Compositions and methods for immune cell trafficking modulation

Abstract

The present invention relates to methods and compositions comprising a delivery vehicle for delivery of tissue trafficking modulators to modulate the trafficking of immune cell to a specific locus. The invention also relates to methods for treating or preventing a disease and disorder, including cancer, an infectious disease and an immunological disorder.

Description

Attorney Docket No.: 046483-6287-00WO COMPOSITIONS AND METHODS FOR IMMUNE CELL TRAFFICKING MODULATION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 661,793, filed June 19, 2024, which is hereby incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AI169633 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “046483- 6287-00WO Sequence Listing.xml,” having a creation date of June 19, 2025, and having a size of 167,067 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0004] The success of mRNA-lipid nanoparticle (LNP) vaccines in managing the COVID-19 pandemic underscores the vast clinical potential of mRNA-LNP therapeutics (Micci L, et al., J Clin Invest.2015;125(12):4497-513; Hoang TN, et al., Cell. 2021;184(2):460-75). Pioneering research has demonstrated the modularity and versatility of mRNA-LNPs not only as vaccines, but also as targeted systems for cell-specific RNA therapeutics. Conjugating antibodies to the surface of mRNA-LNPs enables specific delivery to the lung vasculature or T cells (Rurik J.2022 Science, 375: 91-96; Tombácz et al., 2021 Mol Ther.29:3293-3304). These targeting technologies can overcome limitations in ex vivo CAR-T cell generation by allowing for transient expression of bioactive CAR constructs on T cells in vivo. 1Attorney Docket No.: 046483-6287-00WO

[0005] While antibody and natural ligand-based targeting are highly effective strategies to precisely deliver mRNA-LNPs to some cell populations, their success depends on the availability of both unique, bona fide cell surface markers that undergo internalization upon binding and specific, high affinity antibodies or ligands for targeting.

[0006] Perforin and granzyme B expressing cytotoxic T lymphocytes (CTLs), defined by cell surface expression of CX3CR1 (Nishimura et al., 2002, J Immunol.16:6173-6180; Gerlach et al., 2016, Immunity, 45:1270-1284; Bottcher et al., 2015, Nat Commun.6:8306), the fractalkine receptor, perform an essential role in vascular immune surveillance and clearance of infected cells. However, CTLs are excluded from secondary lymphoid tissue (SLT) due to the absence of cell surface markers required for SLT trafficking, including CD62L (aka L-selectin) and CCR7 (Sallusto et al., 1999, Nature, 401:708-712). This severely limits the effectiveness of strategies to engage CTLs in SLT, as these cells do not efficiently traffic to SLTs (Pino M, et al., PLoS Pathog.2019;15(10); Matloubian M, et al., Nature.2004;427(6972):355-60). Collectively, these features conspire to generate an anatomical niche with limited immunosurveillance enabling the persistence of HIV-1 and its simian counterpart, simian immunodeficiency virus (SIV) (Buggert M, et al., PLoS Pathog. 2018;14(4); Reuter MA, et al., Cell Rep.2017;21(12):3458-70; Fukazawa Y, et al., Nat Med. 2015;21(2):132-9; Perreau M, et al., J Exp Med.2013;210(1):143-56). Importantly, prior work has shown that fully differentiated cytotoxic CD8+ T cells that downregulate CD62L exhibit impaired tumor control in vivo despite potent ex vivo killing and that ectopic expression of degradation-resistant CD62L results in SLT lymphocyte trafficking and enhanced tumor clearance (Gattinoni L, et al., J Clin Invest.2005;115(6):1616-26; Watson HA, et al., Front Immunol.2019;10:1321).

[0007] Type 1 Diabetes (T1D) results in a life-long dependence on exogenous insulin after the autoimmune destruction of pancreatic islet β cells (Atkinson, M. A., et al., Lancet 383, 69–82 (2014)). Immune cell infiltration into the islets is a key mechanism of β cell autoimmunity, as T cell mediated destruction of β cells occurs after a loss of antigenic self- tolerance (Pugliese, A., J. Clin. Invest.127, 2881–2891 (2017)). Genomic susceptibility loci predict several aspects of this process beyond autoantigen presentation may be involved, as certain alleles of key regulatory T cell (Treg) genes, cytokines, and chemokine receptors are 2Attorney Docket No.: 046483-6287-00WO associated with T1D risk (Todd, J. A., et al., Nat. Genet.39, 857–864 (2007); Nejentsev, S., et al., Nature 450, 887–892 (2007); Shapiro, M. R., et al., Immunol. Cell Biol.99, 496–508 (2021); Pociot, F., et al., Diabetes 59, 1561–1571 (2010)). Evidence suggests that the islet microenvironment promotes T cell migration in T1D, as T1D patients have increased frequencies of T cells in proximity to islets and increased expression of proinflammatory chemokines within islets (Wang, Y. J., et al., Cell Metab.29, 769–783.e4 (2019); Uno, S., et al., Endocrine Journal 57, 991–996 (2010); Sarkar, S. A., et al., Diabetes 61, 436–446 (2012); Roep, B. O., et al., Clin. Exp. Immunol.159, 338–343 (2010)). As T cell infiltrate peaks around the time of T1D onset and declines as T1D duration increases, it is critical to intervene in islet proinflammatory signaling that recruits T cells in order to mitigate autoimmune development and preserve β cell mass (Wang, Y. J., et al., Cell Metab.29, 769–783.e4 (2019); Campbell-Thompson, M., et al., Diabetes 65, 719–731 (2016); Coppieters, K. T., et al., J. Exp. Med.209, 51–60 (2012)). Current approaches towards this goal have broad immunosuppressive effects with possible detrimental side effects (Herold, K. C., et al., N. Engl. J. Med.381, 603–613 (2019); Ramos, E. L., et al., N. Engl. J. Med.389, 2151–2161 (2023)). An ideal yet unattained treatment involves agents that promote β cell tolerance and suppress inflammatory conditions with limited off-target effects.

[0008] CD4+ T regulatory (Treg) cell-based strategies are currently being explored for treatment of autoimmune disorders including T1D (Bluestone, J. A., et al., Front. Immunol.14, 1166135 (2023)). While trials centered around autologous polyclonal Tregs demonstrated the safety of Treg based immunotherapy and therapeutic Treg durability in vivo, clinical success has been limited, possibly due to the low frequency of islet antigen-specific Tregs expanded from blood, polyclonal Tregs lacking immunosuppressive activity in the pancreas, or a failure to home to the pancreas (Bluestone, J. A., et al., Front. Immunol.14, 1166135 (2023); Marek- Trzonkowska, N., et al., Diabetes Care 35, 1817–1820 (2012); Bluestone, J. A., et al., Sci. Transl. Med.7, 315ra189 (2015)). Recently, there is a renewed effort to develop therapeutic Tregs expressing islet-specific chimeric antigen receptors (CAR), thus conveying antigen specificity (Bluestone, J. A., et al., Front. Immunol.14, 1166135 (2023); Raffin, C., et al., Nat. Rev. Immunol.20, 158–172 (2020)). However, current CARbased therapies may develop deleterious activities, thus requiring a kill switch requiring further treatment to activate. 3Attorney Docket No.: 046483-6287-00WO Further, the use of viral vectors to insert transgenes is time consuming, costly, and requires safe genomic insertion (Raffin, C., et al., Nat. Rev. Immunol.20, 158–172 (2020)).

[0009] Thus, there is a need in the art for technology that can specifically deliver therapeutic agents that mobilize T cell subsets out of the periphery and into tissue, allowing more tailored therapeutic applications. This invention addresses this unmet need SUMMARY OF THE INVENTION

[0010] The invention relates, in part, to targeted LNP molecules for delivery of tissue trafficking modulators to immune cells to alter the trafficking of the immune cells to specific tissues or cellular niches, and methods of use thereof for treating a disease or disorder in a subject in need thereof.

[0011] In some embodiments, the invention relates to a composition comprising at least one delivery vehicle for delivery of an agent to at least one immune cell, wherein the at least one delivery vehicle comprises at least one nucleic acid molecule encoding a tissue trafficking modulator.

[0012] In some embodiments, the at least one delivery vehicle comprises a lipid nanoparticle (LNP). In some embodiments, the at least one nucleic acid molecule encoding a tissue trafficking modulator is encapsulated in the LNP.

[0013] In some embodiments, the delivery vehicle comprises a targeting ligand specific for binding to a receptor on the cell surface of at least one target immune cell.

[0014] In some embodiments, the target immune cell is a cytotoxic T lymphocyte (CTL), a CX3CR1+ lymphocyte, a vascular cytolytic lymphocyte, a CD16+ NK cell, a monocyte, a macrophage, an activated neutrophil, a regulatory T cell, an activated CD25+ T cell, an early differentiated T cell, an effector memory CD4+ or CD8+ T cell, a NK cell, a B cell, a CD4+ T follicular helper cell, a CD8+ T follicular cytotoxic cell, a tissue homing CD4+ or CD8+ memory T cell, a resident memory CD4+ or CD8+ T cell, or a CD4+ Th17 cell.

[0015] In some embodiments, the target immune cell is a CTL, and the immune cell targeting ligand comprises fractalkine, or a fragment thereof comprising at least the chemokine 4Attorney Docket No.: 046483-6287-00WO binding domain. In some embodiments, the immune cell targeting ligand comprises SEQ ID NO:1 or SEQ ID NO:2.

[0016] In some embodiments, the tissue trafficking modulator is CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69, or a variant or fragment thereof.

[0017] In some embodiments, the tissue trafficking modulator comprises an amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, or SEQ ID NO:61, or a fragment or variant thereof.

[0018] In some embodiments, nucleic acid molecule comprises an RNA molecule comprising a sequence as set forth in SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO:62, or a fragment or variant thereof

[0019] In some embodiments, the target immune cell is an activated CD25+ T cell or a regulatory T cell, and the immune cell targeting ligand comprises IL-2. In some embodiments, the immune cell targeting ligand comprises SEQ ID NO:3.

[0020] In some embodiments, the tissue trafficking modulator is CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4, or a variant or fragment thereof.

[0021] In some embodiments, the tissue trafficking modulator comprises an amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10, or a fragment or variant thereof.

[0022] In some embodiments, the nucleic acid molecule comprises an RNA molecule comprising a sequence as set forth in SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:11, or a fragment or variant thereof.

[0023] In some embodiments, the delivery vehicle further comprises at least one additional agent. In some embodiments, the additional agent is a therapeutic agent, a gene editing agent, a chimeric antigen receptor (CAR) molecule, a cytokine receptor, or an immunosuppressive cytokine. In some embodiments, the additional agent comprises a nucleic 5Attorney Docket No.: 046483-6287-00WO acid molecule encoding a therapeutic agent, a gene editing agent, a chimeric antigen receptor (CAR) molecule, a cytokine receptor, or an immunosuppressive cytokine. In some embodiments, the at least one additional agent comprises a nucleic acid molecule encoding IL- 10 or TGFβ.

[0024] In some embodiments, the nucleic acid molecule encoding a tissue trafficking modulator comprises at least one messenger RNA (mRNA). In some embodiments, the nucleic acid molecule encoding a tissue trafficking modulator is delivered to the target immune cell. In some embodiments, expression of the tissue trafficking modulator alters the trafficking of the immune cell.

[0025] In some embodiments, the invention relates to a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising at least one delivery vehicle, wherein the at least one delivery vehicle comprises at least one nucleic acid molecule encoding a tissue trafficking modulator.

[0026] In some embodiments, the disease or disorder is a cancer, an infectious disease or an immunological disease or disorder. In some embodiments, the disease is HIV. In some embodiments, the disease is diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0028] Figure 1 depicts data demonstrating in vitro delivery of GFP or human CD62L mRNA to rhesus macaque PBMC via fractalkine-targeted LNPs. Cells were treated with or without LNPs as indicated for 48 hours in vitro without additional stimulation. CD62L detected using a human CD62L specific antibody (not cross-reactive with rhesus).

[0029] Figure 2 depicts the results of example experiments demonstrating In vivo delivery of fractalkine-conjugated GFP mRNA-LNPs to murine peripheral blood CX3CR1+ CD8+ T cells. Mice were infected with LCMV Armstrong for 8 days prior to retro-orbital 6Attorney Docket No.: 046483-6287-00WO delivery of mRNA-LNPs. Mice were bled 24 hours after LNP delivery and GFP expression assessed by flow cytometry. Controls include PBS and unconjugated GFP mRNA-LNPs. Data shown representative of n=5 mice / group.

[0030] Figure 3 depicts a NHP study design to characterize homing and retention of CX3CR1+ lymphocytes in SLT following CD62L mRNA therapy.

[0031] Figure 4 depicts a NHP study design to investigate the antiviral function of CD62L transduced CTLs at ART initiation and on ART in combination with Tat-mediated latency reversal.

[0032] Figure 5, comprising Figure 5A through 5G, depicts data demonstrating IL2- LNP targeting. (Figure 5A) IL2-LNP visual representation. (Figure 5B) Plot and (Figure 5C) representative images of IL2-LNP cargo expression (GFP) after in vivo administration in mice. Unconjugated LNP is used as a non-targeted LNP control. (Figure 5D) IL2-LNP cargo expression across immune subsets in vivo. (Figure 5E) IL2-LNP cargo expression in CD25+ CD127-CD4+ T cells and (Figure 5F) FoxP3+ CD4+ T cells in vivo. (Figure 5G) IL2-LNP cargo expression in human PBMCs.

[0033] Figure 6, comprising Figure 6A and Figure 6B, depicts data demonstrating selected chemokine and chemokine receptor mRNA expression determined by CITEseq and Spatial Transcriptomics on matched lymphoid tissues and pancreata from the HPAP consortium. (Figure 6A) Expression levels of various genes are shown grouped by manually annotated cell clusters from a single-cell RNAseq of lymph nodes and spleen across type 1 disease progression (ND, AAb+, T1D). Average expression is scaled by gene and percent expressed is calculated by the percent of cells with at least one UMI of transcripts pertaining to the specified gene. (Figure 6B) Heatmap visualization depicting the relative scaled expression of various CXC- and CC- motif ligands derived from spatial transcriptomics (Visium - 10x Genomics) of whole FFPE pancreas sections from non-diabetic (n = 5), autoantibody positive (AAB+; n = 7), and type 1 diabetic (T1D; n = 8) donors.

[0034] Figure 7 depicts a schema for experimental studies.

[0035] Figure 8 depicts production of IL2 labeled LNPs.

[0036] Figure 9, comprising Figure 9A and Figure 9B, depicts data demonstrating delivery of CD62L mRNA to human cytotoxic effector T cells using Fractalkine (CX3CL1)- 7Attorney Docket No.: 046483-6287-00WO conjugated mRNA LNPs. Figure 9A depicts a baseline assessment of CD62L expression vs. cytotoxic function in primary human CD8+ T cells prior to addition of mRNA-LNPs. Data show that human CX3CR1+ CD8+ T cells express cytolytic molecules (perforin and granzyme B) but are CD62L-. CD62L+ CD8+ T cells do not express cytolytic molecules. Figure 9B depicts data demonstrating that sddition of Fractalkine-conjugated CD62L mRNA LNPs induces expression of CD62L on cytotoxic effector CD8+ T cells. Data shown are gated on perforin+granzymeB+ CD8+ T cells as shown in the center panel of (Figure 9A). Left plot show addition of unconjugated CD62L mRNA LNPs, demonstrating no increase of expression of CD62L on perforin+granzymeB+ CD8 T cells. Middle and left plots show CD62L expression on perforin+granzymeB+ CD8+ T cells 24 hours after addition of fractalkine- conjugated CD62L mRNA LNPs at two different doses. High expression of CD62L is observed on the majority of cells in contrast to the baseline data shown in (Figure 9A) or the unconjugated condition in (Figure 9B).

[0037] Figure 10, comprising Figure 10A and Figure 10B, depicts data demonstrating delivery of CD62L mRNA to human cytotoxic NK cells using Fractalkine (CX3CL1)- conjugated mRNA LNPs. Figure 10A depicts data demonstrating NK cells 24 hours after treatment with unconjugated CD62LmRNA LNPs. Few cells express CD62L (top plot), and CX3CR1-CD62L+ cells are shown not to express perforin and granzyme B (bottom plot). Figure 10B depicts data demonstrating the addition of Fractalkine-conjugated CD62L mRNA LNPs induces expression of CD62L on cytotoxic effector NK cells. Data show two different concentrations of fractalkine-conjugated CD62L mRNA LNPs are shown in the center panel and right columns. Top Row: High expression of CD62L is observed on the majority of NK cells in contrast to the unconjugated condition in (Figure 10A). Transduced cells lose expression of CX3CR1 due to LNP targeting. Bottom Row: CD62L expressing NK cells are now shown to be perforin+granzymeB+ cells after treatment with Fractalkine-conjugated CD62L mRNA LNPs in contrast to the unconjugated condition in (Figure 10A).

[0038] Figure 11, comprising Figure 11A and Figure 11B, depicts data demonstrating fractalkine-conjugated CD62L mRNA LNPs can deliver CD62L mRNA to rhesus macaque CX3CR1+ CD8+ T cells, and expression persists for at least 72 hours in vitro. Data show Fractalkine-conjugated CD62L mRNA LNPs added to fresh rhesus macaque PBMC at 2 8Attorney Docket No.: 046483-6287-00WO different concentrations (0.1 or 1 μg / million cells) for 24 hours (Figure 11A), or 72 hours (Figure 11B). Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. CX3CR1 is down regulated on CD8+ T cells targeted by Fractalkine-conjugated CD62L mRNA LNPs.

[0039] Figure 12 depicts data demonstrating that fractalkine-conjugated CD62L mRNA LNPs can deliver CD62L mRNA to rhesus macaque CX3CR1+ NK cells. Data show Fractalkine-conjugated CD62L mRNA LNPs added to fresh rhesus macaque PBMC at 3 different concentrations. NK cells are gated as CD3-NKG2A+CD8+ cells. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L.

[0040] Figure 13 depicts a NHP study design to test whether CD62L mRNA could be delivered to rhesus macaque CX3CR1+ CD8+ T cells, NK cells, and monocytes using fractalkine-conjugated CD62L mRNA LNPs and enable lymph node trafficking of those cells. Two animals were treated with LNPs as shown, 2 control animals received PBS. Blood and lymph node fine needle aspirates were taken at the indicated times. All four rhesus macaques were infected with Simian Immunodeficiency virus and were receiving daily antiretroviral therapy (ART) prior to ART interruption (ATI). Rhesus macaques were dosed with LNPs 4 days following ATI, and human CD62L expression was measured for 4 days. A fine needle aspirate was obtained from each animal 24 hours after LNP delivery to assess lymph node trafficking of targeted cells.

[0041] Figure 14, comprising Figure 14A through Figure 14C, depicts data demonstrating in vivo expression of human CD62L in rhesus macaque CD8+ T cells after fractalkine-conjugated CD62L mRNA LNPs in peripheral blood. Figure 14A depicts flow cytometry data showing CX3CR1 vs. human CD62L expression patterns on CD8+ T cells prior to and after fractalkine-conjugated CD62L mRNA LNPs delivered IV at 0.5 mg / kg. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. Data from one treated animal are shown. Figure 14B provides a plot depicting the detection level of human CD62L over time after LNP injection in experimental animals (Rjb21 and RUf21) vs control animals (MR79 and RPw20). Figure 14C depicts data demonstrating the loss of CX3CR1 expression after delivery of 9Attorney Docket No.: 046483-6287-00WO fractalkine-conjugated CD62L mRNA LNPs in treated animals but not in control animals. Human CD62L expression is detected for >48 hours.

[0042] Figure 15 depicts data demonstrating the in vivo expression of human CD62L in rhesus macaque effector memory CD8+ T cells after fractalkine-conjugated CD62L mRNA LNPs in lymph nodes. Flow cytometry data showing CX3CR1 vs. human CD62L expression patterns 24 hours after fractalkine-conjugated CD62L mRNA LNPs delivered IV at 0.5 mg / kg on CD3+CD8+CD28-CD95+ cells. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. Data from all four animals is shown; control animals (MR79, RPw20) are on left, treated animals (Rjb21, RUf21) on right.10-11% of CD8+ TEM cells in LN of the fractalkine-conjugated CD62L mRNA LNP treated animals expressed human CD62L.

[0043] Figure 16, comprising Figure 16A and Figure 16B, depicts data demonstrating IV delivery of fractalkine-conjugated CD62L mRNA LNPs enables migration of granzyme B+ effector memory T cells into lymph nodes. Flow cytometry data showing Granzyme B vs. rhesus or human CD62L expression patterns 24 hours after control (Figure 16A) or fractalkine- conjugated CD62L mRNA LNP (Figure 16B) delivered IV at 0.5 mg / kg on CD3+CD8+CD28- CD95+ cells. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. The antibody that recognizes rhesus CD62L cross reacts with human CD62L. Data from all four animals is shown; (Figure 16A) control animals (MR79, RPw20), (Figure 16B) treated animals (Rjb21, RUf21).25-30% of CD8+ TEM cells in LN of the fractalkine-conjugated CD62L mRNA LNP treated animals expressed Granzyme B, whereas <2% of the endogenous rhesus CD62L+ cells in the control animals express Granzyme B.

[0044] Figure 17, comprising Figure 17A through Figure 17C, depicts data demonstrating in vivo expression of human CD62L in rhesus macaque NK cells after fractalkine-conjugated CD62L mRNA LNPs in peripheral blood. Figure 16A depicts flow cytometry data showing CX3CR1 vs. human CD62L expression patterns on NK cells prior to and after fractalkine-conjugated CD62L mRNA LNPs delivered IV at 0.5 mg / kg. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. Data from one treated animal are shown. Figure 10Attorney Docket No.: 046483-6287-00WO 16B shows a plot depicting the detection level of human CD62L on NK cells over time after LNP injection in experimental animals (Rjb21 and RUf21) vs control animals (MR79 and RPw20). Figure 16C depicts data demonstrating a loss of CX3CR1 expression after delivery of fractalkine-conjugated CD62L mRNA LNPs in treated animals but not in control animals. Human CD62L expression is detected for >48 hours.

[0045] Figure 18 depicts data demonstrating in vivo expression of human CD62L in rhesus macaque NK cells after fractalkine-conjugated CD62L mRNA LNPs in lymph nodes. Flow cytometry data showing CX3CR1 vs. human CD62L expression patterns 24 hours after fractalkine-conjugated CD62L mRNA LNPs delivered IV at 0.5 mg / kg on CD3- / HLA-DR- / CD20-NKG2A+ / CD8+ NK cells. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. Data from all four animals is shown; control animals (MR79, RPw20) are on left, treated animals (Rjb21, RUf21) on right.6-10% of NK cells in LN of the fractalkine-conjugated CD62L mRNA LNP treated animals expressed human CD62L.

[0046] Figure 19, comprising Figure 19A and Figure 19B, depicts data demonstrating IV delivery of fractalkine-conjugated CD62L mRNA LNP enables migration of granzyme B+ NK cells into lymph nodes. Flow cytometry data showing Granzyme B vs. rhesus or human CD62L expression patterns 24 hours after control (Figure 19A) or fractalkine-conjugated CD62L mRNA LNP (Figure 19B) delivered IV at 0.5 mg / kg on CD3- / HLA-DR- / CD20- NKG2A+ / CD8+ NK cells. Human CD62L is selectively detected by a human-specific anti- CD62L antibody clone that does not cross-react with endogenous rhesus CD62L. The antibody that recognizes rhesus CD62L cross reacts with human CD62L. Data from all four animals is shown; (Figure 19A) control animals (MR79, RPw20), (Figure 19B) treated animals (Rjb21, RUf21).60-75% of NK cells in LN of the fractalkine-conjugated CD62L mRNA LNP treated animals expressed Granzyme B, whereas <3% of the endogenous rhesus NK CD62L+ cells in the control animals express Granzyme B.

[0047] Figure 20 depicts data demonstrating in vivo expression of human CD62L in rhesus macaque monocytes after fractalkine-conjugated CD62L mRNA LNPs in peripheral blood. Top row shows flow cytometric gating strategy to identify monocytes in the right most flow plot. Monocytes are gated as live CD3- / NKG2A- / HLA-DR+ / CD19- / CD14+ / -CD16+ / - 11Attorney Docket No.: 046483-6287-00WO (but not- / -). Bottom row: human CD62L expression in peripheral blood monocytes. (left) control animals do not show expression of human CD62L. (right) human CD62L expression on 90-95% of circulating monocytes after fractalkine-conjugated CD62L mRNA LNPs delivered IV at 0.5 mg / kg. Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross-react with endogenous rhesus CD62L.

[0048] Figure 21 depicts data demonstrating in vivo expression of human CD62L in rhesus macaque monocytes after fractalkine-conjugated CD62L mRNA LNPs in lymph nodes. Flow cytometry data showing CX3CR1 vs. human CD62L expression patterns 24 hours after fractalkine-conjugated CD62L mRNA LNPs delivered IV at 0.5 mg / kg on Monocytes are gated as live CD3- / NKG2A- / HLA-DR+ / CD19- / CD14+ / -CD16+ / - (but not- / -). Human CD62L is selectively detected by a human-specific anti-CD62L antibody clone that does not cross- react with endogenous rhesus CD62L.

[0049] Figure 22 depicts data demonstrating expression of human CCR7 protein on 293F cells after treatment with unconjugated CCR7 mRNA LNPs. Cells were treated with 1μg of LNPs / 106cells for 48 hours. Two replicates are shown compared to the no LNP control.

[0050] Figure 23, comprising Figure 23A and Figure 23B, depicts data demonstrating depicts data demonstrating delivery of CCR7 mRNA to Cytotoxic CD8+ T cells by Fractalkine-conjugated CCR7 mRNA LNP. Human PBMC were treated with PBS, unconjugated CCR7 mRNA LNP, or fractalkine-conjugated CCR7 mRNA LNP at a dose of 2μg / million cells for 48 hours. Figure 23A shows flow plots depicting CCR7 expression on CX3CR1+ cells after LNP treatment or control. Data show that CCR7 is not expressed at baseline, unconjugated CCR7-LNPs do not effectively deliver mRNA to CX3CR1+ cells, but ~25% of CX3CR1+ cells can express CCR7 after treatment with fractalkine-conjugated CCR7 mRNA LNPs. Figure 23B shows histograms comparing the level of CCR7 on CX3CR1+ cells in the various treatment conditions, demonstrating that fractalkine conjugated CCR7 mRNA LNPs induce higher level expression of CCR7 compared to the control conditions.

[0051] Figure 24 depicts data demonstrating expression of human CXCR5 protein on 293F cells after treatment with unconjugated CXCR5 mRNA LNPs. Cells were treated with 1μg of LNPs / 106cells for 48 hours. Two replicates are shown compared to the no LNP control. 12Attorney Docket No.: 046483-6287-00WO

[0052] Figure 25 depicts data demonstrating expression of human CCR4 protein on 293F cells after transfection of human CCR4 mRNA with lipofectamine. Cells were treated with 0.4μg of RNA106cells for 24 hours.

[0053] Figure 26 depicts data demonstrating expression of mouse CCR4 protein on 293F cells after transfection of mouse CCR4 mRNA with lipofectamine. Cells were treated with 0.4μg of RNA106cells for 24 hours.

[0054] Figure 27 depicts data demonstrating expression of human CCR8 protein on 293F cells after transfection of human CCR8 mRNA with lipofectamine. Cells were treated with 0.4μg of RNA106cells for 24 hours.

[0055] Figure 28 depicts data demonstrating expression of mouse CCR8 protein on 293F cells after transfection of mouse CCR8 mRNA with lipofectamine. Cells were treated with 0.4μg of RNA106cells for 24 hours.

[0056] Figure 29 depicts data demonstrating expression of mouse CXCR2 protein on 293F cells after transfection of mouse CXCR2 mRNA with lipofectamine. Cells were treated with 1μg of RNA106cells for 48 hours. Two replicates are shown compared to the no LNP control.

[0057] Figure 30 depicts data demonstrating expression of human CXCR2 protein on 293F cells after transfection of human CXCR2 mRNA with lipofectamine. Cells were treated with 1μg of RNA / 106cells for 48 hours. No transfection control shown on the left, human CXCR2 mRNA transfected cells shown on the right.

[0058] Figure 31, comprising Figure 31A and Figure 31B, depicts data demonstrating delivery of human CXCR2 mRNA to CD4+ CD25+ regulatory T cells by IL2-conjugated CXCR2 mRNA LNP. Human PBMC were treated with PBS, unconjugated CXCR2 mRNA LNP, or IL2-conjugated CXCR2 mRNA LNP at a dose of 2 μg / million cells for 48 hours. Figure 31A shows flow plots depicting CXCR2 expression on CD25+ cells after LNP treatment or control. Data show that CXCR2 is not expressed on CD4+ CD25+ regulatory T cells at baseline, unconjugated CXCR2-LNPs do not effectively deliver mRNA to CD25+ CD4+ regulatory T cells, but ~34% of CD25+ CD4+ regulatory T cells can express CXCR2 after treatment with IL2-conjugated CXCR2 mRNA LNPs. Figure 31B shows histograms comparing the level of CXCR2 on CD25+ CD4+ regulatory T cells in the various treatment 13Attorney Docket No.: 046483-6287-00WO conditions, demonstrating that IL2 conjugated CXCR2 mRNA LNPs induce higher level expression of CXCR2 compared to the control conditions. DETAILED DESCRIPTION

[0059] The invention relates to targeted LNP molecules for delivery of tissue trafficking modulators to specific cell subsets to alter the trafficking of immune cells to specific tissues or cellular niches.

[0060] In some embodiments, the targeted LNP comprises a targeting domain. In some embodiments, the targeting domain comprising a cytokine ligand or chemokine ligand which targets the LNP to a target cell expressing the cognate receptor for the bound cytokine or chemokine ligand. Thus, in some embodiments, the invention provides compositions comprising LNP molecules comprising a cytokine or chemokine ligand conjugated to the surface of the LNP which serves as a targeting domain, as well as methods of use thereof for delivery of tissue trafficking modulators to an immune cell subset expressing the cognate cytokine or chemokine receptor.

[0061] In some embodiments, the targeted LNP comprises a binding molecule specific for binding to a receptor on a non-immune target cell. In some embodiments, expression of the tissue trafficking modulators by the target cell type increases trafficking of immune cells to the cellular niche of the non-immune cell type.

[0062] In some embodiments, the targeted LNP of the invention serves as a delivery vehicle for uptake of an encapsulated agent (e.g., a mRNA encoding a tissue trafficking modulator, etc.) by a target cell subset. In some embodiments, the delivery vehicle is taken up via endocytosis by the target cell subset. In some embodiments, the encapsulated agent is released from the delivery vehicle inside a target cell. In some embodiments, once released, the agent (e.g., mRNA, etc.) is expressed in the target cell. In some embodiments, the expressed agent modulates the trafficking of the target cell, e.g., by expression of at least one surface receptor which directs a target immune cell to a specific cell, tissue or niche. In some embodiments, the expressed agent modulates the trafficking of immune cells to the target cell, e.g., by expression of at least one ligand which recruits a target immune cell to a specific cell, 14Attorney Docket No.: 046483-6287-00WO tissue or niche. In some embodiments, the LNP further comprises a therapeutic agent or a nucleic acid molecule encoding a therapeutic agent for delivery to the target cell, tissue or niche.

[0063] The invention also relates to methods of treating a disease or disorder in a subject in need thereof, the method comprising the administration of LNP of the invention comprising or encapsulating a tissue trafficking modulator for altering the trafficking of an immune cell. In some embodiments, the LNP further comprises or encapsulates at least one therapeutic agent, wherein the targeted LNP serves as a delivery vehicle for delivery of the therapeutic agent to a specific cell subset, and further wherein uptake and expression of the therapeutic agent is beneficial for the treatment of the disease or disorder. In one embodiment, the at least one therapeutic agent comprises an mRNA molecule. In one embodiment, the at least one therapeutic agent comprises an mRNA molecule encoding a CAR molecule. In one embodiment, the at least one therapeutic agent comprises a gene editor (e.g., a CRISPR Cas9 protein and at least one sgRNA). In one embodiment, the at least one therapeutic agent comprises an mRNA molecule encoding an immunosuppressive agent.

[0064] In some embodiments, the invention provides targeted LNP molecules for delivery to cytotoxic T cells or NK cells. In some embodiments, the targeted LNP comprises fractalkine (CX3CL1) or a fragment or variant thereof for targeted delivery of the LNP to the cytotoxic T cells or NK cells. In some embodiments, the fractalkine targeted LNP comprises or encapsulates a nucleic acid molecule encoding a tissue trafficking modulator for expression on the target T cell or NK cell. In some embodiments, the tissue trafficking modulator comprises CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69, or any combination thereof. Therefore, in one embodiment, the invention provides compositions comprising a fractalkine (CX3CL1)-LNP comprising an mRNA molecule encoding CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69. In one embodiment, the invention provides compositions comprising a fractalkine (CX3CL1)-LNP comprising a combination of mRNA molecules encoding a combination of at least one of CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69 and at least one additional agent. In some 15Attorney Docket No.: 046483-6287-00WO embodiments, the additional agent is an additional tissue trafficking modulator. In some embodiments, the additional agent is a therapeutic agent.

[0065] In some embodiments, the invention provides targeted LNP molecules for delivery to activated CD25+ T cells. In some embodiments, the targeted LNP comprises IL-2 or a fragment or variant thereof for targeted delivery of the LNP to the activated CD25+ T cells. In some embodiments, the IL-2 targeted LNP comprises or encapsulates a nucleic acid molecule encoding a tissue trafficking modulator for expression on the activated CD25+ T cells. In some embodiments, the tissue trafficking modulator comprises a chemokine receptor. In some embodiments, the tissue trafficking modulator comprises CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4, or a combination thereof.

[0066] In some embodiments, the invention provides targeted LNP molecules for delivery to regulatory T cells. In some embodiments, the targeted LNP comprises IL-2 or a fragment or variant thereof for targeted delivery of the LNP to the regulatory T cells (Treg). In some embodiments, the IL-2 targeted LNP comprises or encapsulates a nucleic acid molecule encoding a tissue trafficking modulator for expression on the target Treg. In some embodiments, the tissue trafficking modulator comprises a chemokine receptor. In some embodiments, the tissue trafficking modulator comprises CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4, or a combination thereof. Therefore, in one embodiment, the invention provides compositions comprising an IL-2-LNP comprising an mRNA molecule encoding at least one tissue trafficking modulator. In one embodiment, the invention provides compositions comprising an IL-2-LNP comprising a combination of mRNA molecules encoding a tissue trafficking modulator and at least one additional agent. In some embodiments, the additional agent is an additional tissue trafficking modulator. In some embodiments, the additional agent is an additional chemokine receptor. In some embodiments, the additional agent is an additional immunosuppressive agent. In some embodiments, the immunosuppressive agent comprises IL-10 or TGFβ. In some embodiments, the IL-2-LNP comprises at least one nucleic acid molecule encoding an immunosuppressive agent in addition to at least one nucleic acid molecule encoding a tissue trafficking modulator. In one embodiment, the invention provides compositions comprising an IL-2-LNP comprising a combination of mRNA molecules encoding, at least one chemokine receptor and at least one immunosuppressive chemokine. 16Attorney Docket No.: 046483-6287-00WO

[0067] In one embodiment, the present disclosure provides compositions and methods for treating or preventing a disease or disorder (e.g., cancer, infection, autoimmune diseases etc.) in a subject. In some embodiments, the method comprises modulating the trafficking of a subject’s immune cells (e.g. cytotoxic T cells or NK cells) in vivo, ex vivo or in vitro to a tissue, locus or niche. In one embodiment the tissue, locus or niche comprises a cancer cell or at least one pathogen. In some embodiments, the immune cells proceed to target and destroy disease cells within the tissue, locus or niche, thereby treating or preventing the disease (e.g., cancer, HIV etc.). In some embodiments, the method comprises modulating the trafficking of a subject’s immune cells (e.g. Tregs) in vivo, ex vivo or in vitro to a tissue, locus or niche comprising at least one cell for suppression. In some embodiments, the immune cells proceed to target or suppress disease cells within the tissue, locus or niche, thereby treating, delaying onset of or preventing the disease or disorder (e.g., an autoimmune disease or an immunological disorder). Exemplary diseases and disorders that can be treated using the methods and compositions of the invention include, but are not limited to, cancers, infectious diseases, and immunological disorders. Definitions

[0068] 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.

[0069] As used herein, each of the following terms has the meaning associated with it in this section.

[0070] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0071] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. 17Attorney Docket No.: 046483-6287-00WO

[0072] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen or epitope. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0073] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0074] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0075] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. k and l light chains refer to the two major antibody light chain isotypes.

[0076] By the term “synthetic antibody” as used herein, is meant an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term should also be construed to mean an antibody, which has been generated by the synthesis of an RNA molecule encoding the antibody. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by 18Attorney Docket No.: 046483-6287-00WO transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.

[0077] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0078] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0079] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non- coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0080] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0081] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a 19Attorney Docket No.: 046483-6287-00WO position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0082] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0083] In the context of the present invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N- glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0084] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0085] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject (e.g., a human).

[0086] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA 20Attorney Docket No.: 046483-6287-00WO may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell. For example, in some aspects, the nucleotide sequence comprises an mRNA where some or all of the uridines have been replaced with pseudouridine, 1-methyl pseudouridine, or another modified nucleoside.

[0087] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0088] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0089] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0090] In certain instances, the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA 21Attorney Docket No.: 046483-6287-00WO (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0091] In some embodiments, “pseudouridine” refers to m1acp3Ψ (1-methyl-3-(3- amino-3-carboxypropyl) pseudouridine). In another embodiment, the term refers to m1Ψ (1- methylpseudouridine). In another embodiment, the term refers to Ψm (2’-O- methylpseudouridine. In another embodiment, the term refers to m5D (5- methyldihydrouridine). In another embodiment, the term refer - methylpseudouridine). In another embodiment, the term refers ouridine moiety thatis not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0092] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0093] As used herein, “fragment” is defined as at least a portion of a sequence. For example, in one embodiment, the term “fragment” refers to a portion of the variable region of the immunoglobulin molecule which binds to its target, i.e. the antigen binding region. Some of the constant region of the immunoglobulin may be included. 22Attorney Docket No.: 046483-6287-00WO

[0094] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and a reference peptide can differ in amino acid sequence by at least one substitution, addition, or deletion, or any combination thereof. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.

[0095] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

[0096] By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody or binding fragment thereof, is meant an antibody or binding fragment thereof which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody or binding fragment thereof that specifically binds to an antigen from one species may also bind to that antigen from at least one other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody or binding fragment thereof that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody or binding 23Attorney Docket No.: 046483-6287-00WO fragment thereof as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody or binding fragment thereof, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody or binding fragment thereof specifically binds epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody or binding fragment thereof, will reduce the amount of labeled A bound to the antibody or binding fragment thereof.

[0097] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.

[0098] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, at least one of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0099] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0100] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0101] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a 24Attorney Docket No.: 046483-6287-00WO polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0102] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0103] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains at least one double and / or triple bond), having from one to twenty-four carbon atoms (C1-C24 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1-methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-1-enyl, pent-1- enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless specifically stated otherwise, an alkyl group is optionally substituted.

[0104] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains at least one double (alkenylene) and / or triple bond (alkynylene)), and having, for example, from one to twenty- four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene),one to twelve carbon atoms (C1-C12alkylene), one to eight carbon atoms (C1-C8alkylene), one to six carbon atoms (C1-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, n butylene, ethenylene, propenylene, n butenylene, propynylene, n butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group 25Attorney Docket No.: 046483-6287-00WO through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0105] “Cycloalkyl” or “carbocyclic ring” refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, or having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.

[0106] “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.

[0107] “Heterocyclyl” or “heterocyclic ring” refers to a stable 3 to 18 membered non aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2 oxopiperazinyl, 2 oxopiperidinyl, 2 oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4 piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 oxo thiomorpholinyl, and 1,1 dioxo thiomorpholinyl. Unless specifically stated otherwise, a heterocyclyl group may be optionally substituted. 26Attorney Docket No.: 046483-6287-00WO

[0108] The term “substituted” used herein means any of the above groups (e.g., alkyl, cycloalkyl or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non- hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=O); hydroxyl groups (-OH); alkoxy groups ( O Ra, where Rais C1-C12 alkyl or cycloalkyl); carboxyl groups ( OC(=O) Raor –C(=O)ORa, where Rais H, C1-C12 alkyl or cycloalkyl); amine groups ( N RaRb, where Raand Rbare each independently H, C1-C12alkyl or cycloalkyl); C1-C12alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0109] “Optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0110] As used herein, the term “genome editing vector” refers to a nucleic acid molecule which encodes the components of a genome editing system, such as, but not limited to, a CRISPR / Cas9 protein, a base editor, or a prime editor, and any associated required components, such as an appropriate guide RNA (gRNA). See Kantor et al., “CRISPR-Cas9 DNA Base-Editing and Prime Editing,” Int J Mol Sci, 2020; 21; p.6240, the contents of which are incorporated by reference.

[0111] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered 27Attorney Docket No.: 046483-6287-00WO to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description

[0112] In one embodiment, the invention relates to compositions comprising at least one delivery vehicle conjugated to at least one immune cell targeting ligand further comprising an mRNA molecule encoding at least one tissue trafficking modulator, or a variant or fragment thereof. In some embodiments, the immune cell targeting ligand binds to a receptor on the surface of at least one target immune cell or at least one specific subset of immune cells. In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP). In some embodiments, the delivery vehicle comprises or encapsulates a nucleic acid molecule encoding at least one tissue trafficking modulator. In one embodiment, the agent comprises a messenger RNA (mRNA) molecule encoding at least one tissue trafficking modulator.

[0113] The term “trafficking” as used herein refers to the process by which immune cells migrate to a target location (e.g., tissue, cell, niche etc.) along chemotactic gradients. Therefore, in some embodiments, the invention relates to compositions and methods that alter the chemotactic gradients of a target location (e.g., tissue, cell, niche etc.) to increase recruitment or retention of a specific immune cell subset. In some embodiments, the invention relates to compositions and methods that alter immune cells to recognize and traverse along chemotactic gradients that they would not otherwise traverse (e.g., by expressing receptors which recognize and bind to ligands expressed by or released in a target location (e.g., tissue, cell, niche etc.).

[0114] In some embodiments, the delivery vehicle comprises a targeting ligand. The term “targeting” as used herein generally refers to specific binding between a first molecule attached to the surface of a first particle, delivery vehicle or cell and a second molecule attached to the surface of a second particle, delivery vehicle or cell. For example, in some embodiments, the term targeting is used in reference to a delivery vehicle (e.g., LNP) linked to a ligand which specifically binds to a surface receptor on a specific cell type. In some embodiments, the immune cell targeting ligand that can be incorporated into the targeted LNP 28Attorney Docket No.: 046483-6287-00WO compositions comprises CX3CL1 (“fractalkine”), variants thereof and fragments thereof. In some embodiments, the fragment of CXCL1 comprises at least the chemokine binding domain of fractalkine. In some embodiments, the immune cell targeting ligand comprises at least SEQ ID NO:2. In some embodiments, the immune cell targeting ligand comprises the full length fractalkine, including the mucin-like tail. In some embodiments, the immune cell targeting ligand comprises at least SEQ ID NO:1. In some embodiments, the fractalkine targeted LNP (fractalkine-LNP) comprises at least one mRNA molecule encoding a tissue trafficking modulator for altering the trafficking of the targeted immune cell. In some embodiments, the targeted immune cell is a cytotoxic T cell or a natural killer (NK) cell. In some embodiments, the immune cell targeting ligand that can be incorporated into the targeted LNP compositions comprises IL-2, variants thereof and fragments thereof. In some embodiments, the immune cell targeting ligand comprises at least SEQ ID NO:3. In some embodiments, the IL-2 targeted LNP (IL-2-LNP) comprises at least one mRNA molecule encoding a tissue trafficking modulator for altering the trafficking of the targeted immune cell. In some embodiments, the targeted immune cell is a regulatory T cell.

[0115] In some embodiments, the fractalkine-LNP or the IL-2-LNP comprises at least one mRNA molecule encoding a trafficking modulator for expression on the targeted immune cell. Trafficking modulators include, but are not limited to, ligands for receptors expressed on a target cell and chemokine receptors. For example, in some embodiments, the fractalkine-LNP comprise at least one nucleic acid molecule encoding CD62L wild type, CD62L cleavage- defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69 for modulating the trafficking of a cytotoxic T lymphocyte (CTL) to secondary lymphoid tissue (SLT). In some embodiments, the IL-2-LNP comprises at least one nucleic acid molecule encoding CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4 for modulating the trafficking of a Treg to pancreatic islet cells. However, the invention is not limited to the specific embodiments exemplified herein, but encompasses fractalkine-LNP or IL-2-LNP comprising mRNA molecules encoding receptors, ligands or binding molecules for expression by an immune cell to alter the trafficking of the immune cell to a target cell, tissue or niche comprising the corresponding ligand, receptor or antigen. 29Attorney Docket No.: 046483-6287-00WO

[0116] In one embodiment, the present invention relates to compositions comprising at least one delivery vehicle conjugated to at least one chemokine ligand, wherein the at least one delivery vehicle comprises at least one agent, wherein the at least one chemokine ligand binds to at least one receptor on the surface of at least one target cell, wherein the at least one chemokine ligand comprises fractalkine, or a fragment or variant thereof, wherein the at least one target cell comprises at least one immune cell expressing the cell surface receptor fractalkine, wherein the at least one agent is delivered to the at least one immune cell. In one embodiment, the at least one target immune cell or immune cell subset is vascular cytolytic lymphocyte subsets (e.g. Temra, Tem subsets) of CD4+ and CD8+ T cells, CD16+ NK cells, monocytes / macrophages, or activated neutrophils, or a combination thereof. In some embodiments, the at least one agent comprises an mRNA molecule encoding a trafficking modulator. In some embodiments, the trafficking modulator comprises a CAR molecule for binding to a specific target cell type. In some embodiments, the trafficking modulator comprises a chemokine receptor. In some embodiments, the trafficking modulator comprises CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69 or a fragment or variant thereof. In some embodiments, the at least one delivery vehicle comprises an LNP.

[0117] In one embodiment, the present invention relates to compositions comprising at least one delivery vehicle conjugated to at least one cytokine ligand, wherein the at least one delivery vehicle comprises at least one agent, wherein the at least one cytokine ligand binds to at least one receptor on the surface of at least one target cell, wherein the at least one cytokine ligand comprises IL-2, or a variant or fragment thereof, wherein the at least one target cell comprises at least one immune cell expressing the cell surface receptor CD25, wherein the at least one agent is delivered to the at least one immune cell. In one embodiment, the at least one target immune cell or immune cell subset is T cells or regulatory T cells (Tregs). In some embodiments, the at least one agent comprises at least one mRNA molecule encoding a trafficking modulator. In some embodiments, the trafficking modulator comprises a chemokine receptor. In some embodiments, the trafficking modulator comprises CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4 or a fragment or variant thereof. In some embodiments, the at least one delivery vehicle comprises an LNP. 30Attorney Docket No.: 046483-6287-00WO

[0118] In some embodiments, the tissue trafficking modulator comprises an amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, or SEQ ID NO:61, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, or SEQ ID NO:61 retains the function of altering the trafficking of an immune cell.

[0119] In some embodiments, the tissue trafficking modulator is encoded by a nucleotide sequence as set forth in SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO:62, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO:62 encodes an amino acid sequence which retains the function of altering the trafficking of an immune cell. In some embodiments, the nucleotide sequence is a DNA sequence. In some embodiments, the nucleotide sequence is an RNA sequence.

[0120] In some embodiments, the nucleotide sequence is an RNA molecule transcribed from a plasmid as set forth in SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, or SEQ ID NO:63.

[0121] In some embodiments, the disclosure provides a method of modulating or altering the trafficking of an immune cell to a cell, tissue or cellular niche. In some embodiments, at least one immune cell targeted delivery vehicle is taken up via endocytosis by 31Attorney Docket No.: 046483-6287-00WO at least one target immune cell. In some embodiments, the at least one RNA molecule encoding a tissue trafficking modulator is released from the at least one delivery vehicle inside the at least one target immune cell. Once released, expression of the trafficking modulator modifies the trafficking of the target immune cell to travel along a chemotaxis gradient that it would not otherwise travel. In some embodiments, expression of the trafficking modulator modifies the trafficking of the target immune cell to travel to a specific tissue or cellular niche. In some embodiments, expression of the trafficking modulator modifies the trafficking of the target immune cell to retain the immune cell in a specific tissue or cellular niche for an extended period of time over the time the immune cell would remain in the specific tissue or cellular niche in the absence of the trafficking modulator. In some embodiments, trafficking, or retention, of immune cells to a specific cellular niche is beneficial for the clearance of an infectious disease or pathogen. In some embodiments, trafficking, or retention, of immune cells to a specific cellular niche is beneficial for the treatment or prevention of cancer. In some embodiments, trafficking, or retention. of immune cells to a specific cellular niche is beneficial for the suppression of autoimmune diseases.

[0122] The invention relates, in part, to methods of treating a disease or disorder in a subject in need thereof, the method comprising the administration of a composition comprising at least one immune cell targeted delivery vehicle, wherein the at least one delivery vehicle comprises at least one nucleic acid molecule encoding a trafficking modulator, wherein the at least one chemokine or cytokine ligand binds to at least one receptor on the surface of at least one target cell in the subject, and wherein the nucleic acid molecule encoding a trafficking modulator is delivered to the at least one target cell in the subject, and wherein expression of the trafficking modulator alters the trafficking of the target cell. In some embodiments, the trafficking modulator alters the trafficking of the target cell to a cell, tissue or niche that it would not traffic to in the absence of the trafficking modulator. In some embodiments, the trafficking modulator increases the frequency of trafficking of the target cell type to a cell, tissue or niche that it would not traffic to in the absence of the trafficking modulator. In some embodiments, the trafficking modulator retains the target cell type at a cell, tissue or niche that it would traffic away from in the absence of the trafficking modulator. 32Attorney Docket No.: 046483-6287-00WO

[0123] In some embodiments, the targeted LNP of the invention further comprises at least one additional agent for delivery to the target immune cell. In some embodiments, the at least one additional agent is a therapeutic agent. In some embodiments, the at least one additional agent is a nucleic acid molecule encoding an immunosuppressive chemokine. In some embodiments, at least one additional agent is a nucleic acid molecule encoding a CAR molecule. In some embodiments, at least one additional agent is a gene editing agent.

[0124] In one embodiment, the present disclosure provides compositions and methods for treating or preventing a disease or disorder (e.g., cancer, infection, immune diseases etc.) in a subject by using an immunotherapy approach that involves altering the trafficking of a subject’s immune cells in vivo, ex vivo or in vitro to target and destroy or repress at least one immune cell, cancer cell or at least one pathogen, thereby treating or preventing the disease. Exemplary diseases and disorders that can be treated using the methods and compositions of the invention include, but are not limited to, cancers, infectious diseases, and immunological disorders.

[0125] In one embodiment, the present invention relates to compositions comprising at least one targeted LNP wherein the targeted LNP comprises at least one nucleic acid molecule encoding a trafficking modulator, wherein the targeted LNP binds to at least one receptor on the surface of at least one target cell, and wherein the at least one nucleic acid molecule encoding a trafficking modulator is delivered to the at least one target cell, wherein expression of the trafficking modulator alters the cellular tracking of the at least one immune cell.

[0126] In one embodiment, the present invention relates to compositions comprising at least one IL-2-LNP wherein the IL-2-LNP comprises at least one nucleic acid molecule encoding a trafficking modulator, wherein the IL-2-LNP binds to at least one receptor on the surface of at least one target immune cell, and wherein the at least one nucleic acid molecule encoding a trafficking modulator is delivered to the at least one target immune cell, wherein expression of the trafficking modulator alters the cellular tracking of the at least one target immune cell. In some embodiments, the target immune cell is a regulatory T cell. In some embodiments, the target immune cell is an activated CD25+ T cell.

[0127] In one embodiment, the present invention relates to compositions comprising at least one fractalkine-LNP wherein the fractalkine-LNP comprises at least one nucleic acid 33Attorney Docket No.: 046483-6287-00WO molecule encoding a trafficking modulator, wherein the fractalkine-LNP binds to at least one receptor on the surface of at least one target immune cell, and wherein the at least one nucleic acid molecule encoding a trafficking modulator is delivered to the at least one target immune cell, wherein expression of the trafficking modulator alters the cellular tracking of the at least one target immune cell. In some embodiments, the target immune cell is a CTL.

[0128] In some embodiments, the LNP is administered at a dose ranging between 0.01 µg / million cells to 10 µg / million cells. Typically, dosages which may be administered in a method of the invention to a mammal, such as a human, range in amount from 0.01 μg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In some embodiments, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of body weight of the mammal. In some embodiments, the dosage will vary from about 1 μg to about 1 mg per kilogram of body weight of the mammal. In one embodiment, the nucleic acid molecule encoding a trafficking modulator comprises messenger RNA (mRNA).

[0129] In some embodiments, the LNP comprises a mass ratio of mRNA to ligand ranging between 100:1 to 1:1 to 1:100. In some embodiments, the composition comprises a mass ratio of mRNA to ligand of about 1:2 to about 1:10. In some embodiments, the composition comprises a mass ratio of mRNA to chemokine ligand of about 1:2. In some embodiments, the composition comprises a mass ratio of mRNA to fractalkine of about 1:2. In some embodiments, the composition comprises a mass ratio of mRNA to cytokine of about 1:8. In some embodiments, the composition comprises a mass ratio of mRNA to IL-2 of about 1:8. Delivery Vehicle

[0130] In one embodiment, the present invention relates to nanoparticle (LNP) compositions and the use of the LNP as a delivery vehicle for at least one tissue trafficking modulator. 34Attorney Docket No.: 046483-6287-00WO

[0131] In various embodiments, the composition comprises lipids or a derivative thereof. In various embodiments, the composition comprises a nanoparticle (LNP).

[0132] Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, aldehydes, and polymers (e.g., PEGylated lipids).

[0133] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Chol”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.

[0134] In one embodiment, the LNP comprises at least one cationic lipid, and at least one stabilizing lipid. Stabilizing lipids include neutral lipids and pegylated lipids.

[0135] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0136] In certain embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N- distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N—(N′,N′-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), 35Attorney Docket No.: 046483-6287-00WO dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM®(commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), N,N-dimethyl-2,3-bis(((9Z,12Z,15Z)-octadeca-9,12,15- trien-1-yl)oxy)propan-1-amine (DLenDMA).

[0137] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3- (2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0138] Suitable amino lipids include, but are not limited to, those having the formula: 36Attorney Docket No.: 046483-6287-00WOwherein R1 and R2 are either the same or different and independently optionally substituted C10-C24alkyl, optionally substituted C10-C24alkenyl, optionally substituted C10-C24alkynyl, or optionally substituted C10-C24acyl; R3 and R4 are either the same or different and independently optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl or R3and R4may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5 is either absent or present and when present is hydrogen or C1-C6 alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and independently O, S, or NH.

[0139] In one embodiment, R1and R2are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0140] A representative useful dilinoleyl amino lipid has the formula:wherein n is 0, 1, 2, 3, or 4.

[0141] In one embodiment, the cationic lipid is a DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2). 37Attorney Docket No.: 046483-6287-00WO

[0142] In one embodiment, the cationic lipid component of the LNPs has the structure of Formula (I): or a pharmaceutically acceptable somer thereof, wherein:L12and L are each independently ˗O(C=O)˗, ˗(C=O)O˗ or a carbon-carbon double bond; R1aand R1bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R1ais H or C1-C12alkyl, and R1btogether with the carbon atom to which it is bound is taken together with an adjacent R1band the carbon atom to which it is bound to form a carbon- carbon double bond; R2aand R2bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R2ais H or C1-C12alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon- carbon double bond; R3aand R3bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R3ais H or C1-C12alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon- carbon double bond; R4aand R4bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R4ais H or C1-C12alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon- carbon double bond; R5and R6are each independently methyl or cycloalkyl; R7is, at each occurrence, independently H or C1-C12alkyl; 38Attorney Docket No.: 046483-6287-00WO R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.

[0143] In certain embodiments of Formula (I), at least one of R1a, R2a, R3aor R4ais C1- C12 alkyl, or at least one of L1or L2is –O(C=O)- or –(C=O)O-. In other embodiments, R1aand R1bare not isopropyl when a is 6 or n-butyl when a is 8.

[0144] In still further embodiments of Formula (I), at least one of R1a, R2a, R3aor R4ais C1-C12 alkyl, or at least one of L1or L2is ˗O(C=O)˗ or ˗(C=O)O˗; and R1aand R1bare not isopropyl when a is 6 or n-butyl when a is 8.

[0145] In other embodiments of Formula (I), R8and R9are each independently unsubstituted C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

[0146] In certain embodiments of Formula (I), any one of L1or L2may be ˗O(C=O)˗ or a carbon-carbon double bond. L1and L2may each be ˗O(C=O)˗ or may each be a carbon- carbon double bond.

[0147] In some embodiments of Formula (I), one of L1or L2is ˗O(C=O)˗. In other embodiments, both L1and L2are ˗O(C=O)˗.

[0148] In some embodiments of Formula (I), one of L1or L2is ˗(C=O)O˗. In other embodiments, both L1and L2are ˗(C=O)O˗.

[0149] In some other embodiments of Formula (I), one of L1or L2is a carbon-carbon double bond. In other embodiments, both L1and L2are a carbon-carbon double bond.

[0150] In still other embodiments of Formula (I), one of L1or L2is ˗O(C=O)˗ and the other of L1or L2is ˗(C=O)O˗. In more embodiments, one of L1or L2is ˗O(C=O)˗ and the other of L1or L2is a carbon-carbon double bond. In yet more embodiments, one of L1or L2is ˗(C=O)O˗ and the other of L1or L2is a carbon-carbon double bond.

[0151] It is understood that “carbon-carbon” double bond, as used throughout the specification, refers to one of the following structures: 39Attorney Docket No.: 046483-6287-00WO wherein Raand Rbare, at each occu substituent. For example, in some embodiments Raand Rbare, a, ently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12alkyl.

[0152] In other embodiments, the lipid compounds of Formula (I) have the following structure (Ia):

[0153] In other emb (I) have the following structure (Ib):

[0154] In yet other e ula (I) have thefollowing structure (Ic):Attorney Docket No.: 046483-6287-00WO

[0155] In certain embodiments of the lipid compound of Formula (I), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0156] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0157] In some more embodiments of Formula (I), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0158] In some certain other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16. 41Attorney Docket No.: 046483-6287-00WO

[0159] In some other various embodiments of Formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0160] The sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

[0161] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0162] The substituents at R1a, R2a, R3aand R4aof Formula (I) are not particularly limited. In certain embodiments R1a, R2a, R3aand R4aare H at each occurrence. In certain other embodiments at least one of R1a, R2a, R3aand R4ais C1-C12 alkyl. In certain other embodiments at least one of R1a, R2a, R3aand R4ais C1-C8alkyl. In certain other embodiments at least one of R1a, R2a, R3aand R4ais C1-C6alkyl. In some of the foregoing embodiments, the C1-C8alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0163] In certain embodiments of Formula (I), R1a, R1b, R4aand R4bare C1-C12alkyl at each occurrence.

[0164] In further embodiments of Formula (I), at least one of R1b, R2b, R3band R4bis H or R1b, R2b, R3band R4bare H at each occurrence.

[0165] In certain embodiments of Formula (I), R1btogether with the carbon atom to which it is bound is taken together with an adjacent R1band the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0166] The substituents at R5and R6of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R5or R6is methyl. In certain other embodiments one or both of R5or R6is cycloalkyl for example cyclohexyl. In these 42Attorney Docket No.: 046483-6287-00WO embodiments the cycloalkyl may be substituted or not substituted. In certain other embodiments the cycloalkyl is substituted with C1-C12 alkyl, for example tert-butyl.

[0167] The substituents at R7are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments at least one R7is H. In some other embodiments, R7is H at each occurrence. In certain other embodiments R7is C1-C12 alkyl.

[0168] In certain other of the foregoing embodiments of Formula (I), one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0169] In some different embodiments of Formula (I), R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

[0170] In various different embodiments, an exemplary lipid of Formula (I) can includeAttorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WO O OAttorney Docket No.: 046483-6287-00WO N O NAttorney Docket No.: 046483-6287-00WO

[0171] In some embodiments, the LNP comprises a lipid of Formula (I), at least one agent, and at least one excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (I) is compound I-5. In some embodiments the lipid of Formula (I) is compound I-6.

[0172] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (II): 49Attorney Docket No.: 046483-6287-00WO or a pharmaceutic drug or stereoisomerthereof, wherein: L1and L2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NR, -OC(=O)NRa-, -NRaC(=O)O-, or a direct bond; G1is C1-C2alkylene, –(C=O)- , -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond; G2is –C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NRaor a direct bond; G3is C1-C6 alkylene; Rais H or C1-C12alkyl; R1aand R1bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R1ais H or C1-C12alkyl, and R1btogether with the carbon atom to which it is bound is taken together with an adjacent R1band the carbon atom to which it is bound to form a carbon- carbon double bond; R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2ais H or C1-C12alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon- carbon double bond; R3aand R3bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R3ais H or C1-C12alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon- carbon double bond; 50Attorney Docket No.: 046483-6287-00WO R4aand R4bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon- carbon double bond; R5and R6are each independently H or methyl; R7is C4-C20alkyl; R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[0173] In some embodiments of Formula (II), L1and L2are each independently –O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G1and G2are each independently -(C=O)- or a direct bond. In some different embodiments, L1and L2are each independently –O(C=O)-, -(C=O)O- or a direct bond; and G1and G2are each independently – (C=O)- or a direct bond.

[0174] In some different embodiments of Formula (II), L1and L2are each independently -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRa-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC(=O)O-, -NRaS(O)xNRa-, -NRaS(O)x- or -S(O)xNRa-.

[0175] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following structures (IIA) or (IIB):Attorney Docket No.: 046483-6287-00WO

[0176] In some embodiments of Formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

[0177] In any of the foregoing embodiments of Formula (II), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-.

[0178] In some different embodiments of Formula (II), one of L1or L2is -(C=O)O-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0179] In different embodiments of Formula (II), one of L1or L2is a direct bond. As used herein, a “direct bond” means the group (e.g., L1or L2) is absent. For example, in some embodiments each of L1and L2is a direct bond.

[0180] In other different embodiments of Formula (II), for at least one occurrence of R1aand R1b, R1ais H or C1-C12 alkyl, and R1btogether with the carbon atom to which it is bound is taken together with an adjacent R1band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0181] In still other different embodiments of Formula (II), for at least one occurrence of R4aand R4b, R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0182] In more embodiments of Formula (II), for at least one occurrence of R2aand R2b, R2ais H or C1-C12alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon- carbon double bond.

[0183] In other different embodiments of Formula (II), for at least one occurrence of R3aand R3b, R3ais H or C1-C12alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0184] In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID): 52Attorney Docket No.: 046483-6287-00WO or ,

[0185] In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[0186] In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0187] In certain embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0188] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In 53Attorney Docket No.: 046483-6287-00WO other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0189] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0190] In some certain embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0191] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

[0192] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[0193] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In 54Attorney Docket No.: 046483-6287-00WO some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[0194] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

[0195] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

[0196] The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0197] The substituents at R1a, R2a, R3aand R4aof Formula (II) are not particularly limited. In some embodiments, at least one of R1a, R2a, R3aand R4ais H. In certain embodiments R1a, R2a, R3aand R4aare H at each occurrence. In certain other embodiments at least one of R1a, R2a, R3aand R4ais C1-C12alkyl. In certain other embodiments at least one of R1a, R2a, R3aand R4ais C1-C8alkyl. In certain other embodiments at least one of R1a, R2a, R3aand R4ais C1-C6 alkyl. In some of the foregoing embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0198] In certain embodiments of Formula (II), R1a, R1b, R4aand R4bare C1-C12alkyl at each occurrence.

[0199] In further embodiments of Formula (II), at least one of R1b, R2b, R3band R4bis H or R1b, R2b, R3band R4bare H at each occurrence. 55Attorney Docket No.: 046483-6287-00WO

[0200] In certain embodiments of Formula (II), R1btogether with the carbon atom to which it is bound is taken together with an adjacent R1band the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0201] The substituents at R5and R6of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R5or R6is methyl. In other embodiments each of R5or R6is methyl.

[0202] The substituents at R7of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R7is C6-C16alkyl. In some other embodiments, R7is C6-C9 alkyl. In some of these embodiments, R7is substituted with -(C=O)ORb, –O(C=O)Rb, -C(=O)Rb, -ORb, -S(O)xRb, -S-SRb, -C(=O)SRb, -SC(=O)Rb, -NRaRb, -NRaC(=O)Rb, -C(=O)NRaRb, -NRaC(=O)NRaRb, -OC(=O)NRaRb, -NRaC(=O)ORb, -NRaS(O)xNRaRb, -NRaS(O)xRbor -S(O)xNRaRb, wherein: Rais H or C1-C12 alkyl; Rbis C1-C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R7is substituted with -(C=O)ORbor –O(C=O)Rb.

[0203] In various of the foregoing embodiments of Formula (II), Rbis branched C1-C15alkyl. For example, in some embodiments Rbhas one of the following structures: ; r [0208 9R or R is methyl. In other embodiments, both R8and R9are methyl.

[0205] In some different embodiments of Formula (II), R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they 56Attorney Docket No.: 046483-6287-00WO are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

[0206] In still other embodiments of the foregoing lipids of Formula (II), G3is C2-C4 alkylene, for example C3 alkylene.

[0207] In various different embodiments, the lipid compound has one of the following structures:   N NAttorney Docket No.: 046483-6287-00WO OAttorney Docket No.: 046483-6287-00WO O N N OAttorney Docket No.: 046483-6287-00WO O OAttorney Docket No.: 046483-6287-00WO O OAttorney Docket No.: 046483-6287-00WO O

[0208] In some em o ments, t e compr ses a p o ormu a ( I), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound II-10. In some embodiments, the lipid of Formula (II) is compound II-11. In some embodiments, the lipid of Formula (II) is compound II-12. In some embodiments, the lipid of Formula (II) is compound II-32.

[0209] In some other embodiments, the cationic lipid component of the LNP has the structure of Formula (III): or a pharmaceutically, prodrug or stereoisomer thereof, wherein: one of L1or L2is –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, - C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, ,NRaC(=O)NRa-, -OC(=O)NRa- or - NRaC(=O)O- or a direct bond; G1and G2are each independently unsubstituted C1-C12alkylene or C1-C12alkenylene; 62Attorney Docket No.: 046483-6287-00WO G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Rais H or C1-C12 alkyl; R1and R2are each independently C6-C24alkyl or C6-C24alkenyl; R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or –NR5C(=O)R4; R4is C1-C12alkyl; R5is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0210] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R6is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0211] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0212] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID): wherein y and z are ea

[0213] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some 63Attorney Docket No.: 046483-6287-00WO different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0214] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF): R3G3.

[0215] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ): ; .

[0216] r rangingfrom 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0217] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. 64Attorney Docket No.: 046483-6287-00WO

[0218] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0219] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0220] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6- C24alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: , wherein:R7aand R7bare, at each occurrence, independently H or C1-C12alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0221] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert- butyl, n-hexyl or n-octyl.

[0222] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures: ; ;Attorney Docket No.: 046483-6287-00WO

[0223] In some of the foregoing embodiments of Formula (III), R3is OH, CN, -C(=O)OR4, -OC(=O)R4or –NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0224] In various different embodiments, the cationic lipid of Formula (III) has one of the following structures:Attorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WOAttorney Docket No.: 046483-6287-00WO

[0225] In some embodiments, the LNP comprises a lipid of Formula (III), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7.

[0226] In some other embodiments, the cationic lipid component of the LNP has the structure of Formula (IV): or a pharmaceutic, prodrug or stereoisomer thereof, wherein: Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4, m i o ip is an integer from 1 to 4, wherein when p = 1 , each R is independently C6 to C16 straight-chain alkyl; C6 to C16 branched alkyl; C6 to C16 straight-chain alkenyl; C6 to C16 branched alkenyl; C9 to C16 71Attorney Docket No.: 046483-6287-00WO cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl chain; or C8 to C18 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each R is independently C6 to C14 straight-chain alkyl; C6 to C14 straight-chain alkenyl; C6 to C14 branched alkyl; C6 to C14 branched alkenyl; C9 to C14 cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at the either end or within the alkyl chain; or C8to C16aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 3, each R is independently C6to C12straight-chain alkyl; C6to C12straight-chain alkenyl; C6to C12branched alkyl; C6to C12branched alkenyl; C9to C12cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl chain; or C8 to C14 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each R is independently C6 to C10 straight-chain alkyl; C6 to C10 straight-chain alkenyl; C6 to C10 branched alkyl; C6 to C10 branched alkenyl; C9 to C10 cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0227] Some embodiments specifically include one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. In some embodiments, when p is 1, each R is independently C6to C12, C13, or C14straight-chain alkyl. In some embodiments, each R from a nearest common branch point is the same. In some embodiments, each R is the same.

[0228] In some embodiments, the ionizable cationic lipid has a structure of Formula IVa 72Attorney Docket No.: 046483-6287-00WOt-chain alkyl; C6to C16branched alkyl; C6to C16straight-chain alkenyl; C6to C16branched alkenyl; C9to C16cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl chain; or C8to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,m is a o is an

[0229] In some other embodiments, the cationic lipid component of the LNP has the structure of Formula (V): 73Attorney Docket No.: 046483-6287-00WOm is a o is anp is an integer from 1 to 4, wherein when p = 1 , each R is independently C6to C16straight-chain alkyl; C6to C16 branched alkyl; C6 to C16 straight-chain alkenyl; C6 to C16 branched alkenyl; C9 to C16 cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl chain; or C8to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each R is independently C6 to C14 straight-chain alkyl; C6 to C14straight-chain alkenyl; C6to C14branched alkyl; C6to C14branched alkenyl; C9to C14cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at the either end or within the alkyl chain; or C8 to C16 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, 74Attorney Docket No.: 046483-6287-00WO wherein when p = 3, each R is independently C6 to C12 straight-chain alkyl; C6 to C12 straight-chain alkenyl; C6 to C12 branched alkyl; C6 to C12 branched alkenyl; C9 to C12 cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8 to C14 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each R is independently C6to C10straight-chain alkyl; C6to C10straight-chain alkenyl; C6to C10branched alkyl; C6to C10branched alkenyl; C9to C10cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0230] Some embodiments include one or more species or subgenera based on specific choices of R, X, Y, m, n, o, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. In some embodiments, each R from a nearest common branch point is the same. In some embodiments, each R is the same.

[0231] In some embodiments, the ionizable cationic lipid has a structure of Formula Va wherein R is16straight-chain alkenyl; C6to C16branched alkyl; branched C6to C16alkenyl; C9to C16cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl chain; or C8 to C18 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, 75Attorney Docket No.: 046483-6287-00WO Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,m is a o is an

[0232] In some embodiments, the ionizable cationic lipid has a structure of Formula VI: whereinm io is an integer from 1 to 4, p is an integer from 1 to 4, 76Attorney Docket No.: 046483-6287-00WO wherein when p = 1 , each Rc is independently C8 to C18 straight-chain alkyl; C8 to C18 straight-chain alkenyl; C8 to C18 branched alkyl; C8 to C18 branched alkenyl; C11 to C18 cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C10 to C20 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each Rcis independently C8to C16straight-chain alkyl; C8to C16straight-chain alkenyl; C8to C16branched alkyl; C8to C16branched alkenyl; C11to C16cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at the either end or within the alkyl chain; or C10to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 3, each Rc is independently C8 to C14 straight-chain alkyl; C8 to C14 straight-chain alkenyl; C8 to C14 branched alkyl; C8 to C14 branched alkenyl; C11 to C14 cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C10 to C16 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each Rcis independently C8to C12straight-chain alkyl; C8to C12straight-chain alkenyl; C8to C12branched alkyl; C8to C12branched alkenyl; C11to C12cycloalkyl-alkyl in which the cycloalkyl is C3 to C8 cycloalkyl positioned at either end or within the alkyl; or C10to C14aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0233] Some embodiments include one or more species or subgenera based on specific choices of Rc, W, X, m, n, 0, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of Rc, W, X, m, n, 0, p, and / or carbon chain length, structure, or saturation. In some embodiments, each Rc from a nearest common branch point is the same. In some embodiments, each Rcis the same.

[0234] In some embodiments, the ionizable cationic lipid has a structure of Formula VIa: 77Attorney Docket No.: 046483-6287-00WO wherein Rc is C18 straight-chain alkenyl; C8 to C18 branched alkyl; C8 to C8 y ; 8 y oalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C10to C20aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein W is C=O or CH2,m is an o is an

[0235] With respect to each of the forgoing aspects, in some embodiments, all four R groups are identical. In other embodiments, the two Rcgroups stemming from a first branchpoint are identical to each other and the two Rcgroups from a second branchpoint are identical to each other, but the Rc groups stemming from the first branchpoint are different than the R groups stemming from the second branchpoint.

[0236] With respect to each of the forgoing aspects, some embodiments are limited to one, or a subset, of the alternatives for Rc, W, X, Y, m, n, 0, and / or p, as applicable. Other embodiments specifically exclude one, or a subset, of the alternatives for Rc, W, X, Y, m, n, o, 78Attorney Docket No.: 046483-6287-00WO p, and / or carbon chain length, structure, or saturation, as applicable. Each range of carbon chain length is meant to convey embodiments of all individual lengths and subranges therein.

[0237] With respect to each of the foregoing aspects and embodiments, in some instances Rc is straight-chain alkyl and in further instances the chain is unsubstituted. In still further instances, Rc is C8 or C9 or C10 to C12.

[0238] With respect to each of the foregoing aspects and embodiments, in some / Ninstances X is . With respect to each of the foregoing aspects and embodiments, in someinstances Y isand in other instances Y is NH or N-CH3.

[0239] Ionizable cationic lipids of this disclosure have a branched structure to give the lipid a conical rather than cylindrical shape and such structure helps promote endosomolytic activity. The greater the endosomolytic activity, the more efficient release of the nucleotide cargo.

[0240] To promote biodegradability and minimize the accumulation of ionizable cationic lipids of this disclosure, the fatty acid tails are designed to comprise esters in a position that minimizes steric hinderance of ester cleavage. For example, while a single fatty acid tail will tend to extend away from the ester carbonyl, the presence of two tails leads to the tails extending in opposite directions as this is an energetically favorable conformation. This means one of the tails may extend toward the carbonyl and sterically hinder cleavage of the ester. Accordingly, large branches immediately adjacent to the ester carbonyl were avoided. In positioning the ester(s) within the lipid, consideration was also given to potential degradation products to avoid the generation of toxic compounds, such as formaldehyde.

[0241] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[0242] In certain embodiments, the LNP comprises at least one additional lipid which stabilizes the formation of particles during their formation.

[0243] Suitable stabilizing lipids include neutral lipids and anionic lipids. 79Attorney Docket No.: 046483-6287-00WO

[0244] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0245] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

[0246] In some embodiments, the LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I to VI)) to the neutral lipid ranges from about 2:1 to about 8:1.

[0247] In various embodiments, the LNP further comprises a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton: .

[0248] In certain embodimanalogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I to VI)) to cholesterol ranges from about 2:1 to 1:1.

[0249] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, 80Attorney Docket No.: 046483-6287-00WO diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0250] In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GM1). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

[0251] In some embodiments, the LNP comprises a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid.

[0252] In certain embodiments, the LNP comprises a polyethylene glycol-lipid (pegylated lipid). The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. In some embodiments, a PEG is of 500-5000 or 10DO- 5000 Da molecular weight (MW). In some embodiments, the PEG unit has a MW of 2000 Da. In some instances, the MW2000 PEG-lipid comprises DMG-PEG2000 (1 ,2- dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1 ,2- dipalmitoyl- glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1 ,2- distearoyl-glycero-3- methoxypolyethylene glycol-2000), DOG-PEG2000 (1 ,2-dioleoyl- glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1 ,2-dimyristoyl- glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE- PEG2000 (1 ,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE- PEG2000 (1 ,2-distearoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol- 2000), DGPE-PEG2000 (1 ,2-dioleoyl-glycero-3- phosphoethanolamine-3- methoxypolyethylene glycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da. In some instances, the MW2000 PEG-lipid comprises DMrG- PEG2000 (1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DPrG- PEG2000 (1 ,2-dipalmitoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DSrG- PEG2000 (1 ,2-distearoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DOrG-PEG2000 (1 ,2-dioleoyl-glycero-3- methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1 ,2- dimyristoyl-rac-glycero- 3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), 81Attorney Docket No.: 046483-6287-00WO DPPEr-PEG2000 (1 ,2- dipalmitoyl-rac-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), DSPEr-PEG2000 (1 ,2-distearoyl-rac-glycero-3- phosphoethanolamine-3- methoxypolyethylene glycol-2000), DOPEr-PEG2000 (1 ,2-dioleoyl- rac-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof. Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG- CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c- DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(^-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ^-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(^-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 25:1.

[0253] A PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity. Additionally, a PEG moiety may impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG-moiety can also be functionalized to serve as an attachment point for a targeting moiety. Conjugating a cell- or tissue-specific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted 82Attorney Docket No.: 046483-6287-00WO tissue or cell type. PEG-lipid can thus serve as means for inhibiting LNP binding, and PEG- lipid conjugated to a binding moiety can serve as means for LNP-targeting.

[0254] In some embodiments, the LNP comprises a pegylated lipid having the following structure (VII): or a pharmace or stereoisomer thereof, wherein:R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by at least one ester bond; and z has mean value ranging from 30 to 60.

[0255] In some of the foregoing embodiments of the pegylated lipid (VII), R10and R11are not both n-octadecyl when z is 42. In some other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R10is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R11is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. 83Attorney Docket No.: 046483-6287-00WO

[0256] In various embodiments, z spans a range that is selected such that the PEG portion has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0257] In other embodiments, the pegylated lipid has one of the following structures: .g g g of the pegylated lipid is about 2500 g / mol.

[0258] In certain embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.

[0259] In some embodiments, the LNP comprises a lipid of Formula (I to VI), a nucleoside-modified RNA, a neutral lipid, a steroid and a pegylated lipid. In different embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In still different embodiments, the pegylated lipid is compound IVa.

[0260] Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 84Attorney Docket No.: 046483-6287-00WO 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids.2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[0261] The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III). GENERAL REACTION SCHEME 1 OORN OROHO NH2N OR paredaccording to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-1, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessarily workup and or purification step. 85Attorney Docket No.: 046483-6287-00WO GENERAL REACTION SCHEME 2

[0263] Other emb ) (e.g., compound B-5) canbe prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinum chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and / or purification.

[0264] It should be noted that although starting materials A-1 and B-1 are depicted above as including only saturated methylene carbons, starting materials which include carbon- carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds. 86Attorney Docket No.: 046483-6287-00WO GENERAL REACTION SCHEME 3 OOR O ORO HO NHBrm2HO N SOCl n2Cl C-4 N ' C-8 R R -7 or C9)can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. 87Attorney Docket No.: 046483-6287-00WO GENERAL REACTION SCHEME 4

[0266] Embodiments of the compound of Formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R8, R9, L1, L2, G1, G2, G3, a, b, c and d are as defined herein, and R7’represents R7or a C3-C19alkyl. Referring to General Reaction Scheme 1, compounds of structure D-1 and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of D-1 and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up. A solution of D-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary work up and / or purification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and / or purification. 88Attorney Docket No.: 046483-6287-00WO GENERAL REACTION SCHEME 5 reparedaccording to General Reaction Scheme 5 ( Method E ), wherein R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8, R9, L1, L2, G3, a, b, c and d are as defined herein. Referring to General Reaction Scheme 2, compounds of structure E-1 and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of E-1 (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylic acid and DCC) to obtain E-5 after any necessary work up and / or purification. 89Attorney Docket No.: 046483-6287-00WO GENERAL REACTION SCHEME 6

[0268] G Method F) for preparation of Lip s o o u a . , , a e e a eac on Scheme 6 are as defined herein for Formula (III), and G1’ refers to a one-carbon shorter homologue of G1. Compounds of structure F-1 are purchased or prepared according to methods known in the art. Reaction of F-1 with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester / alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).

[0269] It should be noted that various alternative strategies for preparation of lipids of Formula (III) are available to those of ordinary skill in the art. For example, other lipids of Formula (III) wherein L1and L2are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G1and G2are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G1and G2are different.

[0270] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t- butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R″ (where R″ is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. 90Attorney Docket No.: 046483-6287-00WO Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

[0271] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1000nm) which includes at least one lipid. In some embodiments, the LNP comprises at least one agent that is either organized within inverse lipid micelles and encased within a lipid monolayer envelope or intercalated between adjacent lipid bilayers (e.g. lipid bilayer-agent-lipid bilayer). In some embodiments, the morphology of the LNP is distinct from that of a traditional liposome, characterized by a lipid bilayer surrounding an aqueous core, as the LNP possesses an electron-dense core, where the cationic / ionizable lipids are organized into inverted micelles around the encapsulated agent (e.g. mRNA molecules)(Cullis and Hope, 2017; Guevara et al., 2019b). In various embodiments, the LNP includes a lipid of Formula (I to VI). In some embodiments, the LNP is included in a formulation comprising at least one agent as described herein. In some embodiments, the LNP comprises a cationic lipid (e.g., a lipid of Formula (I to VI)) and at least one excipient selected from a neutral lipid, charged lipid, steroid and lipid-anchored polyethylene glycol (e.g., a pegylated lipid such as a pegylated lipid of structure (VII). In some embodiments, the at least one agent is encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or all of the lipid portion of the at least one LNP, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g., an adverse immune response.

[0272] In various embodiments, the LNP has a mean diameter from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 91Attorney Docket No.: 046483-6287-00WO nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In one embodiment, the LNP has a mean diameter of about 83 nm. In one embodiment, the LNP has a mean diameter of about 102 nm. In one embodiment, the LNP has a mean diameter of about 103 nm. In some embodiments, the LNP is substantially non-toxic. In certain embodiments, the at least one agent, when present in the at least one LNP, is resistant in aqueous solution to degradation by intra- or intercellular enzymes

[0273] The LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated or complexed. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Exemplary lipids are shown elsewhere herein.

[0274] In one embodiment, the LNP comprises at least one cationic lipid, and at least one stabilizing lipid. Stabilizing lipids include neutral lipids, anionic lipids and pegylated lipids.

[0275] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term “cationic or ionizable lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0276] In various embodiments, the LNP comprises a cationic or ionizable lipids, stabilizing lipids, sterol, and a lipid-anchored polyethylene glycol (i.e PEGylated lipids).

[0277] In some embodiments, the LNP comprises an ionic lipid of Formula (I), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments the lipid of Formula (I) is compound I-5. In some embodiments the lipid of Formula (I) is compound I-6.

[0278] In some embodiments, the LNP comprises an ionic lipid of Formula (II), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound II-10. In some embodiments, the lipid of Formula (II) is 92Attorney Docket No.: 046483-6287-00WO compound II-11. In some embodiments, the lipid of Formula (II) is compound II-12. In some embodiments, the lipid of Formula (II) is compound II-32.

[0279] In some embodiments, the LNP comprises an ionic lipid of Formula (III), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7.

[0280] In some embodiments, the LNP comprises an ionic lipid of Formula (IV), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (IV) comprises a lipid of Formula IVa.

[0281] In some embodiments, the LNP comprises an ionic lipid of Formula (V), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (IV) comprises a lipid of Formula Va.

[0282] In some embodiments, the LNP comprises an ionic lipid of Formula (VI), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (IV) comprises a lipid of Formula VIa.

[0283] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[0284] In certain embodiments, the LNP comprises at least one stabilizing lipid (e.g. a neutral or anionic lipid) which helps to encapsulate the agent and stabilize the formation of particles during their formation.

[0285] Any suitable format of the at least one delivery vehicle is contemplated. In some embodiments, the at least one delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes, and lipid nanoparticles. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo include liposomes (e.g., an artificial membrane vesicle) and lipid nanoparticles. 93Attorney Docket No.: 046483-6287-00WO

[0286] The use of lipid formulations, as described above, is contemplated for the introduction of the at least one agent into the host cell (in vitro, ex vivo, or in vivo). In another aspect, the at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, complexed with a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.

[0287] In one embodiment, delivery of the at least one agent comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein. In certain embodiments, delivery of the at least one agent to a subject comprises mixing the at least one agent with a transfection reagent prior to the step of contacting. In another embodiment, a method of the present invention further comprises administering the at least one agent together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0288] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0289] In some embodiments, delivery of the at least one agent comprises liposomes. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an 94Attorney Docket No.: 046483-6287-00WO inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

[0290] In one embodiment, the at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.

[0291] In another embodiment, the transfection reagent forms a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. In some embodiments, the liposomes comprise an internal aqueous space for entrapping water- soluble compounds. In another embodiment, liposomes can deliver the at least one agent to cells in an active form. Conjugation

[0292] In one embodiment, the invention relates to compositions having at least one delivery vehicle conjugated to a targeting domain (e.g., chemokine, cytokine, fractalkine, IL-2, etc.). In some embodiments, the delivery vehicle comprises an LNP. 95Attorney Docket No.: 046483-6287-00WO

[0293] Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, van der Waals interactions and hydrophobic interactions. In one embodiment, the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain.

[0294] In some embodiments, the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the targeting domain. The term “activated polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group. In one embodiment, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer conjugated lipid is an activated pegylated lipid. In one embodiment, the first coupling group is bound to the lipid portion of the pegylated lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid. In one embodiment, the second functional group is covalently attached to the targeting domain.

[0295] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of maleimides, N- hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first coupling group or second coupling group is selected from the group consisting of free amines (–NH2), free sulfhydryl groups (– SH), free hydroxide groups (–OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In one embodiment, the first coupling group is a maleimide. 96Attorney Docket No.: 046483-6287-00WO

[0296] In one embodiment, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art. In one embodiment, the sulfhydryl group is present on a free cysteine residue. In one embodiment, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine. In one embodiment, the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0297] In some embodiments, the polymer conjugated lipid and targeting domain are functionalized with groups used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone.

[0298] It will be appreciated that the at least one chemokine or cytokine ligand, or a variant or fragment thereof, can be conjugated to the surface of the at least one delivery vehicle during or after preparation. In some embodiments, the at least one chemokine or cytokine ligand is conjugated to the surface of the at least one delivery vehicle after the at least one delivery vehicle has been prepared. In other embodiments, the at least one chemokine or cytokine ligand is conjugated to a component (e.g., a lipid, etc.) of an unassembled delivery vehicle before the at least one delivery vehicle has been prepared. Such conjugation means may be carried out by any known means in the art, including any suitable conjugation chemistry already well known in the art and discussed herein.

[0299] In some embodiments, the at least one delivery vehicle or compositions comprising the at least one delivery vehicle may further include at least one additional agent that enhances the localization of the delivery vehicles to a target cell. Such additional agents may include other peptides, aptamers, oligonucleotides, vitamins or other molecules that facilitate the localization of a delivery vehicle to a target cell, but which are not necessarily directly coupled to the delivery vehicle. 97Attorney Docket No.: 046483-6287-00WO Fractalkine-conjugated LNPs

[0300] In some embodiments, mRNA containing LNPs are incubated with micelles containing both mPEG-DSPE and mPEG-DSPE-Maleimide. During the incubation, N- Succinimidyl-S-acetylthioacetate (SATA) 1s mixed with DMSO to make a 0.092 mg / µl SATA / DMSO solution. Recombinant human fractalkine is then incubated with the SATA / DMSO mixture. The fractalkine-SATA mixture is then run through a desalting column, deprotected with NH2OH, and the deprotected protein is then run through a desalting column. The micelle-LNP mixture is added to the functionalized-fractalkine. IL-2-conjugated LNPs

[0301] In some embodiments, mRNA containing LNPs are incubated with micelles containing both mPEG-DSPE and mPEG-DSPE-Maleimide. During the incubation, N- Succinimidyl-S-acetylthioacetate (SATA) is mixed with DMSO to make a 0.092 mg / µl SATA / DMSO solution. Recombinant human IL-2 is then incubated with the SATA / DMSO mixture. The IL-2-SATA mixture is then run through a desalting column, deprotected with NH2OH, and then run through a desalting column. The micelle-LNP mixture is then added to the functionalized-IL-2 mixture. Target Cells

[0302] In some embodiments, the delivery vehicles of the invention are specifically targeted for binding to a surface receptor expressed on at least one target immune cell. In some embodiments, the at least one target cell comprises at least one natural killer (NK) cell, at least one T cell, at least one B cell, at least one monocyte, at least one macrophage, at least one dendritic cell, or at least one neutrophil, or a combination thereof.

[0303] In one embodiment, the T cells that can be targeted using the compositions of the invention comprise T helper cells (CD4+), cytotoxic T cells (also referred to as cytotoxic T lymphocytes, CTL; CD8+ T cells), and memory T cells, including effector memory T cells, for example, TEM cells and TEMRA (CD45RA+) cells, Th17 cells, Tfh (follicular helper) cells, regulatory T cells (Tregs), activated CD25+ T cells and early differentiated T cells. 98Attorney Docket No.: 046483-6287-00WO

[0304] One of ordinary skill in the art will be able to identify at least one appropriate cell surface receptor on any cell type of interest such that the at least one delivery vehicle conjugated to at least one chemokine or cytokine ligand becomes localized to any cell type of interest due to specific and selective interaction between the at least one chemokine or cytokine ligand and the at least one appropriate cell surface receptor on any cell type of interest that will allow the targeting of the at least one delivery vehicle of the present invention to any cell of interest. Delivery vehicles

[0305] In some embodiments, the delivery vehicle comprises or encapsulates at least one agent for delivery to a target immune cell or immune cell subpopulation. In various embodiments, the agent is a nucleic acid. In various embodiments, a nucleic acid is an mRNA, a self-replicating RNA, a siRNA, a miRNA, DNA, a gene editing component (for example, a guide RNA, a tracr RNA, a sgRNA), a gene writing component, an mRNA encoding a gene or base editing protein, a zinc- finger nuclease, a Talen, a CRISPR nuclease, such as Cas9, a DNA molecule to be inserted or serve as a template for repair), and the like, or a combination thereof. In some embodiments, an mRNA encodes a chimeric antigen receptor (CAR). In some embodiments, the agent comprises at least one nucleoside-modified mRNA molecule. In some embodiments, the agent comprises at least one in vitro transcribed (IVT) mRNA molecule. In some embodiments, the mRNA molecule encodes a chimeric antigen receptor (CAR), at least one RNA interference (RNAi) component (siRNA, antisense polynucleotide, shRNA, miRNA), components of a CRISPR-Cas9 system, at least one isolated polypeptide, at least one antibody functional fragment, at least one imaging agent, or a combination thereof. Nucleoside-modified RNA

[0306] In one aspect, the at least one agent comprises at least one nucleoside-modified nucleic acid, wherein the at least one nucleoside-modified nucleic acid comprises at least one nucleoside-modified RNA, wherein the at least one nucleoside-modified RNA comprises as least one nucleoside-modified mRNA molecule. In one embodiment, the nucleoside-modified mRNA encodes a CAR molecule, antibody, peptide, polypeptide and / or protein. 99Attorney Docket No.: 046483-6287-00WO

[0307] For example, in one embodiment, the composition comprises at least one nucleoside-modified RNA. Nucleoside-modified mRNA have particular advantages over non- modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent Nos.8,278,036, 8,691,966, and 8,835,108, each of which is incorporated by reference herein in its entirety. In certain embodiments, nucleoside- modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy.

[0308] In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. For example, when expressing a protein by delivering the encoding mRNA, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery.

[0309] In certain embodiments, the at least one nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the at least one mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0310] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it is shown that the presence of 100Attorney Docket No.: 046483-6287-00WO pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). Similar effects as described for pseudouridine have also been observed for RNA containing 1-methyl-pseudouridine.

[0311] In some embodiments, the at least one nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule. For example, in some embodiments, the composition is purified to remove double-stranded contaminants. In some instances, a preparative HPLC purification procedure is used to obtain pseudouridine- containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). Administering HPLC-purified, pseudourine- containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0312] In some embodiments, the at least one nucleoside-modified nucleic acid molecule is purified using non-HPLC methods. In some instances, the nucleoside-modified nucleic acid molecule is purified using chromatography methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC). An exemplary FPLC-based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. Exemplary purification procedures are also described in U.S. Patent Application Publication No. US2016 / 0032316, which is hereby incorporated by reference in its entirety.

[0313] In one embodiment, the at least one nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in certain embodiments, the at least one nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the at least one nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the at least one nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0314] In one embodiment, the at least one nucleoside-modified RNA of the invention comprises at least one modified nucleoside. In one embodiment, the at least one modified nucleoside is m1acp3Ψ (1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the at least one modified nucleoside is m1Ψ (1-methylpseudouridine). In another 101Attorney Docket No.: 046483-6287-00WO embodiment, the at least one modified nucleoside is Ψm (2'-O-methylpseudouridine. In another embodiment, the at least one modified nucleoside is m5D (5-methyldihydrouridine). In another embodiment, the at least one modified nucleoside is m3Ψ (3-methylpseudouridine). In another embodiment, the at least one modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the at least one modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the at least one modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0315] In another embodiment, the at least one nucleoside that is modified in the at least one nucleoside-modified RNA the present invention is uridine (U). In another embodiment, the at least one modified nucleoside is cytidine (C). In another embodiment, the at least one modified nucleoside is adenosine (A). In another embodiment, the at least one modified nucleoside is guanosine (G).

[0316] In another embodiment, the at least one modified nucleoside of the present invention is m5C (5-methylcytidine). In another embodiment, the at least one modified nucleoside is m5U (5-methyluridine). In another embodiment, the at least one modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the at least one modified nucleoside is s2U (2-thiouridine). In another embodiment, the at least one modified nucleoside is Ψ (pseudouridine). In another embodiment, the at least one modified nucleoside is Um (2'-O- methyluridine).

[0317] In other embodiments, the at least one modified nucleoside is m1A (1- methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2- methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m1I (1- methylinosine); m1Im (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2'-O- methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm 102Attorney Docket No.: 046483-6287-00WO (5,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine); k2C (lysidine); m1G (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ0(7- cyano-7-deazaguanosine); preQ1 (7-aminomethyl-7-deazaguanosine); G+(archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5- methoxycarbonylmethyluridine); mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cmnm5Um (5-carboxymethylaminomethyl-2'-O- methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6- dimethyladenosine); Im (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2'-O- dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,2'-O-dimethyladenosine); m62Am (N6,N6,O-2'- trimethyladenosine); m2,7G (N2,7-dimethylguanosine); m2,2,7G (N2,N2,7-trimethylguanosine); m3Um (3,2'-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O- methylcytidine); m1Gm (1,2'-O-dimethylguanosine); m1Am (1,2'-O-dimethyladenosine); τm5U (5-taurinomethyluridine); τm5s2U (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); or ac6A (N6-acetyladenosine).

[0318] In another embodiment, the at least one nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above modifications. In another 103Attorney Docket No.: 046483-6287-00WO embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[0319] In another embodiment, between 0.1% and 100% of the residues in the at least one nucleoside-modified mRNA of the present invention are modified (e.g. either by the presence of pseudouridine or a modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0320] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 104Attorney Docket No.: 046483-6287-00WO 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0321] In some embodiments, the at least one agent comprises a purified preparation of at least one single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of at least one single-stranded nucleoside modified RNA is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, or at least 91%, or at least 92%, or at least 93 % or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0322] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of the given nucleotide that is modified is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%. 105Attorney Docket No.: 046483-6287-00WO

[0323] In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0324] In another embodiment, the at least one nucleoside-modified RNA of the present invention is translated in the at least one target cell more efficiently than an unmodified RNA molecule with the same sequence. In another embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3-fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200- fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000-fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts. 106Attorney Docket No.: 046483-6287-00WO

[0325] In another embodiment, the at least one nucleoside-modified RNA of the present invention exhibits significantly less innate immunogenicity than an unmodified in vitro-synthesized RNA molecule of the same sequence. In another embodiment, the at least one modified RNA molecule exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In another embodiment, innate immunogenicity is reduced by a 3-fold factor. In another embodiment, innate immunogenicity is reduced by a 4-fold factor. In another embodiment, innate immunogenicity is reduced by a 5-fold factor. In another embodiment, innate immunogenicity is reduced by a 6-fold factor. In another embodiment, innate immunogenicity is reduced by a 7-fold factor. In another embodiment, innate immunogenicity is reduced by a 8-fold factor. In another embodiment, innate immunogenicity is reduced by a 9- fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.

[0326] In another embodiment, “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity. In another embodiment, the term refers to a fold decrease in innate immunogenicity (e.g., 1 of the fold decreases enumerated above). In another embodiment, the term refers to a decrease such that an effective amount of the nucleoside-modified RNA can be administered without triggering a detectable innate immune response. In another embodiment, the term refers to a decrease such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the modified RNA. In another embodiment, the decrease is such that the nucleoside-modified RNA can be 107Attorney Docket No.: 046483-6287-00WO repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the modified RNA. In vitro transcribed mRNA

[0327] In one embodiment, the at least one agent of the invention comprises at least one in vitro transcribed (IVT) RNA molecule, wherein the at least one IVT RNA is messenger RNA (mRNA). In one embodiment, the at least one in vitro transcribed (IVT) RNA encodes at least one CAR molecule, antibody, peptide, polypeptide and / or protein.

[0328] In one embodiment, the at least one mRNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.

[0329] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5' and 3' UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0330] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that induce or enhance an adaptive immune response in an organism. Exemplary 108Attorney Docket No.: 046483-6287-00WO genes are genes which are useful for a short-term treatment, or where there are safety concerns regarding dosage or the expressed gene.

[0331] In various embodiments, a plasmid is used to generate a template for in vitro transcription of mRNA.

[0332] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. In some embodiments, the mRNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation of the transcribed mRNA.

[0333] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the mRNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed mRNA based on properties of UTRs that are well known in the art.

[0334] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some mRNA transcripts, but does not appear to be required for all mRNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments the 5' UTR can be derived from an mRNA virus whose mRNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA. 109Attorney Docket No.: 046483-6287-00WO

[0335] To enable synthesis of mRNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an mRNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one exemplary embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0336] In an exemplary embodiment, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized RNA which is effective in eukaryotic transfection when it is polyadenylated after transcription.

[0337] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0338] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However, polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.

[0339] Poly(A) tails of mRNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the mRNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated 110Attorney Docket No.: 046483-6287-00WO into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the mRNA.

[0340] 5' caps also provide stability to mRNA molecules. In an exemplary embodiment, mRNAs produced by the methods include a 5' cap-1 structure. Such cap-1 structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes (CellScript, Madison, WI). Alternatively, 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)). Expression Vectors

[0341] In one embodiment, the at least one agent comprises at least one expression vector. In one embodiment, the at least one expression vector comprises at least one promoter / regulatory sequence upstream of at least one coding sequence wherein the at least one promoter / regulatory sequence directs the expression of the at least one coding sequence, wherein the coding sequence encodes at least one mRNA molecule.

[0342] In one embodiment, the at least one expression vector comprises at least one promoter / regulatory sequence upstream of at least one coding sequence wherein the at least one promoter / regulatory sequence directs the expression of the at least one coding sequence, wherein the coding sequence encodes at least one mRNA molecule, wherein the at least one mRNA molecule encodes at least one peptide, at least one polypeptide, and / or at least one protein. In one embodiment, the promoter / regulatory sequence comprises an enhancer. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous nucleic acid into at least one target cell with concomitant expression of the exogenous nucleic acid in the at least one target cell such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0343] In one embodiment, the promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences 111Attorney Docket No.: 046483-6287-00WO located upstream of the coding segment and / or exon. Such a promoter can be referred to as “endogenous.” Similarly, the enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0344] A skilled artisan will appreciate the importance of employing a promoter and / or enhancer that effectively directs the expression of the DNA segment in the at least one target cell. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous. Chimeric Antigen Receptors (CARs)

[0345] In one embodiment, the composition of the invention can be administered in combination with at least one additional agent. In some embodiments, at least one additional agent comprises an mRNA molecule encoding a CAR. In one embodiment, the mRNA 112Attorney Docket No.: 046483-6287-00WO molecule encoding a CAR is translated into CAR protein in the at least one target cell. In one embodiment, the CAR protein is localized to the surface of the at least one target cell.

[0346] In one embodiment, the CAR comprises an antigen binding domain which specifically binds to at least one autoantigen, marker of at least one cancer cell or at least one pathogen. In some embodiments, once bound to the at least cancer cell or at least one pathogen, the at least one modified target cell facilitates the destruction of the at least one cancer cell or at least one pathogen (e.g., by phagocytosis, T cell-mediated cytotoxicity, etc.), thereby treating or preventing a disease or disorder (e.g., cancer, etc.) in the subject.

[0347] In some embodiments, once bound to the at least autoantigen, the at least one modified target cell protects a cell expressing the autoantigen from destruction by other immune cells, thereby treating or preventing an autoimmune disease or disorder in the subject.

[0348] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial cell receptor that is engineered to be expressed on an immune effector cell, such as an NK cell, a macrophage, a B cell, or a dendritic cell, and specifically bind an antigen on at least one cancer cell or at least one pathogen. CARs may be used as a therapy with adoptive cell transfer. Generally, immune cells of interest, are removed from a patient and modified so that they express the receptors specific to a particular form of antigen. In some embodiments, the CARs have specificity to at least one autoantigen, cancer antigen or at least one pathogen antigen. CARs may also comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising an antigen binding region that specifically binds to at least one autoantigen, cancer antigen or at least one pathogen antigen.

[0349] In various embodiments, the CARs contemplated herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element otherwise referred to as an antigen binding domain. In some embodiments, the extracellular domain also comprises a hinge domain. In certain embodiments, the intracellular domain or otherwise the cytoplasmic domain comprises, a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen. 113Attorney Docket No.: 046483-6287-00WO

[0350] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term "spacer domain" generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise up to 5 amino acids, or 10 amino acids, or 20 amino acids, or 30 amino acids, or 40 amino acids, or 50 amino acids, or 60 amino acids, or 70 amino acids, or 80 amino acids, or 90 amino acids, or 100 amino acids, or 110 amino acids, or 120 amino acids, or 130 amino acids, or 140 amino acids, or 150 amino acids, or 160 amino acids, or 170 amino acids, or 180 amino acids, or 190 amino acids, or 200 amino acids, or 210 amino acids, or 220 amino acids, or 230 amino acids, or 240 amino acids, or 250 amino acids, or 260 amino acids, or 270 amino acids, or 280 amino acids, or 290 amino acids, or 300 amino acids.

[0351] The extracellular domain, transmembrane domain, and intracellular domain can be derived from any desired source of such domains. CAR antigen binding domain

[0352] The antigen binding domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. In one embodiment, the antigen binding domain may consist of an Ig heavy chain which may in turn be covalently associated with Ig light chain by virtue of the presence of CHI and hinge regions, or may become covalently associated with other Ig heavy / light chain complexes by virtue of the presence of hinge, CH2 and CH3 domains. In the latter case, the heavy / light chain complex that becomes joined to the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct. Depending on the function of the antibody, the desired structure and the signal transduction, the entire chain may be used or a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed.

[0353] In various embodiments, the CAR antigen binding domain may be humanized or comprise a fully human sequence. 114Attorney Docket No.: 046483-6287-00WO CAR transmembrane domain

[0354] With respect to the transmembrane domain, a CAR of the disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0355] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, for example, but not limited to between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In another embodiment, the linker comprises a glycine-serine doublet. CAR intracellular domain

[0356] In various embodiments, the cytoplasmic domain or otherwise the intracellular domain of a CAR may be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines. The term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire 115Attorney Docket No.: 046483-6287-00WO intracellular domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0357] Examples of intracellular domains for use in the CARs of the disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0358] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0359] Primary intracellular signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0360] Examples of ITAMs containing primary intracellular signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling molecule in the CAR of the invention comprises an intracellular signaling sequence derived from CD3 zeta.

[0361] In another embodiment, the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell 116Attorney Docket No.: 046483-6287-00WO surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD2, CD27, CD28, 4-1BB (CD137), Ox40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0362] The intracellular signaling sequences within the intracellular domain of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a suitable linker in some embodiments.

[0363] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB.

[0364] In various embodiments, the CAR can be a “first generation,” “second generation,” “third generation,” “fourth generation” or “fifth generation” CAR (see, for example, Sadelain et al., Cancer Discov.3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech.8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res.13:5426-5435 (2007); Gade et al., Cancer Res.65:9080-9088 (2005); Maher et al., Nat. Biotechnol.20:70-75 (2002); Kershaw et al., J. Immunol.173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother.32:169-180 (2009)), each of which are incorporated by reference in its entirety).

[0365] “First generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3ζ-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. 117Attorney Docket No.: 046483-6287-00WO

[0366] “Second-generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to augment T cell potency and persistence (Sadelain et al., Cancer Discov.3:388-398 (2013)). CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co- stimulatory molecules, for example, CD28, 4-1BB, ICOS, OX40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

[0367] “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3ζ signaling domain. Preclinical studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol.1(9):1577-1583 (2012)).

[0368] “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4-1BB domains, and activation, for example, by comprising a CD3ζ activation domain.

[0369] “Fourth generation” CARs provide co-stimulation, for example, by CD28 or 4- 1BB domains, and activation, for example, by a CD3ζ signaling domain in addition to a constitutive or inducible chemokine component.

[0370] “Fifth generation” CARs provide co-stimulation, for example, by CD28 or 4- 1BB domains, and activation, for example, by a CD3ζ signaling domain, a constitutive or inducible chemokine component, and an intracellular domain of a cytokine receptor, for example, IL-2Rβ.

[0371] In various embodiments, the CAR can be included in a multivalent CAR system, for example, a DualCAR or “TandemCAR” system. Multivalent CAR systems include systems or cells comprising multiple CARs and systems or cells comprising bivalent / bispecific CARs targeting more than one antigen. 118Attorney Docket No.: 046483-6287-00WO

[0372] In the embodiments disclosed herein, the CARs generally comprise an antigen binding domain, a transmembrane domain and an intracellular domain, as described above. In a particular non-limiting embodiment, the antigen-binding domain is an scFv.

[0373] In one embodiment, the antigen binding domain of the CAR molecule is a targeting domain, wherein the targeting domain directs the cell expressing the CAR to at least one cancer cell or at least one pathogen For example, in one embodiment, the targeting domain comprises an antibody, antibody fragment, or peptide that specifically binds to an antigen (e.g., a self-antigen or a foreign antigen) thereby directing the cell expressing the CAR to at least one cancer cell or at least one pathogen, wherein the at least one cancer cell or at least one pathogen expresses the antigen.

[0374] In one embodiment, the antigen binding domain of the CAR molecule of the invention can be generated to be reactive to any desirable antigen of interest, or fragment thereof, including, but not limited to a tumor antigen, a foreign antigen (e.g, a bacterial antigen, a viral antigen, etc.) or a self-antigen. In some embodiments, the antigen is an HIV antigen.

[0375] In some embodiments, the antigen comprises a foreign antigen wherein the foreign antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen or fragment thereof, or variant thereof. CRISPR-Cas system

[0376] In some embodiments, at least one additional agent comprises a gene editing molecule. In one embodiment, the at least one agent comprises components of a CRISPR-Cas system, or an mRNA molecule encoding the same. In one embodiment, the components of a CRISPR-Cas system comprise a guide RNA (gRNA) molecule, wherein the gRNA is designed to bind to a genomic region of choice in the at least one target cell based on the nucleotide sequence of the gRNA, and an mRNA molecule, wherein the mRNA molecule encodes a CRISPR-associated (Cas) peptide, and wherein the gRNA and Cas peptide form a complex to induce mutations within the genomic region of choice. In one embodiment, the Cas peptide is any Cas peptide known in the art to function effectively in a genome editing CRISPR-Cas system (e.g., Cas3, Cas5, Cas9, etc.). In one embodiment, the components of a CRISPR-Cas system comprise a guide gRNA molecule and a Cas peptide. 119Attorney Docket No.: 046483-6287-00WO

[0377] In one embodiment, a CRISPR-Cas system is designed to incorporate an exogenous nucleic acid into the genome of the at least one target cell. In one embodiment, the components of a CRISPR-Cas system further comprise single stranded (ssDNA) or double stranded DNA (dsDNA), wherein the ssDNA or dsDNA comprises the exogenous nucleic acid to be incorporated into the genome of the at least one target cell. In one embodiment, such incorporation comprises without limitation, homologous recombination mediated incorporation of the exogenous nucleic acid. In one embodiment, the exogenous nucleic acid encodes at least one mRNA, and wherein the at least one mRNA encodes at least one peptide, polypeptide and / or protein. In one embodiment, the exogenous nucleic acid comprises a dsDNA molecule, wherein the dsDNA molecule comprises a promoter, gene body and any regulatory DNA element required for the expression of the at least one gene of interest in the at least one target cell.

[0378] In one embodiment, the exogenous nucleic acid incorporated into the genome of the at least one target cell restores normal function to the at least one target cell, wherein the incorporated exogenous nucleic acid supplements the at least one target cell with a functional gene and / or genomic region.

[0379] In an embodiment, a CRISPR-Cas system is designed to mutate any genomic region of choice in the at least one target cell of the present invention, wherein the mutation comprises a deletion of at least one nucleotide in the genome of the at least one target cell, and wherein the mutation alters the level of expression of at least one gene of interest in the at least one target cell, wherein the alteration comprises an increase or a decrease in level of expression of the at least one gene of interest, wherein the increase or decrease in level of expression comprises an increase or decrease in expression of mRNA and / or protein encoded by the at least one gene of interest. Polypeptides

[0380] In one embodiment, the at least one additional agent comprises at least one isolated peptide, or an mRNA molecule encoding the same. In one embodiment, the at least one isolated peptide modulates a target in the at least one target cell. For example, in one embodiment, the at least one isolated peptide of the invention inhibits or activates a target 120Attorney Docket No.: 046483-6287-00WO directly by binding to the target thereby modulating the normal functional activity of the target. In one embodiment, the at least one isolated peptide of the invention modulates the target by competing with endogenous proteins. In one embodiment, the at least one isolated peptide of the invention modulates the activity of the target by acting as a transdominant negative mutant. Antibodies

[0381] In one embodiment, at least one additional agent comprises at least one antibody functional fragment, or an mRNA molecule encoding the same, wherein the at least one antibody functional fragment comprises an antibody fragment, immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, a genetically engineered single chain FV molecule (Ladner et al, U.S. Pat. No.4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including fragments and chimeras, may be prepared using methods known to those skilled in the art. In one embodiment,the at least one antibody functional fragment is selected from the group consisting of a Fab, F(ab2)′, F(ab)2′ and scFV.

[0382] In one embodiment, the at least one agent comprises a recombinant nucleic acid sequence encoding the at least one antibody functional fragment. In one embodiment, the at least one agent comprises an mRNA molecule encoding the at least one antibody functional fragment. In one embodiment, the at least one agent comprises a recombinant nucleic acid sequence encoding the at least one antibody functional fragment, wherein the at least one antibody functional fragment is selected from the group consisting of a Fab, F(ab2)′, F(ab)2′ and scFV. In one embodiment, the at least one agent comprises an mRNA molecule encoding the at least one antibody functional fragment, wherein the at least one antibody functional fragment is selected from the group consisting of a Fab, F(ab2)′, F(ab)2′ and scFV.

[0383] Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest. The polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Suitable carriers that may be chemically coupled to peptides 121Attorney Docket No.: 046483-6287-00WO include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled polypeptide may then be used to immunize the animal (e.g., a mouse, a rat, or a rabbit). RNA interference

[0384] RNA interference (RNAi) is a phenomenon in which the introduction of double- stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No.6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173- 178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the present invention also includes methods of decreasing levels of PTPN22 using RNAi technology. siRNA

[0385] In one embodiment, the at least one additional agent comprises at least one siRNA, wherein the at least one siRNA decreases the level of expression of a target gene in the at least one target cell.

[0386] Following the generation of the at least one siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the at least one 122Attorney Docket No.: 046483-6287-00WO siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrawal et al., 1987, Tetrahedron Lett.28:3539-3542; Stec et al., 1985 Tetrahedron Lett.26:2191-2194; Moody et al., 1989 Nucleic Acids Res.12:4769-4782; Eckstein, 1989 Trends Biol. Sci.14:97- 100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp.97-117 (1989)).

[0387] Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine. Antisense Polynucleotide

[0388] In one embodiment, the at least one additional agent comprises at least one antisense polynucleotide. In some embodiments, the at least one antisense polynucleotide decreases the level of expression of a target gene in the at least one target cell. The incorporation of a desired polynucleotide into a vector and the choice of vectors are well- known in the art as described in, for example, Sambrook et al. (2012), and in Ausubel et al. (1997), and elsewhere herein.

[0389] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0390] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem.172:289). 123Attorney Docket No.: 046483-6287-00WO Short Hairpin RNA (shRNA)

[0391] In one embodiment, the at least one additional agent comprises at least one short hairpin RNA (shRNA). In one embodiment, the at least one shRNA decreases the level of expression of a target gene in the at least one target cell. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. miRNA

[0392] In one embodiment, the at least one additional agent comprises at least one micro RNA (miRNA) or at least one mimic of a miRNA. In one embodiment, the at least one miRNA or at least one mimic of a miRNA decreases the level of expression of a target gene in the at least one target cell.

[0393] MiRNAs are small non-coding RNA molecules that are capable of causing post- transcriptional silencing of specific genes in cells by the inhibition of translation or through degradation of the targeted mRNA. A miRNA can be completely complementary or can have a region of non-complementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity. A miRNA can inhibit gene expression by repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity. The disclosure also can include double-stranded precursors of miRNA. A miRNA or pri-miRNA can be 18- 100 nucleotides in length, or from 18-80 nucleotides in length. Mature miRNAs can have a length of 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MiRNA precursors typically have a length of about 70-100 nucleotides and have a hairpin conformation. miRNAs are generated in vivo from pre- miRNAs by the enzymes Dicer and Drosha, which specifically process long pre-miRNA into functional miRNA. The hairpin or mature microRNAs, or pri-microRNA agents featured in the disclosure can be synthesized in vivo by a cell-based system or in vitro by chemical synthesis. 124Attorney Docket No.: 046483-6287-00WO Imaging Agents

[0394] In one embodiment, the at least one additional agent comprises at least one imaging agent, or mRNA molecule encoding the same. Imaging agents are materials that allow the at least one delivery vehicle to be visualized after exposure to a cell or tissue. Visualization includes imaging for the naked eye, as well as imaging that requires detecting with instruments or detecting information not normally visible to the eye, and includes imaging that requires detecting of photons, sound or other energy quanta. Examples include stains, vital dyes, fluorescent markers, radioactive markers, enzymes or plasmid constructs encoding markers or enzymes. Many materials and methods for imaging and targeting that may be used in the delivery vehicle are provided in the Handbook of Targeted delivery of Imaging Agents, Torchilin, ed. (1995) CRC Press, Boca Raton, Fla.

[0395] Visualization based on molecular imaging typically involves detecting biological processes or biological molecules at a tissue, cell, or molecular level. Molecular imaging can be used to assess specific targets for gene therapies, cell-based therapies, and to visualize pathological conditions as a diagnostic or research tool. Imaging agents that are able to be delivered intracellularly are particularly useful because such agents can be used to assess intracellular activities or conditions. Imaging agents must reach their targets to be effective; thus, in some embodiments, an efficient uptake by cells is desirable. A rapid uptake may also be desirable to avoid the RES, see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001).

[0396] Further, imaging agents should provide high signal to noise ratios so that they may be detected in small quantities, whether directly, or by effective amplification techniques that increase the signal associated with a particular target. Amplification strategies are reviewed in Allport and Weissleder, Experimental Hematology 1237-1246 (2001), and include, for example, avidin-biotin binding systems, trapping of converted ligands, probes that change physical behavior after being bound by a target, and taking advantage of relaxation rates. Examples of imaging technologies include magnetic resonance imaging, radionuclide imaging, computed tomography, ultrasound, and optical imaging.

[0397] Many imaging techniques and strategies are known, e.g., see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001); such strategies may be adapted 125Attorney Docket No.: 046483-6287-00WO to use with delivery vehicles. Suitable imaging agents include, for example, fluorescent molecules, labeled antibodies, labeled avidin:biotin binding agents, colloidal metals (e.g., gold, silver), reporter enzymes (e.g., horseradish peroxidase), superparamagnetic transferrin, second reporter systems (e.g., tyrosinase), and paramagnetic chelates.

[0398] In some embodiments, the at least one imaging agent comprises a magnetic resonance imaging contrast agent. Examples of magnetic resonance imaging contrast agents include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N′,N″N′″-tetracetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), 1,4,7,10-tetraazacyclododecane-N,N′, N″,N′″-tetraethylphosphorus (DOTEP), 1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid (DOTA) and derivatives thereof (see U.S. Pat. Nos.5,188,816, 5,219,553, and 5,358,704). In some embodiments, the at least one imaging agent is an X-Ray contrast agent. X-ray contrast agents already known in the art include a number of halogenated derivatives, especially iodinated derivatives, of 5-amino-isophthalic acid. Small molecules

[0399] In various embodiments, the at least one additional agent comprises at least one small molecule. When the at least one agent comprises at least one small molecule, the at least one small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art. In one embodiment, the at least one small molecule comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.

[0400] Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art, as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for 126Attorney Docket No.: 046483-6287-00WO lead discovery vs. biased structures for lead development. In some embodiments of the invention, the agent is synthesized and / or identified using combinatorial techniques.

[0401] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores. In some embodiments of the invention, the agent is synthesized via small library synthesis.

[0402] The at least one small molecule described herein may be present as salts even if salts are not depicted, and it is understood that the invention embraces all salts and solvates of the agents depicted here, as well as the non-salt and non-solvate form of the agents, as is well understood by the skilled artisan. In some embodiments, the salts of the agents of the invention are pharmaceutically acceptable salts.

[0403] Where tautomeric forms may be present for any of the agents described herein, each and every tautomeric form is intended to be included in the present invention, even though only one or some of the tautomeric forms may be explicitly depicted. For example, when a 2-hydroxypyridyl moiety is depicted, the corresponding 2-pyridone tautomer is also intended.

[0404] The invention also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms of the agents described. The recitation of the structure or name herein is intended to embrace all possible stereoisomers of agents depicted. All forms of the agents are also embraced by the invention, such as crystalline or non- crystalline forms of the agent. Compositions comprising an agent of the invention are also intended, such as a composition of substantially pure agent, including a specific stereochemical form thereof, or a composition comprising mixtures of agents of the invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture.

[0405] The invention also includes any or all active analog or derivative, such as a prodrug, of any agent described herein. In one embodiment, the agent is a prodrug. In one embodiment, the small molecules described herein are candidates for derivatization. As such, 127Attorney Docket No.: 046483-6287-00WO in certain instances, the analogs of the small molecules described herein that have modulated potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and drug development. Thus, in certain instances, during optimization new analogs are designed considering issues of drug delivery, metabolism, novelty, and safety.

[0406] In some instances, the at least one small molecule described herein are derivatives or analogs of known agents, as is well known in the art of combinatorial and medicinal chemistry. The analogs or derivatives can be prepared by adding and / or substituting functional groups at various locations. As such, the small molecules described herein can be converted into derivatives / analogs using well known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. Also, the linking atoms or groups can be modified into longer or shorter linkers with carbon backbones or hetero atoms. Also, the ring groups can be changed so as to have a different number of atoms in the ring and / or to include hetero atoms. Moreover, aromatics can be converted to cyclic rings, and vice versa. For example, the rings may be from 5-7 atoms, and may be carbocyclic or heterocyclic.

[0407] As used herein, the term “analog,” “analogue,” or “derivative” is meant to refer to a chemical compound or molecule made from a parent compound or molecule by at least one chemical reaction. As such, an analog can be a structure having a structure similar to that of the small molecule agents described herein or can be based on a scaffold of a small molecule agents described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative of any of a small molecule inhibitor in accordance with the present invention can be used to treat a disease or disorder.

[0408] In one embodiment, the at least one small molecule described herein can independently be derivatized, or analogs prepared therefrom, by modifying hydrogen groups independently from each other into other substituents. That is, each atom on each molecule can be independently modified with respect to the other atoms on the same molecule. Any traditional modification for producing a derivative / analog can be used. For example, the atoms and substituents can be independently comprised of hydrogen, an alkyl, aliphatic, straight chain aliphatic, aliphatic having a chain hetero atom, branched aliphatic, substituted aliphatic, cyclic 128Attorney Docket No.: 046483-6287-00WO aliphatic, heterocyclic aliphatic having at least one hetero atom, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptide, polypeptide, combinations thereof, halogen, halo- substituted aliphatic, and the like. Additionally, any ring group on a compound can be derivatized to increase and / or decrease ring size as well as change the backbone atoms to carbon atoms or hetero atoms. Other agents

[0409] In some embodiments, the at least one additional agent is at least one therapeutic agent, at least one diagnostic agent, at least one contrast agent, at least one labeling agent, at least one detection agent, or at least one disinfectant, or an mRNA molecule encoding the same. In some embodiments, the at least one additional agent comprises substances with biological activities which are not typically considered to be active ingredients, such as fragrances, sweeteners, flavorings and flavor enhancer agents, pH adjusting agents, effervescent agents, emollients, bulking agents, soluble organic salts, permeabilizing agents, anti-oxidants, colorants or coloring agents, and the like.

[0410] In one embodiment, the at least one delivery vehicle comprises at least one additional therapeutic agent. The present invention is not limited to any particular therapeutic agent, but rather encompasses any suitable therapeutic agent that can be included within the delivery vehicle for delivery to an immune cell. In some embodiments, the at least one therapeutic agent comprises an anti-viral agent, an anti-bacterial agent, an anti-oxidant agent, a thrombolytic agent, a chemotherapeutic agent, an anti-inflammatory agent, an immunogenic agent, an antiseptic, an anesthetic, an analgesic, a pharmaceutical agent, a small molecule, a peptide, a nucleic acid, or the like. Therapeutic Methods

[0411] The present invention relates in part to methods of treating a disease or disorder in a subject in need thereof, the method comprising the administration of a composition comprising at least one immune cell targeted delivery vehicle comprising at least one nucleic acid molecule encoding a tissue trafficking modulator. In one embodiment, the at least one 129Attorney Docket No.: 046483-6287-00WO delivery vehicle comprises an LNP. In one embodiment, the at least one nucleic acid molecule encoding a tissue trafficking modulator is mRNA.

[0412] In some embodiments, the delivery vehicle comprises a targeted delivery vehicle, wherein the targeting molecule binds to at least one receptor on the surface of at least one target immune cell in the subject, and wherein the at least one tissue trafficking modulator is delivered to the at least one target immune cell in the subject. In some embodiments, the target cell is an immune cell and expression of the tissue trafficking modulator alters the trafficking of the target immune cell. For example, expression of a tissue trafficking modulator in a CTL or Treg alters the trafficking of the CTL or Treg.

[0413] In some embodiments, the invention provides methods for altering or modulating the trafficking of at least one cytotoxic T cell or NK cell. In some embodiments, the method comprises administering a targeted LNP comprising a fractalkine (CX3CL1) targeting domain, or a fragment or variant thereof. In some embodiments, the fractalkine targeted LNP comprises or encapsulates a nucleic acid molecule encoding a tissue trafficking modulator for expression on the target T cell or NK cell. In some embodiments, the tissue trafficking modulator comprises CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69, or any combination thereof. Therefore, in one embodiment, the method comprises administering a fractalkine (CX3CL1)-LNP comprising an mRNA molecule encoding CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69. In one embodiment, the method comprises administering a fractalkine (CX3CL1)-LNP comprising a combination of mRNA molecules encoding at least one of CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69 and at least one additional agent. In some embodiments, the additional agent is an additional tissue trafficking modulator. In some embodiments, the additional agent is a therapeutic agent. In some embodiments, expression of CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69 alters trafficking of at least one CTL or NK cell to secondary lymphatic tissue (SLT). Therefore, in some embodiments, the disclosure provides methods for increasing CTL or NK cell trafficking to SLT comprising administering a fractalkine (CX3CL1)-LNP comprising an mRNA 130Attorney Docket No.: 046483-6287-00WO molecule encoding CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69 to a subject.

[0414] In some embodiments, the invention provides methods for altering or modulating the trafficking of at least one activated CD25+ T cell or regulatory T cell (Treg). In some embodiments, the method comprises administering a targeted LNP comprising an IL-2 targeting domain, or a fragment or variant thereof. In some embodiments, the IL-2 targeted LNP comprises or encapsulates a nucleic acid molecule encoding a tissue trafficking modulator for expression on the target activated CD25+ T cell or Treg. In some embodiments, the tissue trafficking modulator comprises a chemokine receptor. In some embodiments, the tissue trafficking modulator comprises CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4, or a combination thereof. Therefore, in one embodiment, the method comprises administering an IL-2-LNP comprising an mRNA molecule encoding at least one tissue trafficking modulator. In one embodiment, the method comprises administering a IL-2-LNP comprising a combination of mRNA molecules encoding at least one tissue trafficking modulator and at least one additional agent. In some embodiments, the additional agent is an additional tissue trafficking modulator. In some embodiments, the additional agent is a nucleic acid molecule encoding a CAR. In some embodiments, the additional agent is an additional chemokine receptor. In some embodiments, the additional agent is an additional immunosuppressive agent. In one embodiment, the invention provides compositions comprising an IL-2-LNP comprising a combination of mRNA molecules encoding, at least one chemokine receptor and at least one immunosuppressive chemokine. In some embodiments, the immunosuppressive chemokine comprises IL-10 or TGFβ. In some embodiments, expression of CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4 alters trafficking of at least one Treg to pancreatic islet cells. Therefore, in some embodiments, the disclosure provides methods for increasing Treg trafficking to pancreatic islet cells comprising administering an IL-2-LNP comprising an mRNA molecule encoding at least one of CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4 to a subject.

[0415] In one embodiment, the present disclosure provides compositions and methods for treating or preventing disease (e.g., cancer, infection, immunological disease, etc.) in a subject by using an approach that involves modulating the trafficking a subject’s immune cells 131Attorney Docket No.: 046483-6287-00WO in vivo to target and destroy or suppress at least one disease associated cell, thereby treating or preventing the disease (e.g., cancer, infection, autoimmune disease, etc.). In one embodiment, the method involves targeting regulatory or cytotoxic immune cells to at least one disease associated cell through delivery and expression of a tissue trafficking modulator on the immune cell population which specifically binds to a ligand present in a specific cell niche to recruit the cytotoxic immune cells to a specific tissue or cellular niche. Exemplary diseases and disorders that can be treated using the methods and compositions of the invention include, but are not limited to, cancers, infectious diseases, and immunological disorders.

[0416] The following are non-limiting examples of cancers that can be treated or prevented by the disclosed methods: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytotna / malignant glioma, cervical cancer, childhood visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous cancer, cutaneous T- cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney (renal cell) cancer, Langerhans cell cancer, langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocvtoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, 132Attorney Docket No.: 046483-6287-00WO metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (nonmelanoma), skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer , stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, supratentorial primitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0417] In some embodiments, the present invention features methods for treating or preventing an autoimmune disease including, but not limited to, rheumatoid arthritis / seronegative arthropathies, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosis, iridoeyelitis / uveitistoptic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / Wegener's gramilornatosis, sarcoidosis, including, but not limited to, rheumatoid arthritis / seronegative arthropathies, osteoarthritis, inflammatory bowel disease, systemic lupus 133Attorney Docket No.: 046483-6287-00WO erythematosis, iridoeyelitis / uveitistoptic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / Wegener's gramilornatosis, sarcoidosis, myocarditis, postmyocardial infarction syndrome, postpericardiotomy syndrome, subacute bacterial endocarditis (SBE), anti- glomerular basement membrane nephritis, interstitial cystitis, lupus nephritis, autoimmune hepatitis, primary biliary cholangitis(PBC), primary sclerosing cholangitis, antisynthetase syndrome, alopecia areata, autoimmune angioedema, autoimmune progesterone dermatitis, autoimmune urticaria, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus, epidermolysis bullosa acquisita, erythema nodosum, gestational pemphigoid, hidradenitis suppurativa, lichen planus, lichen sclerosus, linear IgA disease (LAD), morphea, pemphigus vulgaris, pityriasis lichenoides et varioliformis acuta, Mucha- Habermann disease, psoriasis, systemic scleroderma, vitiligo, Addison's disease, autoimmune polyendocrine syndrome (APS) type 1, autoimmune polyendocrine syndrome (APS) type 2, autoimmune polyendocrine syndrome (APS) type 3, autoimmune pancreatitis (AIP), diabetes mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis, Graves' disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmune enteropathy, Coeliac disease, Crohn's disease, microscopic colitis, ulcerative colitis, antiphospholipid syndrome(APS, APLS), aplastic anemia, autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune thrombocytopenic purpura, cold agglutinin disease, essential mixed cryoglobulinemia, Evans syndrome, pernicious anemia, pure red cell aplasia, thrombocytopenia, adiposis dolorosa, adult-onset Still's disease, ankylosing spondylitis, CREST syndrome, drug-induced lupus, enthesitis- related arthritis, eosinophilic fasciitis Felty syndrome, IgG4-related disease, juvenile arthritis, Lyme disease (chronic), mixed connective tissue disease (MCTD), palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, Schnitzler syndrome, undifferentiated connective tissue disease (UCTD), dermatomyositis, fibromyalgia, inclusion body myositis, myositis, myasthenia gravis, neuromyotonia, paraneoplastic cerebellar degeneration, polymyositis, acute disseminated encephalomyelitis (ADEM), acute motor axonal neuropathy, anti-N-methyl-D-aspartate (Anti-NMDA) receptor encephalitis, balo concentric sclerosis, Bickerstaff's encephalitis, chronic inflammatory demyelinating 134Attorney Docket No.: 046483-6287-00WO polyneuropathy, Guillain–Barré syndrome, Hashimoto's encephalopathy, idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome, multiple sclerosis, pattern II, Oshtoran Syndrome, pediatric autoimmune neuropsychiatric disorder associated with streptococcus (PANDAS), progressive inflammatory neuropathy, restless leg syndrome, stiff person syndrome, sydenham chorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, intermediate uveitis, ligneous conjunctivitis, Mooren's ulcer, neuromyelitis optica, opsoclonus myoclonus syndrome, optic neuritis, scleritis, Susac's syndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune inner ear disease(AIED), Ménière's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis (EGPA), giant cell arteritis, granulomatosis with polyangiitis (GPA), IgA vasculitis (IgAV), Kawasaki's disease, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, microscopic polyangiitis (MPA), polyarteritis nodosa (PAN), polymyalgia rheumatic, urticarial vasculitis, vasculitis, and primary immune deficiency.

[0418] In some embodiments, the present invention features methods for treating or preventing an infection or an infectious disease. In one embodiment, the present invention features methods for treating or preventing a bacterial infection or a disease or disorder associated therewith. The bacterium can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0419] The bacterium can be a gram-positive bacterium or a gram-negative bacterium. The bacterium can be an aerobic bacterium or an anerobic bacterium. The bacterium can be an autotrophic bacterium or a heterotrophic bacterium. The bacterium can be a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, a halophile, or an osmophile.

[0420] The bacterium can be an anthrax bacterium, an antibiotic resistant bacterium, a disease-causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella 135Attorney Docket No.: 046483-6287-00WO bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. The bacterium can be a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0421] In one embodiment, the present invention features methods for treating or preventing a viral infection or a disease or disorder associated therewith. In some embodiments, the virus is from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae (including SARS and SARS-CoV-2), Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The viral antigen can be from human immunodeficiency virus (HIV), Chikungunya virus (CHIKV), dengue fever virus, papilloma viruses, for example, human papillomoa virus (HPV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster, a.k.a., chickenpox), cytomegalovirus (CMV), for example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and mouth disease virus, lassa virus, arenavirus, or a cancer causing virus.

[0422] In one embodiment, the present invention features methods for treating or preventing a parasitic infection or a disease or disorder associated therewith. In some embodiments, the parasite is a protozoa, helminth, or ectoparasite. The helminth (i.e., worm) can be a flatworm (e.g., flukes and tapeworms), a thorny-headed worm, or a round worm (e.g., pinworms). The ectoparasite can be lice, fleas, ticks, and mites.

[0423] The parasite can be any parasite causing any one of the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, 136Attorney Docket No.: 046483-6287-00WO Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

[0424] The parasite can be Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.

[0425] In one embodiment, the present invention features methods for treating or preventing a fungal infection or a disease or disorder associated therewith. In some embodiments, the fungus is Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0426] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of a disease or disorder that is already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of a disease or disorder do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing a disease or disorder, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder.

[0427] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a disease or disorder, encompasses administering to a subject a composition as a preventative measure against the development of, or progression of, a disease or disorder. As more fully discussed elsewhere herein, methods of modulating the level or 137Attorney Docket No.: 046483-6287-00WO activity of a gene, or gene product, encompass a wide plethora of techniques for modulating not only the level and activity of polypeptide gene products, but also for modulating expression of a nucleic acid, including either transcription, translation, or both.

[0428] To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate composition to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0429] One of skill in the art will appreciate that the compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and / or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result. In various embodiments, any of the compositions of the invention described herein can be administered alone or in combination with at least one modulator of an additional molecule associated with a disease or disorder.

[0430] Administration of the compositions of the invention to a human patient can be by any route, including but not limited to intravenous, intranodal, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol, etc.), buccal (e.g., sub-lingual, etc.), topical (i.e., both skin and mucosal surfaces, including airway surfaces, etc.), intrathecal, intraarticular, intraplural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginal administration, by perfusion through a regional catheter, or by direct intralesional injection. In one embodiment, the compositions of the invention are administered by intravenous push or intravenous infusion given over defined period (e.g., 0.5 to 2 hours). The compositions of the invention can be delivered by peristaltic means or in the form of a depot, although the most suitable route in any given case will depend, as is well known in the art, on such factors as the species, age, gender and overall condition of the subject, the nature and severity of the condition being treated and / or on the nature of the particular composition (i.e., dosage, formulation) that is being administered. In particular 138Attorney Docket No.: 046483-6287-00WO embodiments, the route of administration is via bolus or continuous infusion over a period of time, once or twice a week. In other particular embodiments, the route of administration is by subcutaneous injection given in at least one site (e.g. thigh, waist, buttocks, arm), optionally once or twice weekly. In one embodiment, the compositions, and / or methods of the invention are administered on an outpatient basis.

[0431] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.

[0432] The method comprises administering a combination of composition in any suitable ratio. For example, in one embodiment, the method comprises administering two individual compositions at a 1:1 ratio. However, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed. Pharmaceutical Compositions

[0433] The formulations of the pharmaceutical compositions (e.g., comprising at least one delivery vehicle) described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., at least one delivery vehicle) into association with a carrier or at least one other accessory ingredient, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0434] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary 139Attorney Docket No.: 046483-6287-00WO pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0435] Pharmaceutical compositions (e.g., comprising at least one delivery vehicle) that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0436] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0437] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0438] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise at least one additional pharmaceutically active agents.

[0439] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0440] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. 140Attorney Docket No.: 046483-6287-00WO Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non- surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0441] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment, the active ingredient of a formulation for parenteral administration is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0442] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a 141Attorney Docket No.: 046483-6287-00WO biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0443] In various embodiments, the targeted delivery vehicles may be administered to a subject such that the delivery vehicle contacts the targeted cell in vivo. In other embodiments, the cell may be contacted with the delivery vehicles ex vivo and then transferred back to a subject in need with adoptive cell transfer. In this embodiment, cells are removed from a patient and modified ex vivo by contacting them with the herein disclosed delivery vehicles.

[0444] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. At least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0445] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (having a particle size of the same order as particles comprising the active ingredient).

[0446] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable 142Attorney Docket No.: 046483-6287-00WO carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment, the active ingredient of a formulation for parenteral administration is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0447] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0448] As used herein, “additional ingredients” include, but are not limited to, at least one of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; 143Attorney Docket No.: 046483-6287-00WO fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference. EXPERIMENTAL EXAMPLES

[0449] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0450] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure. Example 1: Modulating Trafficking of CTLs

[0451] A method for the efficient delivery of mRNA, encoding for any protein of interest, to specific immune cell subset based on their expression of surface receptors has recently been developed. Briefly, mRNA-lipid nanoparticles (LNPs), like those used in the SARS-CoV-2 vaccine, are modified using SATA-maleimide conjugation chemistry to display a protein of interest on their exterior surface (Howard MD, et al., MolPharm.2014;11(7):2262- 70). This technique has been applied to covalently attach soluble fractalkine to mRNA-LNPs to directly target CTLs. The data presented herein show that fractalkine-targeted mRNA-LNPs specifically and efficiently deliver their mRNA to CX3CR1+ leukocytes in vitro, even when used to treat mixed immune cell populations, as well as in murine CTLs in vivo. Additionally, an mRNA construct that expresses high levels of the cell trafficking receptor CD62L has been 144Attorney Docket No.: 046483-6287-00WO designed that, when delivered using fractalkine-targeted mRNA-LNPs, results in high level CD62L surface expression in human, mouse, cynomolgus and rhesus macaque cells in vitro. Therefore, without being bound by theory, it is hypothesized that CX3CR1+ effector cells can be trafficked into SLT from peripheral blood following fractalkine-targeted CD62L mRNA- LNP delivery. Fractalkine-targeted LNPs with CD62L mRNA to redirect and retain CTLs in SLT, alone or in combination with FTY720 (Fingolimod)

[0452] Accumulating evidence indicates that HIV- specific immune surveillance in SLT is limited due to the inability of vascular cytotoxic CD8+ T cells to traffic into HIV- infected SLTs. Those CD8+ T cells that do migrate into SLTs, composed primarily of naïve and central memory CD8+ T cells, do not express key effector proteins, most notably perforin and granzyme B, limiting their ability to eliminate infected cells. This differential migratory capability is largely based upon expression patterns of CD62L and CCR7, which are highly expressed on naïve and central memory T cells but lost on cells that differentiate to the cytotoxic state. Re-expression of CD62L (with or without CCR7) on vascular cytotoxic CD8+ T cells enables them to traffic into SLTs.

[0453] An mRNA-LNP based strategy was developed targeted to cytotoxic lymphocyte subsets (including CD8+ T cells, CD4+ T cells, and natural killer, NK, cells) through fractalkine receptor (CX3CR1). Using SATA-maleimide chemistry, fractalkine, the ligand for CX3CR1, was conjugated to mRNA-LNPs encoding green fluorescent protein (GFP) or human CD62L. These fractalkine targeted mRNA-LNPs were delivered to human and non-human primate (NHP) peripheral blood CD8+ T cells in vitro and exceptionally high efficiency was found in targeted expression of GFP or human CD62L within CX3CR1+ CD8+ T cells (Figure 1), CD4+ T cells, and NK cells. After further development, dosing, and titrations, these studies were expanded in vivo into LCMV-infected mice, again finding exceptionally potent delivery to CX3CR1+ CD8+ T cells in the blood (Figure 2) and spleen. Together these data provide the rationale for testing this strategy in rhesus macaques (RMs) to determine whether delivery of CD62L mRNA to CX3CR1+ cytolytic CD8+ T cells enables these cells to traffic into lymphoid tissues. 145Attorney Docket No.: 046483-6287-00WO

[0454] Fractalkine-targeted LNPs deliver human CD62L mRNA to CX3CR1+ CD8+ T cells after i.v. administration. Expression of CD62L occurs within hours of delivery and is detectable on CX3CR1+ lymphocytes in the blood by 24-hr post-administration. Following expression, a decrease in CX3CR1+ lymphocytes is seen in the vasculature and a corresponding increase in the SLT. Because the mutant CD62L construct is shedding deficient, maintenance of human CD62L on cells that traffic into SLT is seen. Because CX3CR1 is expressed by some CD4+ T cells and all CD16+ NK cells, these populations similarly expressing human CD62L in the blood, and traffic into SLT. NHP Study Design

[0455] A pilot study is performed to establish the safety and dosing of targeted LNP delivery, and the pharmacodynamics of mRNA expression with a GFP fluorescent reporter in healthy RMs. To increase expression, LNP administration is repeated as a dose escalation with a mCherry fluorescent reporter. Once an LNP dosing regimen is established, the targeted delivery of CD62L mRNA to CX3CR1+ lymphocytes is studied to demonstrate their homing to and retention in SLT (Figure 3). Briefly, healthy RMs are intravenously (i.v.) infused with fractalkine-targeted LNPs with CD62L mRNA at 0.25-2 mg / kg as 3-doses with a 2-day interval. Specifically, the mRNA is a mutant human CD62L that encodes the CD62L functional region linked to the transmembrane portion of CD62P, rendering it resistant to cleavage by extracellular proteases. If CTLs are substantially enriched in lymph node (LN) within 1-week of mRNA administration relative to baseline, then animals are euthanized by barbiturate overdose to harvest other SLTs, such as spleen and thymus, at a post-mortem necropsy. Otherwise, within 4-weeks animals will initiate therapy with oral / gavage FTY720 (Millipore Sigma) at 0.5 mg / kg administered daily for up to 28-days (Pino M, et al., PLoS Pathog.2019;15(10):e1008081; Matloubian M, et al., Nature.2004;427(6972):355-60; Pino M, et al., Nat Commun.2022;13(1):5055). After < 3 days of lead-in with FTY720, the macaques are reinfused with fractalkine-targeted LNPs with CD62L mRNA and are euthanized within 28-days. Longitudinal tissue collections include LN biopsies, LN fine needle aspirates (FNAs) and blood, including collections at 6-hr post-mRNA administration to monitor the peak response. 146Attorney Docket No.: 046483-6287-00WO T cell function:

[0456] Enhancements of CTL function are measured in PBMCs and LNMCs using a redirected killing assay to quantify the bulk cytolytic killing capacity of CD8+ T cells. For the redirected killing assay, FcγR+ P815 cells (“targets”) are labelled with CellTrace, loaded with an anti-CD3 mAb, and are cultured alone or in the presence of resting CD8+ T cells (“effectors”) at multiple ratios (1:3 and 1:6) in duplicate. Effector killing is measured as the frequency of live P815 cells expressing active caspase-3 by flow cytometry relative to background (Buggert M, et al., Cell.2020;183(7):1946-61). After 6hr, the culture is analyzed for expression of IL-2, TNF, IFN-γ, and CD107a in CD4+ and CD8+ T cells by flow cytometry and is reported relative to mock-stimulated controls (Harper J, et al., Nat Med. 2020;26(4):519-28; Harper J, et al., J ClinInvest.2022;132(8)). Tissue-resident CTLs and their impact on viral control in SIV-infected, ART-treated RMs

[0457] The antiviral activity of SLT-resident CTLs was examined when fractalkine- targeted LNPs with CD62L mRNA are administered in acute infection with antiretroviral therapy (ART) initiation and during suppressive ART in combination with an LRA (i.e. anti- CD4-targeted LNPs with Tat mRNA; Figure 4). Briefly, 6 RMs lacking MHC controller haplotypes (i.e. Mamu- A*01, -B*08, and -B*17) are experimentally infected with 500 infectious units (IU) of genetically barcoded SIVmac239M as an i.v. bolus. RMs then initiate ART at 3 weeks post- infection (WPI), which is administered as daily, subcutaneous (s.c.) injections (Loffredo JT, et al., J Virol.2007;81(16):8827-32; Loffredo JT, et al., JImmunol. 2009;182(12):7763-75; Mothe BR, et al., J Virol.2003;77(4):2736-40; Zhang ZQ, et al., J Virol.2002;76(24):12845-54). The ART regimen is composed of 2.5 mg / kg dolutegravir (DTG), 5.1 mg / kg tenofovir disoproxil fumarate (TDF), and 30 mg / kg emtricitabine (FTC) co- formulated in 15% kleptose (Roquette) (Del Prete GQ, et al., AIDS Res Hum Retroviruses. 2016;32(2):163-8) and is considered the standard of care for NHP studies (Gele T, et al., Pharmaceutics.2022;14(11)). These ART drugs have been used in a great number of animals (Pino M, et al., Nat Commun.2022;13(1):5055; Harper J, et al., Nat Commun. 147Attorney Docket No.: 046483-6287-00WO 2021;12(1):2866; Harper J, et al., Nat Med.2020;26(4):519-28; Harper J, et al., J ClinInvest. 2022;132(8); Strongin Z, et al., J Virol.2020;94(14); Statzu M, et al., Nat Microbiol. 2023;8(2):299-308; McBrien JB, et al., Nature.2020;578(7793):154-9). Within 3-days of ART initiation, RMs are i.v. infused with a fractalkine-targeted LNP with either CD62L mRNA (n=3) or an empty mRNA construct (n=3) at 0.25-2 mg / kg as 3-doses with a 2-day interval. Upon attaining viral suppression, all RMs with repeat therapy with fractalkine-targeted LNP with either CD62L or empty mRNA, but now in combination with an anti-CD4-targeted LNP with Tat mRNA, also at 0.25-2 mg / kg as 3-doses with a 2-day interval. Within 6-weeks of receiving the Tat mRNA, all macaques will undergo an ATI to monitor viral rebound kinetics. All animals are then euthanized within 8-weeks following ATI by barbiturate overdose.

[0458] Delivery of CD62L to CTL at ART initiation allows a greater reduction in the size of the SIV reservoir in lymphoid tissues compared to the control animals. This reservoir is further reduced upon the addition of the CD4-targeted Tat mRNA LNPs in combination with the fractalkine- targeted CD62L mRNA LNPs during therapy as compared to the control. This translates to a delay in time to rebound after removal of ART, and an overall decrease in the complexity of the reservoir through barcode analysis. It is possible that CD62L+ CTLs trafficking into the LN may not enter lymphoid follicles and clear reservoirs in T follicular helper (TFH) cells due to the absence of CXCR5. Recent single cell work profiling the HIV reservoir in ART-treated PLWH indicates that TFH cells are not the predominant lymphoid tissue reservoir under ART. These issues are addressed through the spatial imaging studies. T cell function:

[0459] Enhancements of CTL function are measured in PBMCs and LNMCs using a redirected killing assay to quantify the bulk cytolytic killing capacity of CD8+ T cells (as described in Aim 1) and with SIV peptide stimulations to quantify the expression of effector proteins in antigen-specific CD4+ and CD8+ T cells. Peptide stimulations are performed by incubating resting mononuclear cells with SIV Gag peptide pools (BEI Resource) in the presence of anti-CD28 and anti-CD49D mAbs for co-stimulation at 37°C to which a protein transport inhibitor cocktail is added after 2 hr. After 6hr, the culture is analyzed for expression of IL-2, TNF, IFN-γ, and CD107a in CD4+ and CD8+ T cells by flow expression of IL-2, 148Attorney Docket No.: 046483-6287-00WO TNF, IFN-γ, and CD107a in CD4+ and CD8+ T cells by flow cytometry, and CD107a in CD4+ and CD8+ T cells by flow cytometry and is reported relative to mock-stimulated controls (Harper J, et al., Nat Med.2020;26(4):519-28; Harper J, et al., J ClinInvest. 2022;132(8)). Virologic Assays:

[0460] The impact the size of the viral reservoir and the efficacy Tat-induced latency reversal is gauged via the plasma viral loads, which are supplemented with measures of intact viral genomes and cell - associated viral DNA and RNA. Levels of cell-associated SIV-RNA and -DNA are quantified using 10-replicate reaction RT-qPCR and qPCR assays, respectively, using genomic CCR5 normalization and a single copy limit of detection (Hansen SG, et al., Nature.2017;547(7661):123-4). Spatial Phenotyping Analyses:

[0461] Exploratory in situ imaging analyses are performed in LN using dedicated FFPE blocks. in situ hybridization techniques targeting SIVmac239nucleic acids will be used to identify cells harboring viral DNA (vDNA+) and expressing vRNA via DNAscope and RNAscope, respectively, and for cellular microenvironment analysis (Deleage C, et al., Pathog Immun.2016;1(1):68-106), which in combination with immunohistochemistry (IHC) approaches allows the discrimination of the absolute enrichment of infected cells by anatomical localization (i.e. B cell follicle, T cell zone, medullary cords, etc.) and phenotype (McGary CS, et al., Immunity.2017;47(4):776-88 e5). As part of her work in ERASE HIV, Dr. Deleage has recently optimized a PhenoCycler Fusion (Akoya Biosciences) panel for NHP specimens that permits spatial phenotyping analyses for up to 70-parameters, enabling us to investigate the impact of mRNA therapy on different cell types (i.e. NK cells, B cells, TFH, CD8+T cells); their differentiation and activation status and inflammatory environment (perforin, Granzyme B, MxA, Ki-67, STAT3, Bcl-6, ect.); and their co-localization with infected cells when multiplexed with DNAscope and RNAscope techniques. Viral barcode analyses: 149Attorney Docket No.: 046483-6287-00WO

[0462] The enumeration of viral barcode abundance is performed using the MiSeq platform (Illumina) (Fennessey CM, et al., PLoS Pathog.2017;13(5)). Mixed-effects modelling and statistical analyses of viral kinetics and diversity during rebound are employed to understand how immune-mediated control modulates the host-viral equilibrium. As infection seeds a diverse reservoir, individual viral recrudescence events at ATI (i.e. rebound founder lineages) bear a unique barcode. By comparing the size of the rebound founder populations with the growth rate, the impact of the interventions on the time is estimated between successive rebound events (Fennessey CM, et al., PLoS Pathog.2017;13(5); Pinkevych M, et al., Elife. 2019;8). Example 2: Treg-targeted LNPs to promote Treg trafficking to islets

[0463] Tregs, which express high levels of the high affinity IL2 receptor CD25, will be targeted using IL2-conjugated LNPs. mRNA cargoes within the IL-2 LNPs will modify Treg function in the islet environment by conveying tissue specificity through chimeric antigen receptor (CAR) expression and by increasing expression of chemokine receptors that promote tissue trafficking. Treg immunosuppressive function is maintained by expression of anti- inflammatory cytokine mRNAs within the targeted LNPs.

[0464] Without being bound by theory, it is anticipated that IL2-LNP targeting successfully induces Treg specific expression of all proposed constructs. Studies with human cells are predicted to show that exogenous DPP6 CAR expression will allow a high percentage of DPP6 CAR+ T cells to recognize and respond to human islet cells. Expressing DDP6 CAR in Tregs will enhance their suppressive responses in the presence of islet cells and suppress DPP6 CAR+ Tconv inflammatory responses. Further, expression of exogenous chemokine receptors on Tregs will increase Treg trafficking towards chemokine ligand gradient. There is a synergistic effect if targeted Tregs express DPP6, at least one chemokine receptor, and at least one immunosuppressive chemokine. This would confer islet antigen specificity, enhanced chemotactic ability, and increased immunosuppressive ability to a large percentage of Tregs. Due to the potentially high amount of mRNA cargo permutations, lead candidates will only be co-formulated from human studies. In mice, it is predicted that all planned mRNA cargo delivered via IL2-LNP will be highly skewed towards Tregs specifically. Further, Tregs 150Attorney Docket No.: 046483-6287-00WO expressing either DPP6 CAR, exogenous chemokine receptor, or exogenous immunosuppressive chemokines delays diabetes onset in NOD mice. Like in the proposed human in vitro studies, certain combinations of co-formulated mRNA cargoes increases targeted Treg tissue specificity, trafficking ability, and immunosuppressive effects in vivo, but only the lead combinatorial candidate from the human in vitro studies is tested further.

[0465] The major issues with in vivo gene therapeutic approaches are targeted delivery, efficiency, and off target effects. The approach described here mitigates many of these issues. First, IL2 mRNA LNPs are highly skewed towards CD25 expressing CD4+ Treg. As shown in preliminary data, CD25+ Treg preferentially take up the IL2 conjugated LNPs in vivo compared to other cells that may express CD25, reaching 60% in vivo targeting of CD25+ cells with over a 5-fold preference over unconjugated LNPs. The inherent transiency of mRNA LNP based approaches is a distinct advantage when there are concerns of off-target effects, as this strategy does not permanently modify the host. However, this transiency also may require repeated administration to maintain a beneficial effect. Finally, this strategy minimizes off- target effects in two ways. First, co-formulation of mRNAs expressing suppressive cytokines in tandem with the CARs or chemokine receptors ensures a regulatory function irrespective of the expressing lymphocyte subset. Secondly, LNPs are naturally uptaken by the liver. The IL2 based targeting strategy reduces LNP delivery to hepatocytes. Moreover, the proteins that are expressed have little to no functional impact on hepatocyte function during the tr...

Claims

1. Attorney Docket No.: 046483-6287-00WO CLAIMS What is claimed is:

1. A composition comprising at least one immune cell targeted delivery vehicle, wherein the at least one delivery vehicle comprises at least one nucleic acid molecule encoding a tissue trafficking modulator.

2. The composition of claim 1, wherein the at least one delivery vehicle comprises a lipid nanoparticle (LNP).

3. The composition of claim 2, wherein the at least one nucleic acid molecule encoding a tissue trafficking modulator is encapsulated in the LNP.

4. The composition of any of claims 1 to 3, wherein the delivery vehicle comprises a targeting ligand specific for binding to a receptor on the cell surface of at least one target immune cell.

5. The composition of claim 4, wherein the target immune cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a CX3CR1+ lymphocyte, a vascular cytolytic lymphocyte, a CD16+ NK cell, a monocyte, a macrophage, an activated neutrophil, a regulatory T cell, an activated CD25+ T cell, an early differentiated T cell, an effector memory CD4+ or CD8+ T cell, a NK cell, a B cell, a CD4+ T follicular helper cell, a CD8+ T follicular cytotoxic cell, a tissue homing CD4+ or CD8+ memory T cell, a resident memory CD4+ or CD8+ T cell, and a CD4+ Th17 cell.

6. The composition of claim 5, wherein the target immune cell is a CTL, and further wherein the immune cell targeting ligand comprises fractalkine or a fragment thereof comprising at least the chemokine binding domain. 198 Attorney Docket No.: 046483-6287-00WO 7. The composition of claim 6, wherein the immune cell targeting ligand comprises SEQ ID NO:1 or SEQ ID NO:

2.

8. The composition of claim 6, wherein the tissue trafficking modulator is selected from the group consisting of CD62L wild type, CD62L cleavage-defective mutant, CCR7, CCR2, CCR9, CXCR2, CXCR5 or CD69, or a variant or fragment thereof.

9. The composition of claim 8, wherein the tissue trafficking modulator comprises an amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, or SEQ ID NO:61, or a fragment or variant thereof.

10. The composition of claim 8, wherein the nucleic acid molecule comprises an RNA molecule comprising a sequence as set forth in SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO:62, or a fragment or variant thereof 11. The composition of claim 5, wherein the target immune cell is an activated CD25+ T cell or a regulatory T cell, and further wherein the immune cell targeting ligand comprises IL-2.

12. The composition of claim 11, wherein the immune cell targeting ligand comprises SEQ ID NO:

3. 199 Attorney Docket No.: 046483-6287-00WO 13. The composition of claim 11, wherein the tissue trafficking modulator is selected from the group consisting of CCR4, CCR8, CXCR1, CXCR2, CXCR3, or CXCR4, or a variant or fragment thereof.

14. The composition of claim 13, wherein the tissue trafficking modulator comprises an amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10, or a fragment or variant thereof.

15. The composition of claim 13, wherein the nucleic acid molecule comprises an RNA molecule comprising a sequence as set forth in SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:11, or a fragment or variant thereof.

16. The composition of any one of claims 1 to 15, wherein the delivery vehicle further encapsulates at least one additional agent.

17. The composition of claim 16, wherein the at least one additional agent is selected from the group consisting of a therapeutic agent, a gene editing agent, a cytokine receptor, and an immunosuppressive cytokine.

18. The composition of claim 16, wherein the at least one additional agent comprises a nucleic acid molecule encoding a therapeutic agent, a gene editing agent, a cytokine receptor, or an immunosuppressive agent.

19. The composition of claim 16, wherein the at least one additional agent comprises a nucleic acid molecule encoding at least one chimeric antigen receptor (CAR).

20. The composition of claim 16, wherein the at least one additional agent comprises a combination of a nucleic acid molecule encoding Cas9 and at least one sgRNA. 200 Attorney Docket No.: 046483-6287-00WO 21. The composition of claim 16, wherein the at least one additional agent comprises a nucleic acid molecule encoding an immunosuppressive agent selected from the group consisting of IL-10 and TGFβ.

22. The composition of any of claims 1 to 21, wherein the nucleic acid molecule encoding a tissue trafficking modulator comprises at least one messenger RNA (mRNA).

23. The composition of any of claims 1 to 22, wherein the nucleic acid molecule encoding a tissue trafficking modulator is delivered to the target cell.

24. The composition of any of claims 1 to 23, wherein expression of the tissue trafficking modulator alters the trafficking of at least one immune cell.

25. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1 to 24.

26. The method of claim 25 wherein the disease or disorder is selected from the group consisting of cancer, an infectious disease or an immunological disease or disorder.

27. The method of claim 25, wherein the disease is HIV.

28. The method of claim 25, wherein the disease is diabetes. 201

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