The BioNV of claim 1, wherein the HLH or the HLH-associated disease is selected from Table 1, and / or the BioNV targeting moiety and the target cell are disease-matched and selected from Table 1.

5. 10. The BioNV of any one of the preceding claims, wherein the first cell surface marker is or comprises one or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, or a cell surface marker in Table 1.

6. The BioNV of claim 5 , wherein the first cell surface marker is or includes CD34, CD56, and / or CD16.

7. The BioNV of any one of claims 1 or 3-6, wherein the gene editing payload comprises one or more gene editors and / or nucleic acids encoding one or more gene editors.

8. 8. The BioNV of claim 7, wherein the one or more gene editors are a site-specific endonuclease, CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Casomega, a transposase, and an ortholog or homolog thereof.

9. The BioNV of claim 8, wherein the gene editor is a CRISPR / Cas nuclease.

10. The BioNV of claim 8, wherein the gene editor is a site-specific endonuclease.

11. The BioNV of claim 8, wherein the gene editing payload comprises one or more gRNAs and / or nucleic acids encoding one or more gRNAs.

12. The BioNV of claim 11, wherein the one or more gRNAs are targeted to one or more mutations in one or more genes in Table 2 and / or one or more mutations in Table 3, Table 4, or Table 5.

13. The BioNV of claim 12, wherein the one or more genes are STX11, PRF1, UNC13D, STXBP2, RAB27A, RHOG, LYST, CD27, SH2D1A, CTPS1, ITK, MAGT1, RASGRP1, STAT1, STAT2, IFNGR1 / 2, IL7RA, RAG1 / 2, CD3E, WAS, ORAI1, BTK, CYBB / CYBA / NCF1, LIPA, GALT, GBA, CTSA, MUT, COG6, MVK, BTD, HADHA, SLC7A7, SUMF1, PCCA / PCCB, XIAP, CDC42, and NLRC4.

14. 10. The BioNV of any one of the preceding claims, wherein the gene editing payload comprises one or more nucleic acids encoding functional versions of one or more genes in Table 2.

15. The BioNV of any one of the preceding claims, wherein the gene editing payload comprises at least two gRNAs targeting regions adjacent to one or more genes in Table 2 and / or one or more mutations in Table 3, Table 4, or Table 5.

16. The HLH or the HLH-associated disease is HLH, wherein the gene editing payload targets a mutant RHOG gene; FLH4, wherein the gene editing payload targets a mutant STX11 gene; FLH2, and the gene editing payload targets a mutant PRF1 gene; FLH3, and the gene editing payload targets a mutant UNC13D gene; FLH5, wherein the gene editing payload targets a mutant STXBP2 gene; Grischeri syndrome type 2, wherein the gene editing payload targets a mutant RAB27A gene; Chediak-Higashi syndrome, wherein the gene editing payload targets a mutant LYST gene; PID and / or EBV, and the gene editing payload targets a mutant CD27 gene, an ITK gene, a MAGT1 gene, a RASGRP1 gene, a STAT1 gene, a STAT2 gene, an IL7RA gene, or a RAG1 / 2 gene; XLP-1 / EBV, and the gene editing payload targets a mutant SH2D1A gene; immunodeficiency 24 (IMD24), wherein the gene editing payload targets a mutant CTPS1 gene; immunodeficiency 27A / B (IMD27A / B), wherein the gene editing payload targets a mutant IFNGR1 / 2 gene; immunodeficiency 18 (IMD18) and / or EBV, and the gene editing payload targets a mutant CD3E gene; Wiskott-Aldrich syndrome, wherein the gene editing payload targets a mutant WAS gene; immunodeficiency 9 (IMD9), wherein the gene editing payload targets a mutant ORAI1 gene; X-linked agammaglobulinemia (XLA), wherein the gene editing payload targets a mutant BTK gene; X-linked chronic granulomatous disease (CGDX), wherein the gene editing payload targets a mutant CYBB / CYBA / NCF1 gene; Wolman disease, wherein the gene editing payload targets a mutant LIPA gene; galactosemia I, wherein the gene editing payload targets a mutant GALT gene; the patient is Gaucher disease type 1, and the gene editing payload targets a mutant GBA gene; galactosialidosis, wherein the gene editing payload targets a mutant CTSA gene; methylmalonic aciduria, wherein the gene editing payload targets a mutant MUT gene; Shaheen syndrome, wherein the gene editing payload targets a mutated COG6 gene; mevalonic aciduria, wherein the gene editing payload targets a mutant MVK gene; a biotin deficiency disorder, wherein the gene editing payload targets a mutant BTD gene; the gene editing payload targets a mutant HADHA gene; lysinuric protein intolerance and orotic aciduria, wherein the gene editing payload targets a mutant SLC7A7 gene; multisulfatase deficiency and metachromatic leukodystrophy, wherein the gene editing payload targets a mutant SUMF1 gene; propionic acidemia and multicarboxylase deficiency, wherein the gene editing payload targets a mutant PCCA / PCCB gene; lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, wherein the gene editing payload targets a mutant XIAP gene; Takeuchi-Kozaki syndrome and neonatal onset severe multisystem autoinflammatory diseases, wherein the gene editing payload targets a mutant CDC42 gene; The BioNV of any one of the preceding claims, wherein the autoinflammation is associated with familial cold autoinflammatory syndrome 4 and infantile enterocolitis, and the gene editing payload targets a mutant NLRC4 gene.

17. The BioNV of any one of claims 2 to 6, wherein the granzyme protein is selected from granzyme A, B, H, K, and M.

18. The BioNV of any one of claims 2 to 6 or 17, wherein the one or more of perforin, granzyme, and p53 comprise a modification.

19. The BioNV of any one of claims 2 to 6 or 17 to 18, wherein the modification includes one or more of amino acid mutation, fusion, addition of a fluorescent label, PEGylation, and post-translational modification (PTM).

20. The BioNV of any one of claims 2 to 6 or 17 to 19, wherein the one or more nucleic acids are DNA or RNA.

21. The BioNV of claim 20, wherein the RNA is mRNA, optionally modified mRNA, or circular RNA.

22. The BioNV of claim 20, wherein the DNA is a plasmid or vector.

23. The BioNV of any one of claims 2 to 6 or 17 to 22, further comprising one or more caspase proteins and / or one or more nucleic acids encoding one or more caspase proteins.

24. The BioNV of claim 23, wherein the one or more caspase proteins are caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10.

25. The BioNV of any one of the preceding claims, wherein the BioNV is derived from a hypoimmunogenic modified cell.

26. The BioNV of any one of the preceding claims, wherein the hypoimmunogenic cells are differentiated cells derived from stem cells, induced pluripotent stem cells (iPSCs), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or any modified cells thereof.

27. The BioNV of claim 16, wherein the hypoimmunogenic modified cell is an iPSC.

28. The BioNV of claim 26, wherein the differentiated cell is a T cell, a helper T cell, a T memory cell, a γδ T cell, an NK cell, a monocyte, or a macrophage.

29. The BioNV of any one of the preceding claims, wherein the BioNV is substantially devoid of one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and cytokine release syndrome (CRS) proteins.

30. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated expression and / or activity of β2-macroglobulin (B2M) protein, and / or expression and / or activity of MHC class I protein.

31. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated expression and / or activity of a CIITA protein, and / or expression and / or activity of an MHC class II protein.

32. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished HLA-A protein expression and / or activity.

33. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished HLA-B protein expression and / or activity.

34. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated HLA-C protein expression and / or activity.

35. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished HLA-E or HLA-G protein expression and / or activity.

36. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated HLA-F protein expression and / or activity.

37. 10. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished T-cell alpha constant (TRAC) protein expression and / or activity.

38. 10. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished T-cell beta constant (TRBC) protein expression and / or activity.

39. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated PD-1 protein expression and / or activity, or wherein the BioNV has PD-1 protein expression and / or activity.

40. 10. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated IL-4 protein expression and / or activity.

41. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated IL-6 protein expression and / or activity.

42. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished IL-10 protein expression and / or activity.

43. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or abolished IL-16 protein expression and / or activity.

44. 10. The BioNV of any one of the preceding claims, wherein the BioNV has SerpinB9 protein expression and / or activity, or wherein the BioNV has reduced or eliminated SerpinB9 protein expression and / or activity.

45. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of CD34 protein.

46. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of a CCL2 protein.

47. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of PD-L1 protein.

48. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of H2-M3 protein.

49. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of CD47 protein.

50. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of CD24 protein.

51. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of a chimeric CD24 / CD47 protein.

52. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of CD200 protein.

53. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of a chimeric CD24 / CD200 protein or expression and / or activity of a chimeric CD47 / CD200 protein.

54. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of CTLA-4 protein.

55. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of MFG-E8 protein.

56. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of an NCAM protein.

57. 10. The BioNV of any one of the preceding claims, wherein said BioNV has expression and / or activity of an α-phagocyte integrin protein.

58. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of a FasL protein.

59. The BioNV of any one of the preceding claims, wherein the BioNV has reduced or eliminated protein expression of three or more immunogenic proteins, four or more immunogenic proteins, five or more immunogenic proteins, six or more immunogenic proteins, seven or more immunogenic proteins, eight or more immunogenic proteins, nine or more immunogenic proteins, ten or more immunogenic proteins, eleven or more immunogenic proteins, or twelve or more immunogenic proteins.

60. The BioNV of any one of the preceding claims, wherein the BioNV has expression and / or activity of three or more immune defense proteins, four or more immune defense proteins, five or more immune defense proteins, six or more immune defense proteins, seven or more immune defense proteins, eight or more immune defense proteins, nine or more immune defense proteins, or ten or more immune defense proteins.

61. The BioNV of any one of the preceding claims, wherein the BioNV is allogeneic.

62. The BioNV of any one of the preceding claims, wherein the BioNV does not induce a harmful immune response in a subject to which it is administered.

63. 10. The BioNV of any one of the preceding claims, wherein the BioNV substantially lacks the protein and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G.

64. 10. The BioNV of any one of the preceding claims, wherein the BioNV substantially lacks the protein and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, PD-1, and HLA-E or HLA-G.

65. 10. The BioNV of any one of the preceding claims, wherein the BioNV substantially lacks the protein and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, PD-1, HLA-E, or HLA-G, and one or more of IL-4, IL-6, IL-10, and IL-16.

66. The BioNVs bind to membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4; and 10. The BioNV of any one of the preceding claims, comprising any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

67. The BioNVs are selected from the group consisting of membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4; and 10. The BioNV of any one of the preceding claims, comprising any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

68. The BioNV of any one of the preceding claims, wherein the BioNV has a membrane-embedded CD200 protein and is substantially devoid of either CD24 or CD47 protein.

69. 10. The BioNV of any one of the preceding claims, wherein the targeting agent is one or more of a chimeric antigen receptor (CAR), a viral epitope-recognizing receptor (VERR), a viral ligand, a viral receptor, or an antibody or antibody format selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), a diabody, a nanobody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

70. 70. The method of claim 69, wherein the VERR or viral ligand is a gp120 / gp41 complex.

71. The BioNV of claim 69, wherein the targeting agent is an scFv.

72. The BioNV of claim 59, wherein the targeting agent is a CAR, optionally comprising an scFv antigen-binding portion.

73. The BioNV of Claim 72, wherein the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or fragments and / or combinations thereof.

74. The BioNV of Claim 72, wherein the CAR comprises an intracellular domain comprising an intracellular signaling domain of the CD3ζ chain, and / or optionally further comprising one or more costimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40.

75. The BioNV of claim 72, wherein the CAR is activated, and optionally the CAR is activated via its target, through another receptor and / or a virus.

76. The BioNV of any one of the preceding claims, wherein the BioNV is between about 10 nm and about 1200 nm in size.

77. The BioNV of claim 76, wherein the BioNV is about 10 nm to about 100 nm in size.

78. The BioNV of claim 76, wherein the BioNV is about 100 nm to about 200 nm in size.

79. The BioNV of claim 76, wherein the BioNV is about 200 nm to about 500 nm in size.

80. The BioNV of claim 76, wherein the BioNV is about 500 nm to about 1200 nm in size.

81. 10. The BioNV of any one of the preceding claims, wherein the BioNV is stored at or suitable for storage at about -80°C.

82. 10. The BioNV of any one of the preceding claims, wherein the BioNV is lyophilized or suitable for lyophilization and / or suitable for formulation as a pharmaceutical composition.

83. A pharmaceutical composition comprising the biomimetic nanovesicles (BioNVs) of any one of claims 1 to 82 and one or more pharmaceutically acceptable excipients.

84. A method for treating hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease, comprising administering to a subject in need of treatment a BioNV described in any one of claims 1 to 82, or a pharmaceutical composition described in claim 83.

85. The HLH or HLH-related disease is HLH, FLH4, FLH2, FLH3, FLH5, Griscelli syndrome type 2, Chedik-Higashi syndrome, PID, EBV, PID and EBV, XLP-1, XLP-5 and EBV, immunodeficiency 24 (IMD24), immunodeficiency 27A / B (IMD27A / B), immunodeficiency 18 (IMD18), Wiskott-Aldrich syndrome, immunodeficiency 9 (IMD9), X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (CGDX), Wolman disease, or galactosemia. I, Gaucher disease type 1, galactosialidosis, methylmalonic aciduria, Shaheen syndrome, mevalonic aciduria, biotin deficiency, LCHAD deficiency, lysinuric protein intolerance and orotic aciduria, multisulfatase deficiency and metachromatic leukodystrophy, propionic acidemia and multicarboxylase deficiency, lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, Takeuchi-Kosaki syndrome and severe multisystem autoinflammatory disease of neonatal onset, familial cold autoinflammatory syndrome 4, and autoinflammatory syndrome with infantile enteritis, or 85. The method of claim 84, wherein the HLH or the HLH-associated disease is selected from Table 1, and / or the BioNV targeting moiety and the target cell are matched to the disease and selected from Table 1.

86. 86. The method of claim 84 or 85, wherein the BioNV targets one or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3.

87. The method of claim 86, wherein the BioNV targets CD34, CD56, and / or CD16.

88. The HLH or the HLH-associated disease is HLH, wherein the gene editing payload targets a mutant RHOG gene; FLH4, wherein the gene editing payload targets a mutant STX11 gene; FLH2, and the gene editing payload targets a mutant PRF1 gene; FLH3, and the gene editing payload targets a mutant UNC13D gene; FLH5, wherein the gene editing payload targets a mutant STXBP2 gene; Grischeri syndrome type 2, wherein the gene editing payload targets a mutant RAB27A gene; Chediak-Higashi syndrome, wherein the gene editing payload targets a mutant LYST gene; PID and / or EBV, and the gene editing payload targets a mutant CD27 gene, an ITK gene, a MAGT1 gene, a RASGRP1 gene, a STAT1 gene, a STAT2 gene, an IL7RA gene, or a RAG1 / 2 gene; XLP-1 / EBV, and the gene editing payload targets a mutant SH2D1A gene; immunodeficiency 24 (IMD24), wherein the gene editing payload targets a mutant CTPS1 gene; immunodeficiency 27A / B (IMD27A / B), wherein the gene editing payload targets a mutant IFNGR1 / 2 gene; immunodeficiency 18 (IMD18) and / or EBV, and the gene editing payload targets a mutant CD3E gene; Wiskott-Aldrich syndrome, wherein the gene editing payload targets a mutant WAS gene; immunodeficiency 9 (IMD9), wherein the gene editing payload targets a mutant ORAI1 gene; X-linked agammaglobulinemia (XLA), wherein the gene editing payload targets a mutant BTK gene; X-linked chronic granulomatous disease (CGDX), wherein the gene editing payload targets a mutant CYBB / CYBA / NCF1 gene; Wolman disease, wherein the gene editing payload targets a mutant LIPA gene; galactosemia I, wherein the gene editing payload targets a mutant GALT gene; the patient is Gaucher disease type 1, and the gene editing payload targets a mutant GBA gene; galactosialidosis, wherein the gene editing payload targets a mutant CTSA gene; methylmalonic aciduria, wherein the gene editing payload targets a mutant MUT gene; Shaheen syndrome, wherein the gene editing payload targets a mutated COG6 gene; mevalonic aciduria, wherein the gene editing payload targets a mutant MVK gene; a biotin deficiency disorder, wherein the gene editing payload targets a mutant BTD gene; the gene editing payload targets a mutant HADHA gene; lysinuric protein intolerance and orotic aciduria, wherein the gene editing payload targets a mutant SLC7A7 gene; multisulfatase deficiency and metachromatic leukodystrophy, wherein the gene editing payload targets a mutant SUMF1 gene; propionic acidemia and multicarboxylase deficiency, wherein the gene editing payload targets a mutant PCCA / PCCB gene; lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 2, wherein the gene editing payload targets a mutant XIAP gene; Takeuchi-Kozaki syndrome and neonatal onset severe multisystem autoinflammatory diseases, wherein the gene editing payload targets a mutant CDC42 gene; 88. The method of any one of claims 84 to 87, wherein the autoinflammation is associated with familial cold autoinflammatory syndrome 4 and infantile enterocolitis, and the gene editing payload targets a mutant NLRC4 gene.

89. The method of any one of claims 84-88, wherein administering the BioNV delivers a gene editing payload comprising one or more gene editors and / or nucleic acids encoding one or more gene editors.

90. 90. The method of Claim 89, wherein the one or more gene editors are a site-specific endonuclease, CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Casomega, transposase, and orthologs or homologs thereof.

91. 91. The method of Claim 90, wherein the gene editor is a CRISPR / Cas nuclease.

92. 91. The method of Claim 90, wherein the gene editor is a site-specific endonuclease.

93. The method of any one of claims 84 to 92, wherein administering the BioNV delivers one or more gRNAs and / or nucleic acids encoding one or more gRNAs.

94. 94. The method of Claim 93, wherein the one or more gRNAs are targeted to one or more mutations in one or more genes in Table 2, and / or one or more mutations in Table 3, Table 4, or Table 5.

95. The method of any one of claims 84 to 94, wherein administering the BioNV results in nucleotide repair of one or more mutations.

96. 96. The method of claim 95, wherein the one or more mutations are in one or more of the following genes: STX11, PRF1, UNC13D, STXBP2, RAB27A, RHOG, LYST, CD27, SH2D1A, CTPS1, ITK, MAGT1, RASGRP1, STAT1, STAT2, IFNGR1 / 2, IL7RA, RAG1 / 2, CD3E, WAS, ORAI1, BTK, CYBB / CYBA / NCF1, LIPA, GALT, GBA, CTSA, MUT, COG6, MVK, BTD, HADHA, SLC7A7, SUMF1, PCCA / PCCB, XIAP, CDC42, and NLRC4.

97. The method of any one of claims 84 to 96, wherein administration of the BioNV results in excision of the gene editing payload of a genomic sequence.

98. 98. The method of any one of claims 84-97, wherein administration of the BioNV results in excision of the genomic sequence by the gene editing payload and replacement of the genomic sequence with a functional gene.

99. 99. The method of claim 98, wherein the functional gene is one or more of STX11, PRF1, UNC13D, STXBP2, RAB27A, RHOG, LYST, CD27, SH2D1A, CTPS1, ITK, MAGT1, RASGRP1, STAT1, STAT2, IFNGR1 / 2, IL7RA, RAG1 / 2, CD3E, WAS, ORAI1, BTK, CYBB / CYBA / NCF1, LIPA, GALT, GBA, CTSA, MUT, COG6, MVK, BTD, HADHA, SLC7A7, SUMF1, PCCA / PCCB, XIAP, CDC42, and NLRC4.

100. The method of any one of claims 84 to 99, wherein administration of the BioNV results in expression of functional perforin protein.

101. The method of any one of claims 84 to 100, wherein administration of the BioNV results in exocytosis of functional perforin protein.

102. 88. The method of any one of claims 84-87, wherein administration of the BioNV results in delivery of one or more of perforin, granzymes, p53, caspases, and one or more nucleic acids encoding these proteins to one or more diseased cells.

103. 103. The method of claim 102, wherein the granzyme is selected from granzymes A, B, H, K, and M.

104. 104. The method of claim 102 or 103, wherein the one or more of perforin, granzyme, p53, and caspase comprise a modification.

105. 105. The method of claim 104, wherein the modification comprises one or more of an amino acid mutation, a fusion, a fluorescent label, PEGylation, and a post-translational modification (PTM).

106. 103. The method of claim 102, wherein the one or more nucleic acids are DNA or RNA.

107. The BioNV of claim 106, wherein the RNA is mRNA, optionally modified mRNA, or circular RNA.

108. The BioNV of claim 106, wherein the DNA is a plasmid or vector.

109. The method of any one of claims 84-87 or 102-108, wherein administering the BioNV delivers one or more caspase proteins and / or one or more nucleic acids encoding one or more caspase proteins.

110. 110. The method of claim 109, wherein the one or more caspase proteins are caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10.

111. The method of any one of claims 84 to 87 or 102 to 110, wherein administration of the BioNV activates one or more of caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10.

112. The method of any one of claims 84-87 or 102-111, wherein administering the BioNV results in targeted apoptosis.

113. 113. The method of any one of claims 84 to 112, further comprising administering one or more additional therapeutic agents.

114. 90. The method of claim 89, wherein the one or more additional therapeutic agents comprise an anti-inflammatory agent, a cytokine therapy reagent, an immunosuppressant, an anti-infective agent, a monoclonal antibody, and an analgesic agent.

115. 115. The method of any one of claims 84-114, wherein said administering comprises providing one or more doses of BioNV of at least about 1 ng / kg to at least about 10 mg / kg.

116. 116. The method of any one of claims 84-115, wherein the administration is intravenous, intramuscular, or parenteral.

117. 1. A method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH2 in a subject, the method comprising: administering to the subject biomimetic nanovesicles (BioNVs), wherein the BioNVs comprise a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, and the BioNVs comprise: The method is configured to deliver a gene editing payload comprising at least one gRNA and one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the gene editing payload is capable of correcting at least one mutation in the PRF1 gene, and wherein correction of the at least one mutation results in expression of a functional perforin protein.

118. 1. A method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH3 in a subject, the method comprising: administering to the subject biomimetic nanovesicles (BioNVs), wherein the BioNVs comprise a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, and the BioNVs comprise: The method is configured to deliver a gene editing payload comprising at least one gRNA and one or more gene editors and / or one or more nucleic acids encoding the one or more gene editors, wherein the gene editing payload is capable of correcting at least one mutation in the UNC13D gene, and wherein correction of the at least one or more mutations results in exocytosis of functional perforin protein.

119. 1. A method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH2 in a subject, the method comprising: administering to the subject biomimetic nanovesicles (BioNVs), wherein the BioNVs comprise a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, and the BioNVs comprise: the method is configured to deliver a gene editing payload comprising at least two gRNAs, one or more gene editors, and / or one or more nucleic acids encoding the one or more gene editors, and a functional PRF1 nucleic acid sequence, wherein the gene editing payload is capable of excising a mutated PRF1 genomic sequence and replacing the mutated PRF1 genomic sequence with the functional PRF1 nucleic acid sequence, resulting in expression of a functional perforin protein.

120. 1. A method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH3 in a subject, the method comprising: administering to the subject biomimetic nanovesicles (BioNVs), wherein the BioNVs comprise a targeting moiety targeted to at least a first cell surface marker of a hematopoietic cell, and the BioNVs comprise: The method is configured to deliver a gene editing payload comprising at least two gRNAs, one or more gene editors, and / or one or more nucleic acids encoding the one or more gene editors, and a functional UNC13D nucleic acid sequence, wherein the gene editing payload is capable of excising a mutated UNC13D genomic sequence and replacing the mutated UNC13D genomic sequence with the functional UNC13D nucleic acid sequence, resulting in exocytosis of functional perforin protein.

121. 121. The method of any one of claims 117-120, wherein the first cell surface marker is or comprises one or more of CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD25, CD27, CD28, CD30, CD32a, CD34, CD38, CD45RA, CD56, CD69, CD91, CD95, CD147, CD160, CD231, CD257, CXCR3, CCR7, CCR5, LAG-3, CEACAM1, PLXNB2, HLA-DR, PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3.

122. 122. The method of any one of Claims 117-121, wherein the one or more gene editors is a site-specific endonuclease, CRISPR / Cas nuclease, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Casomega, a transposase, and an ortholog or homolog thereof.

123. 123. The method of any one of claims 117-122, wherein said administering comprises providing one or more doses of BioNV of at least about 1 ng / kg to at least about 10 mg / kg, and said administering is intravenous, intramuscular, or parenteral.

124. 124. The method of any one of claims 117 to 123, further comprising administering one or more additional therapeutic agents.

125. 125. The method of claim 124, wherein the one or more additional therapeutic agents comprise one or more cytokine therapy reagents, immunosuppressants, anti-infective agents, monoclonal antibodies, and analgesics.

126. A method for treating hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease in a subject, the method comprising administering biomimetic nanovesicles (BioNVs) to the subject in need of such treatment, the BioNVs comprising a targeting moiety that targets at least a first cell surface marker of a hematopoietic cell, the BioNVs being configured to deliver perforin protein and / or a nucleic acid encoding a perforin protein, and administration of the BioNVs resulting in targeted apoptosis of one or more cells.

127. A method for treating hemophagocytic lymphohistiocytosis (HLH) or an HLH-associated disease in a subject, the method comprising administering biomimetic nanovesicles (BioNVs) to the subject in need of such treatment, the BioNVs comprising a targeting moiety that targets at least a first cell surface marker of a hematopoietic cell, the BioNVs being configured to deliver one or more granzyme proteins and / or nucleic acids encoding one or more granzyme proteins, and administration of the BioNVs resulting in targeted apoptosis of one or more cells.

128. 1. An ex vivo method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH2 in a subject, said method comprising: isolating cells from the subject; and contacting the cells with biomimetic nanovesicles (BioNVs), wherein the BioNVs are configured to deliver a gene editing payload capable of correcting at least one mutation in the PRF1 gene; and reintroducing the cells into the subject.

129. 1. An ex vivo method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH3 in a subject, said method comprising: isolating cells from the subject; and contacting the cells with biomimetic nanovesicles (BioNVs), wherein the BioNVs are configured to deliver a gene editing payload capable of correcting at least one mutation in the UNC13D gene; and reintroducing the cells into the subject.

130. 1. An ex vivo method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH2 in a subject, said method comprising: isolating cells from the subject; and contacting the cell with a biomimetic nanovesicle (BioNV), wherein the BioNV is configured to deliver a gene editing payload capable of excising a mutant PRF1 genomic sequence and replacing the mutant PRF1 genomic sequence with the functional PRF1 nucleic acid sequence; and reintroducing the cells into the subject.

131. 1. An ex vivo method of treating hemophagocytic lymphohistiocytosis (HLH) or FLH3 in a subject, said method comprising: isolating cells from the subject; and contacting the cells with biomimetic nanovesicles (BioNVs), wherein the BioNVs are configured to deliver a gene editing payload capable of excising a mutant UNC13D genomic sequence and replacing the mutant UNC13D genomic sequence with the functional UNC13D nucleic acid sequence; and reintroducing the cells into the subject.

132. A host cell for producing biomimetic nanovesicles (BioNVs) according to any one of claims 1 to 82, comprising: the host cell comprises one or more membrane-embedded targeting agents; the host cell substantially lacks the protein and / or activity of one or more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, CIITA, IL-6, TRAC, TRBC, IL-4, IL-6, IL-10, IL-16, and PD-1; and The host cell comprises the protein and / or activity of one or more membrane-embedded proteins including one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 CTLA-4, CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200.

133. 133. The host cell of claim 132, wherein the host cell is a mammalian host cell, optionally a human host cell.

134. 134. The host cell of claim 133, wherein the human host cell is a human female fibroblast reprogrammed induced pluripotent stem cell (iPSC).

135. 135. The host cell of any one of claims 132-134, wherein the host cell is gene edited to be B2M- / - and CIITA- / -.

136. 136. The host cell of any one of claims 132 to 135, wherein the host cell does not elicit a harmful immune response in a subject to which it is administered, or elicits a significantly reduced harmful immune response.

137. 137. The host cell of any one of claims 132-136, wherein the targeting agent is one or more of a chimeric antigen receptor (CAR), a viral epitope-recognizing receptor (VERR), a viral ligand, a viral receptor, or an antibody or antibody format selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), a diabody, a nanobody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

Images