Multi-channel microfluidics to make next-generation lnp

Abstract

Provided herein are methods to generate lipid nanoparticles (LNPs) having a core and shell and compositions generated thereby. The LNP have various advantages over prior LNP including decreased heterogeneity, increased functionality, and increased ability to deliver cargoes.

Description

[0001] MULTI-CHANNEL MICROFLUIDICS TO MAKE NEXT-GENERATION LNP

[0002] Statement of Government Support

[0003] This invention was made with government support under HL153510, HL160694, HL164594 and HL157189 aw arded by the National Institutes of Health. The government has certain rights in the invention.

[0004] Background of the Invention

[0005] The billion-patient success of the COVID-19 mRNA-lipid nanoparticle (LNP) vaccine showed the unparalleled ability of LNPs to drive expression of chosen proteins. (PMID:34181394) This has led to »$10B investment by industry and governmental funders in developing LNPs therapeutics. Nearly all of these programs focus on mRNA cargo. However. mRNA has two fundamental limitations: i) a half-life of hours, which precludes treatment of chronic diseases (PMID: 26264835); ii) no promoter region, so an inability7to control cell-type-specific protein expression, thus leading to most protein being expressed in off-target cell types (PMID:30374059).

[0006] DNA can solve both of these problems, as it has intrinsic intracellular stability for years and has promoters which can drive exquisite cell-type-specificity. Unfortunately, in the 20 years of LNP development, DNA-loaded LNPs (DNA-LNPs) w ere never advanced because they suffered from tw o different problems: a) DNA-LNPs w ere found to be very toxic, killing naive mice within 2 days; b) DNA-LNPs expressed protein at levels many orders of magnitude less than that of mRNA-LNPs. We recently solved the first problem (PMID: 38915627). We began by showing that the toxicity7of DNA-LNPs is caused by detection of cytosolic DNA by the cGAS-STING pathway, which then activates massive inflammation. Next we loaded DNA-LNPs with nitrated lipids, which are mammals’ natural negative feedback inhibitor of STING. DNA-LNPs loaded with such STING-inhibiting lipids (e.g., nitro-oleic acid) brought the mortality of DNA-LNPs from 100% to 0%. Further, these STING-inhibiting DNA-LNPs stably express protein in vivo for 6 months. However, STING-inhibition alone was not sufficient to fix the second problem of DNA-LNPs, that they have protein expression levels 1,000- to 10,000-fold lower than the peak of mRNA-LNPs. Here we set out to solve that problem.

[0007] What is needed are compositions and methods to protect cargo DNA when it is loaded into LNPs or similar nanocarrier systems.

[0008] Summary of the Invention

[0009] Provided herein in one aspect is a method for generating lipid nanoparticles (LNP) encapsulating a cargo. The method includes mixing a first solution comprising one or more condensing agents with a second solution comprising the cargo, whereby the cargo is condensed, then mixing the condensed cargo with a third solution comprising one or more lipids in an organic solvent, thereby generating LNP having a core containing the cargo with a lipid shell.

[0010] In another aspect is provided is a method for generating lipid nanoparticles (LNP) encapsulating a cargo, the method comprising: in a nanoparticle formulation system comprising a first channel, a second channel, a third channel, and a fourth channel, (a) providing a first solution comprising one or more condensing agents and an organic solvent or a buffer in the first channel; (b) providing a second solution comprising the cargo and a buffer in a second channel; (c) providing a third solution comprising one or more lipids in an organic solvent and a fourth solution comprising one or more polymers, proteins, or peptides in a buffer, the third solution and fourth solution in the third channel; (d) mixing the first solution and the second solution; (e) introducing the mixture of (d) with the third solution and fourth solution to form the LNP. In certain embodiments, the first solution comprises a cationic lipid with or without a helper lipid, ionizable lipid, amphiphilic lipid, protein, peptide, or polymer.

[0011] In another aspect, a method for generating lipid nanoparticles (LNP) encapsulating a cargo is provided, the method comprising: in a nanoparticle formulation system comprising a first channel, a second channel, and a third channel, (a) providing a first solution comprising one or more condensing agents and an organic solvent or a buffer in the first channel; (b) providing a second solution comprising the cargo and a buffer in a second channel; (c) providing a third solution comprising one or more lipids in an organic solvent in the third channel; (d) mixing the first solution and the second solution; (e) introducing the mixture of (d) with the third solution to form the LNP.

[0012] In one embodiment, the first solution comprises a cationic lipid, optionally DOTAP, DOPSA, DOTMA, DMRIE, or N-Tetamine-pLys40, and a helper lipid, optionally DOPE. In other embodiments, the first solution comprises DOTAP at a lipid molar % of 1.96% and DOPE at a lipid molar % of 6.4%.

[0013] In one embodiment, the cargo comprises DNA, RNA, peptide, protein, protein- DNA complex, peptide-DNA complex, lipid-DNA complex, proteolysis targeting chimera (PROTAC), or small molecule.

[0014] In one embodiment, the third solution comprises cholesterol, ionizable lipid, and / or pegylated lipid. In other embodiments, the third solution comprises cholesterol at a lipid molar % of 38.7%. ionizable lipid (e.g., SM102) at a lipid molar % of 45.3%. DMG-PEG at a lipid molar % of 1.5% diluted with 100% ethanol. In another embodiment, the third solution compromises cholesterol at a lipid molar % of 37.5%, ionizable lipid (e.g., SM102) at a lipid molar % of 44.65%. DSPC at a lipid molar % of 8. 1% DMG-PEG at a lipid molar % of 1.37%. In other embodiments, the third solution comprises (a) a cationic lipid in an amount from about 0 to about 80% of total lipid present in the particle; (b) a non-cationic lipid in an amount from about 20% to about 99.9% of the total lipid present in the particle, wherein the non-cationic lipid comprises a mixture of a phospholipid and a cholesterol or derivative thereof; (c) a polyethylene glycol (PEG)-lipid conjugate in an amount from about 0. 1 to about 10% of the total lipid in the particle, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 5000 to about 20000 daltons. In certain embodiments, the third solution further comprises a nitro-oleic acid (NOA) (STING-inhibitory lipid) at a lipid molar % of 0.18%.

[0015] In one embodiment, the flow rate ratio is from 0.1:0.1 : 100 - 100:100:0.1. In other embodiments, the LNPs have a diameter of about 150nm. In other embodiments, the LNPs have a poly dispersity index (PDI) of about 0.1. In other embodiments, the LNPs have a neutral surface charge.

[0016] In another aspect, a LNP made by the method of any of the above is provided.

[0017] In another aspect, a LNP comprising a core and a shell is provided, wherein the core comprises a cargo and a condensing agent, wherein the shell comprises an ionizable lipid, helper lipid, cholesterol, and / or PEG.

[0018] In another aspect, a method of delivering a cargo to a subject is provided, comprising administering an LNP to the subject.

[0019] Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.

[0020] Brief Description of the Drawings

[0021] FIG. 1 depicts an overview of the core-then-shell method.

[0022] FIGs. 2A-2B shows a Zeta view (FIG. 2A) of standard LNP versus core-then- shell (CTS) LNP and differential scanning calorimetry (DSC) (FIG. 2B) of 2-channel versus 3 -channel LNPs.

[0023] FIGs. 3A-20E show a comparison of 2-channel versus 3-channel formulations. Standard LNPs are standard citrate LNP made by 2-channel cartridge. CTS LNPs are core-then-shell LNP in TBS buffer, made by a 3-channel cartridge. FIG. 3A shows a comparison of siRNA LNP between 2-channel and 3-channel. FIG. 3B shows a comparison of mRNA raw expression over 24 hours between 2-channel and 3-channel. FIG. 3C shows a comparison of mRNA hela expression over 24 hours between 2- channel and 3-channel. FIG. 3D shows a comparison of DNA raw expression between 2- channel and 3-channel. FIG. 3E shows a comparison of DNA hela expression between 2- channel and 3-channel.

[0024] FIG. 4 demonstrates core-then-shell (CTS) loading mRNA expression with different cores. Regular CTS has a core containing DOTAP and DOPE and the shell has SMI 02. The new core has SMI 02 in the core and no SMI 02 in the shell. The shell only contains cholesterol, helper lipid and PEG lipid.

[0025] FIG. 5A shows IVIS Spectrum in vivo imaging of mice comparing standard LNP and CTS LNP with a PBS control. Mice were injected with 5ug of DNA loaded LNP and IVIS was measured 24 hours after injection. The total flux (photons / s) is plotted on FIG. 5B.

[0026] FIG. 6A shows screenshots of nanoparticle tracking analysis (NT A) for detection of mRNA loading videos of 2-channel LNPs versus 3-channel LNPs. The percentage of nanoparticles detected with mRNA fluorescence is plotted on FIG. 6B.

[0027] FIG. 7A shows screenshots of nanoparticle tracking analysis (NTA) videos for detection of mRNA loading of 3-channel LNPs versus 3-channel LNPs with an ionizable lipid core. The percentage of nanoparticles detected with mRNA fluorescence is plotted on FIG. 7B.

[0028] FIGs. 8A-8L demonstrate improving transfection by optimizing the buffer used during LNP synthesis to ensure DNA stability . DNA-LNPs have 4 major hurdles to high protein expression: DNA structural perturbations during the LNP synthesis (#1); upon cell uptake and endosomal escape, the DNA can be recognized by intracellular DNA sensors such as cGAS-STING (#2) and cleaved by DNases (#3); finally, the DNA must travel through the cytosol (#4) and cross into the nucleus to engage in transcription (#5) (FIG. 8 A). The current study aims to employ structural modifications of DNA-LNPs to overcome these 5 hurdles. FIG. 8B demonstrates how testing how the buffer used during DNA-LNP synthesis can change the fraction of DNA in the B-form, which is optimal for transcription. DNA was dissolved in either the most common buffer for mRNA-LNP synthesis, citrate pH 4, or TBS buffers pH 6 with 4mM Mg2+, then subjected to CD spectroscopy. The ellipticity’ signal at 275 nm correlates with B-form DNA, and highest for the TBS buffers containing Mg2+, and lowest for citrate. FIG. 8C depicts a schematic illustration of making standard LNP: LNP is formed by mixing of lipid mixture and DNA plasmid in aqueous buffer, followed by dialysis in PBS containing Mg2+in correspondence to Mg2+concentration in the aqueous buffer, unless otherwise noted. FIG. 8D shows cryo-EM of DNA-LNPs in citrate or optimized TBS buffers. A macrophage-like cell line (RAW 264.7) was transfected with DNA-LNPs encoding luciferase, synthesized with the listed buffers (FIGS. 8E-8F). By far the highest expression was seen with TAE buffer containing Mg2+and dialyzed against Mg2+ containing PBS. FIGs. 8G-8I compare DNA expression in macrophages using 4 buffering molecules at pH 5 (FIG. 8G) and pH 6 (FIG. 8H) and with added Mg2+(FIG. 81), showing TBS with Mg2+at pH 6 is the optimal buffer. Using an epithelial cell line (HeLa) produces the same general trends as seen in macrophages, with the exception that in the presence of Mg2+, TAE outperforms TBS (FIGs. 8J-8L).

[0029] FIG. 9A depicts a CD spectrum which show s highest 275 nm signal in TBS pH 6 buffer with 4mM Mg2+. Increasing Mg2+concentration in citrate buffer reduces DNA expression (FIG. 9B). Expression of optimal TBS-pDNA-LNP is only -5% of lipofectamine in macrophages and HeLa cells (FIGs. 9C-9D).

[0030] FIGs. 10A-10L demonstrates multi-stage mixing (MSM) to produce core-then- shell (CTS) improves the encapsulation of nucleic acids. Instead of the standard single mixing stage LNPs have used for decades, core-then-shell LNPs are made via multistage mixing (FIG. 10A). First, in the Core Stage, the DNA (aqueous buffer) is mixed via a microfluidic with an organic solvent containing a cationic molecule and helper lipid. This condenses the DNA into a “core.” Immediately upon core formation, the cores are mixed with standard LNP lipids in the organic phase, to form the “shell”. CTS LNPs are larger than standard (Std) LNPs, but have a lower poly dispersity index (PDI) (FIG. 10B). CTS LNPs are closer to neutral, measuring the bulk nanoparticle population on a ZetaSizer (FIG. IOC). CTS- and standard-LNP has similar DNA entrapment efficiency around 90% (FIG. 10D). FIGs. 10E-10G shows that to evaluate the distribution of nucleic acids within a population of LNPs, we used Nanoparticle Tracking Analysis (NTA) to trace particles using light scattering (right panels) and simultaneously measure DNA loading into each particle via fluorescently labeled DNA (left panels). In FIG. 10F and FIG. 10G, w e plot the distribution of nanoparticle sizes, as measured by light scattering (solid lines) or DNA fluorescence (dashed). FIG. 10F shows that in standard LNPs. a low fraction of particles contain detectable DNA, while FIG. 10G shows that for CTS-LNPs a very high fraction of nanoparticles contain DNA. FIG. 10H depicts quantification of FIGs. 10F-10G, showing that in standard LNPs, only -50% of LNPs have detectable DNA, while 83% of CTS LNPs have detectable DNA. FIGs. 10I-K show NTA evaluation of mRNA payload in standard LNP vs CTS LNP. FIG. 10L depicts quantification of FIGs. 10J-10K, showing that in standard LNPs, only 5% of LNPs have detectable RNA. while 98% of CTS LNPs have detectable RNA.

[0031] FIG. 11 A demonstrates that the activated partial thromboplastin time (aPTT) measures how long it takes to generate fibrin after the intrinsic pathway is activated. CTS LNP treatment has the same aPTT as Std LNPs. CTS LNP only has 1.69% DOTAP, so even if LNPs made with CTS formulation but with 2-channel system (mixing all the lipids in one channel and DNA in another channel, then formulating LNP as standard LNP procedure), the aPTT is still significant less than cationic DOTAP LNP containing 50% DOTAP. Prothrombin time (PT) measures the time it takes to generate fibrin after extrinsic pathway is activated (FIG. 11B). CTS LNP and 2-channel LNP made with CTS LNP formulation treatment has the same PT as Std LNPs, while cationic DOTAP LNP significantly reduces PT. Biodistribution of In-111 labeled LNP shows cationic DOTAP LNPs greatly accumulate in the lungs, but CTS LNPs are uptaken primarily in liver and spleen (FIG. 11C). Biodistribution if 1-125 labeled fibrinogen deposition in major organs shows no difference among cationic DOTAP LNP, CTS LNP and CTS LNP made with 2-channel (FIG. 1 ID).

[0032] FIG. 12A-12G demonstrates multi-stage mixing dramatically changes the internal stmcture of DNA-LNPs, producing a distinct core vs shell. Cryo-EM of core-then-shell (CTS) vs standard LNPs shows that CTS-LNPs made via multi-stage mixing (MSM) display a clear core and shell (FIG. 12A). Using a ZetaView to evaluate the distribution of zeta potentials within a population of nanoparticles (FIG. 12B). We primarily compared two LNP syntheses: the CTS lipid formulation (containing <2% DOTAP), made either with MSM or single-stage (traditional) mixing. Single-stage mixing (green) produces two very' separate populations, one positive and one negative; by contrast, MSM (blue) produces CTS LNPs with a single peak, similar to the standard LNP formulation (no DOTAP). This shows MSM is critical to forming CTS LNPs. LNPs were subjected to differential scanning calorimetry, in which the LNPs are heated and the molar heat capacity measured (FIG. 12C). Standard LNPs (black line) display a broad and short peak, consistent with an amorphous structure and / or a diverse population of structures. By contrast, CTS-LNPs display two tall, sharp peaks, consistent with a uniform population (as shown by the ZetaView data) with two separate phases that are each highly ordered at the nanoscale. FIG. 12D depicts primary SAXS scattering profiles of CTS and standard LNPs displayed as log-log plots. Standard LNPs show characteristic high scattering intensity (Ip = 6.73 cm ) indicative of uniform internal organization, while CTS LNPs exhibit intermediate intensity (Ip = 0.46 cm ') suggesting more complex structural arrangements. FIG. 12E depicts shape distribution function analysis of standard LNPs reveals well-defined structural parameters (Rg = 156 A, Dmax = 400 A), consistent with traditional LNP architecture. The smooth distribution profile indicates homogeneous internal organization. SASVIEW Analysis shows core-shell sphere model fit with poly dispersity term (FIG. 12F). Multiple Lorentz peak fitting analysis reveals distinct peak patterns between standard and CTS LNPs, providing additional evidence for their different internal organizations (FIG. 12G-12H). Standard LNPs show characteristics of uniform lipid organization, while CTS LNPs display features consistent with multiple structural domains.

[0033] FIG. 13 A depicts a schematic illustration of in-house made microfluidic chip. FIG. 13B demonstrates the size of each formulated CTS LNPs. FIG. 13C shows PDI of each LNPs. FIG. 13D shows DNA entrapment efficiency of each LNP formulation. FIG. 13E shows pDNA expression in HeLa cells, demonstrated as % of lipofectamine positive control. Condition ‘CTS LNP’ is made using dilution cartridges with 3 channels in NanoAssemblr™ Ignite™ nanoparticle formulation systems.

[0034] FIGs. 14A-14K demonstrate core- then-shell LNPs enhance transfection by orders of magnitude via improving endosomal escape and rapidly localizing DNA to the nucleus. In vitro transfection with the listed LNPs in RAW macrophages (FIGs. 14A- 142C) and HeLa epithelial cells (FIGs. 14D-14F) shows that comparing with lipofectamine, a gold standard benchmark for in vitro transfection. CTS-DNA-LNPs express 10-fold higher than standard DNA-LNPs in RAW macrophages and 100-fold higher in HeLa cells, and equivalent to mRNA-LNPs. In addition, CTS LNP has significant transfection efficiency than Std LNP. Not only for DNA, CTS-mRNA-LNPs also enhance mRNA expression, compared to standard mRNA-LNPs. To measure endosomal escape events, HeLa cells were transfected with plasmids containing GFP- labeled galectin-9, which forms puncta around endosomes that have opened (FIG. 14G). The Gal-9-GFP puncta per cell were higher in CTS LNP compared to standard LNPs, and reached a similar level to the gold-standard lipofectamine (FIGs. 14H-14I). To measure DNA uptake and import into the nucleus, DNA was fluorescently labeled with SYBR-Green, loaded into CTS-LNPs, and given to HeLa cells (FIGs. 14J-14K). The uptake into the nucleus via CTS-LNPs is on a very similar time course to that of lipofectamine.

[0035] FIGs. 15A-15O demonstrate co-loading a STING-inhibitor into core-then-shell (CTS) DNA-LNPs enables high-level, long-term expression of DNA-LNPs in primary cells in culture and in vivo. The cGAS-STING pathway detects cytosolic DNA and thereafter activates pathways involved in inflammation and repression of protein translation (FIGs. 15A-15B). To measure STING activity, we stained RAW macrophages with phospho-STING antibody, and found that CTS-LNPs activate STING much more than standard LNPs, and to the same extent as lipofectamine. STING induces expression of IFN-P, which activates inflammation and represses translation (FIG. 15C). C- 178 is a small molecule analog of the endogenous negative feedback on STING, nitrated lipids. CTS-DNA-LNPs massively increase expression of IFN- , but this is completely inhibited by co-loading C-178 into the CTS-LNPs (FIG. 15D). In both RAW macrophages and HeLa epithelial cells, C-178 co-loading improves protein expression (FIGs. 15E-15F). Lipofectamine is the gold standard for in vitro transfection, but is inefficient with most primary cells (FIGs. 15G-J). Here we measured expression, via GFP plasmids, in mouse embryonic fibroblasts (FIGs. 15G-15H), and mouse primary cortical neurons (FIGs. 15I-15J). In both cases, CTS-LNPs co-loaded with C-178 had markedly higher transfection efficiency than lipofectamine. Standard and CTS-LNPs, without any STING inhibitor (e g., C-178), were IV-injected into mice and luciferase expression was measured via IVIS at 24 hours (FIG. 15K). CTS showed expression markedly above standard LNPs, but the CTS-LNPs’ expression was likely inhibited by STING expression. CTS-LNPs without STING inhibition had 100% mortality7within 3 days, but CTS-LNPs co-loaded with C-178 had 0% mortality (FIG. 15M). After IV injection, standard-DNA-LNPs with or without STING inhibitor NOA drive expression that is several orders of magnitude lower than mRNA-LNPs, though we recently showed that DNA-LNPs drive expression for 6 months, while LNP-delivered mRNA decays in days (FIG. 15N). However, CTS-DNA-LNPs with co-loaded C-178 show peak expression levels orders of magnitude higher than standard-DNA-LNPs. with the dose 5 times lower than standard-DNA-LNPs, and comparable to mRNA-LNPs’ peak.

[0036] FIGs. 16A-16N shows multi-stage mixing (MSM) to produce core-then-shell (CTS) improves the encapsulation of nucleic acids. FIG. 16A. Schematic illustration of making CTS LNP. Instead of the standard single mixing stage LNPs have used for decades, CTS LNPs are made via multi-stage mixing. First, in the Core Stage, the DNA (aqueous buffer) is mixed via a microfluidic with an organic solvent containing a cationic lipid and helper lipid. This condenses the DNA into a “core.” Immediately upon core formation, the cores are mixed with standard LNP lipids in the organic phase, to form the “shell”. FIG. 16B. Representative Cryo-EM shows that CTS-LNPs created using multi-stage mixing (MSM) display distinct core and shell phases. The white bar shown denotes a length of 25 nm. FIG. 16C. Schematic illustration of in-house made microfluidic chip. FIG. 16D. Dynamic Light Scattering (DLS). CTS LNPs are larger than standard (Std) LNPs, but have a lower poly dispersity index (PDI). FIG. 16E. ^-Potential Analysis using ZetaSizer. CTS LNPs are closer to neutral, measuring the bulk nanoparticle population. FIG. 16F. DNA entrapment efficiency : CTS LNP and standard LNP have similar DNA entrapment efficiency around 90%. FIGs. 16G-16I. DNA cargo payload distribution in LNPs. To evaluate the distribution of DNA within a population of LNPs, we used Nanoparticle Tracking Analysis (NTA) to trace particles using light scattering (right panels) and simultaneously measure DNA loading into each particle via fluorescently labeled DNA (left panels). In FIGs. 16H and 161, w e plot the distribution of nanoparticle sizes, as measured by light scattering (solid lines) or DNA fluorescence (dashed). FIG. 16H shows that in standard LNPs, a low fraction of particles contain detectable DNA, while FIG. 161 show's that for CTS LNPs a very high fraction of nanoparticles contain DNA. FIG. 16J shows quantification of FIGs. 16H-16I, showing that in standard LNPs. only -50% of LNPs have detectable DNA, while 83% of CTS . RNA cargo payload distribution in LNPs. NTA evaluation of mRNA payload in standard LNP vs CTS LNP shows similar trend as in DNA LNP. FIG. 16N. Quantification of FIGs. 16L-16M, showing that in standard LNPs, only 5% of LNPs have detectable RNA, while 98% of CTS LNPs have detectable RNA.

[0037] FIGs. 17A-17I demonstrate multi-stage mixing dramatically changes the internal structure of DNA-LNPs, producing a distinct core vs shell. FIG. 17A. ^-Potential Analysis. We compared the ^-potentials of two LNP formulations: the CTS lipid formulation (containing <2% DOTAP). made either with MSM or single-stage (traditional) mixing. Single-stage mixing produces two very separate populations with ver7low magnitude potentials, one positive and one negative; by contrast, MSM produces CTS LNPs with a single broad distribution of potentials betw een 0 mV and -50 mV. like seen with the standard LNP formulation (no DOTAP. black). This demonstrates that MSM is critical to creating CTS LNPs. FIG. 17B. Differential Scanning Calorimetry (DSC). The molar heat capacity' of CTS and standard LNP particles were determined. Standard LNPs (black line) display a broad profile and short peak, consistent with modestly stable population. In contrast. CTS-LNPs shows two sharp peaks with a larger magnitude, consistent with a more uniform and w ell-ordered population of higher stability. FIG. 17C. Dynamic Light Scattering (DLS). Particle size distributions, normalized for intensity, as determined by DLS are shown as lognormal distributions, with CTS and standard LNPs (shown in black). CTS particles had a determined hydrodynamic radius (Rh) of 151 A ± 2.3 (poly dispersity index (PDI) = 0.14) compared to 80.2 A ± 2.5 (PDI = 0.28) for the standard formulation. FIG. 17D. Radius of Gyration Analysis (Rg) using Size-exclusion Chromatography In-line with Multiangle Light Scattering (SEC-MALS). Shown is a double-y plot, where the left axis provides the normalized refractive index for eluant as a function of retention time for CTS particles (line), while the right axis provides on a log-scale the radius of gyration (Rg, in nm) of the CTS particles as determined using 18-angle light scattering (circles). Across the peak region analyzed, a Rg of 39.5 nm ± 5.5% was determined. A similar analysis could not be performed for standard LNP particles, as the particles were well below the size threshold needed for reliable determination of Rg. FIG. 17E. Mass profiles using SEC-MALS. Shown is a double-y plot, where the left axis provides the normalized refractive index for eluant as a function of retention time for standard (black line) and CTS particles, while the right axis provides on a log-scale the determined molar mass. Across the peak regions analyzed, a Mwof 23.7 MDa ± 2.1% was determined for CTS particles and 5.4 MDa ± 3.1% for the standard LNP particles. FIG. 17F. Synchrotron Size-Exclusion Chromatography in-line with Small-Angle X-ray Scattering (SEC-SAXS). Log-linear plots of SAXS data from standard LNPs (black line) and CTS particles, showing the decay of scattering intensity as a function of the scattering vector q (where < / =4asinO / Z). Characteristic first-order Braggs peak features are observed between ^=0. 1-0.2 A'1. FIG. 17G. Multiple Lorentz Function analysis of Braggs peaks from SAXS of CTS Particles. Shown are the SAXS data in a linear-linear plot. Multiple Lorentz fits were applied to deconvolute the data and are shown as dotted lines. The assignment of the cubosome phase peaks with the spacing of <7=62.8 A and corresponding peaks at integral values of <7*^2 and q*^3. The overlapping peak at <7=0.125 A'1(<7=50.2 A) could additionally be assigned to corresponding integral peaks integral values of q*^2 and q*^3. FIG. 17H. Multiple Lorentz Function analysis of Braggs peaks from SAXS of standard LNP Particles. Shown as black line are the SAXS data in a linear-linear plot. Multiple Lorentzian models were applied to deconvolute the data and are shown as dotted lines. The assignment of the H|| phase peaks with the spacing of d=54.6 A and corresponding peaks at integral values of <7*^3 and q*^4. The overlapping peak at <7=0.127 A'1(d=49.5 A) could be related to a corresponding integral peaks <7*^2, suggesting an additional cubosome phase. FIG. 171. SASVIEW Analysis. Fitting of I vs q data shows core-shell sphere (light grey line) and core-shell ellipsoid model (dark grey lines) fits with poly dispersity7term. Residuals of the fits are shown in the lower panel. In this fitting, scattering length densities (SLDs) were fixed to calculated values and the shell diameter to that of a lipid bilayer (54 A). The SLDs for the component parts of the DNA LNP formulations used in this fitting w ere calculated using MULCh. In the core-shell sphere model, a core radius of 101.8 A ± 42.1 w as determined (x2=2.7). In the core-shell ellispsoidal model fit, an equatorial core radius of 86.5 A ± 86.9 and a long axis radius of 136.7 A ± 332.5 was determined (%2=2.4). FIGs. 18A-18L shows core-then-shell LNPs enhance transfection by orders of magnitude via improving endosomal escape and rapidly localizing DNA to the nucleus. FIG. 18 A. DNA LNPs have 5 major hurdles to high protein expression: DNA structural perturbations during the LNP synthesis (#1); Upon cell uptake and endosomal escape, the DNA can be recognized by intracellular DNA sensors such as cGAS-STING (#2) and cleaved by DNases (#3); Finally, the DNA must travel through the cytosol (#4) and cross into the nucleus to engage in transcription (#5). The current study aims to employ structural modifications of DNA LNPs to overcome these hurdles. FIGs. 18B- 18G) In vitro transfection with the listed LNPs in RAW macrophages (FIGs. 18B, 18D, 18F) and HeLa epithelial cells (FIGs. 18C, 18E, 18G) shows that comparing with lipofectamine, a gold standard benchmark for in vitro transfection. CTS DNA LNPs express 10-fold higher than standard DNA LNPs in RAW macrophages and 100-fold higher in HeLa cells, and equivalent to mRNA-LNPs. In addition, CTS LNP has significant transfection efficiency than Std LNP. Not only for DNA, CTS mRNA LNPs also enhance mRNA expression, compared to standard mRNA-LNPs. FIG. 18H. Endosome escape events. To measure endosomal escape events, HeLa cells were transfected with plasmids containing GFP-labeled galectin-9, which forms puncta around endosomes that have opened. FIG. 181. Representative image of Gal -9 reporter cells after LNP or lipofectamine treatment. FIG. 18 J. Quantification of Gal-9 puncta. The Gal-9- GFP puncta per cell were higher in CTS LNP compared to standard LNPs, and reached a similar level to the gold-standard Lipofectamine. FIG. 18K. Representative image of time lapse of DNA uptake in HeLa cells. FIG. 18L. Quantification of DNA uptake. To measure DNA uptake and import into the nucleus, DNA was fluorescently labeled with SYBR-Green, loaded into CTS-LNPs, and given to HeLa cells. The uptake into the nucleus via CTS LNPs is on a very similar time course to that of Lipofectamine. Scale bar denotes 50 pm.

[0038] FIGs. 19A-19O demonstrates co-loading a STING-inhibitor into core-then-shell (CTS) DNA LNPs enables high-level, long-term expression of DNA LNPs in primary cells in culture and in vivo. FIG. 19A. Representative image of phosphate-STING (p- STING) signal in RAW macrophages after LNP or lipofectamine treatment. The cGAS- STING pathway detects cytosolic DNA and thereafter activates pathways involved in inflammation and repression of protein translation. To measure STING activity, we stained RAW macrophages with phospho-STING antibody. FIG. 19B. Quantification of p-STING. We found that CTS LNPs activate STING much more than standard LNPs. and to the same extent as Lipofectamine. FIG. 19C. Schematic illustration of cGAS- STING activation pathway and C-178 function in inhibition of STING. STING induces expression of IFN-P, which activates inflammation and represses translation. C-178 is a small molecule analog of the endogenous negative feedback on STING. FIG. 19D. IFN- level. CTS DNA LNPs massively increase expression of IFN-P by RAW macrophages, but this is completely inhibited by co-loading C-178 into the CTS LNPs. FIGs. 19E-19F. In vitro transfection of CTS LNP co-loading with C-178. In both RAW macrophages and HeLa epithelial cells, C-178 co-loading improves protein expression. FIGs. 19G-19J. Primary cell transfection by CTS LNPs. Lipofectamine is the gold standard for in vitro transfection, but is inefficient with most primary cells. Here we measured expression, via GFP plasmids, in mouse embryonic fibroblasts (FIG. 19G, 19H), and mouse primary cortical neurons (FIG. 191, 19J). In both cases, CTS LNPs co-loaded with C-178 had markedly higher transfection efficiency than Lipofectamine, and preserved neuron morphology. FIG. 19K. Representative images from In Vivo Imaging System (IVIS). Standard and CTS LNPs, without any STING inhibitor (e.g., C-178), were IV-injected into mice and luciferase expression was measured via IVIS at 24 hours. FIG. 19L. IVIS image quantification. CTS showed expression markedly above standard LNPs, but the CTS LNPs’ expression was likely inhibited by STING expression. FIG. 19M. Animal survival. CTS LNPs without STING inhibition had 100% mortality within 3 days, but CTS LNPs co-loaded with C-178 had 0% mortality. FIG. 19N. Day 1 IVIS image quantification. After IV injection, standard DNA LNPs drive expression that is several orders of magnitude lower than mRNA LNPs, though we recently showed that these DNA LNPs drive expression for more than 3 months, while LNP-delivered mRNA decays in days. However, CTS DNA LNPs with co-loaded C-178 show peak expression levels orders of magnitude higher than standard-DNA-LNPs. with the dose 5 times lower than standard-DNA-LNPs, and comparable to mRNA LNPs’ peak. FIG. 190. Longitudinal quantification of DNA expression in vivo. Scale bar denotes 50 pm.

[0039] FIGs. 20A-20D. FIG. 20A. Activated partial thromboplastin time (aPTT) measures how long it takes to generate fibrin after the intrinsic pathway is activated. CTS LNP treatment has the same aPTT as Std LNPs. CTS LNP only has 1.69% DOTAP, so even if LNPs made with CTS formulation but with 2-channel system (mixing all the lipids in one channel and DNA in another channel, then formulating LNP as standard LNP procedure), the aPTT is still significant less than cationic DOTAP LNP containing 50% DOTAP. FIG. 20B. Prothrombin time (PT) measures the time it takes to generate fibrin after extrinsic pathway is activated. CTS LNP and 2-channel LNP made with CTS LNP formulation treatment has the same PT as Std LNPs, while cationic DOTAP LNP significantly reduces PT. FIG. 20C. Biodistribution of In-111 labeled LNP shows cationic DOTAP LNPs greatly accumulate in the lungs, but CTS LNPs are uptaken primarily in liver and spleen. FIG. 20D. Biodistribution of 1-125 labeled fibrinogen deposition in major organs shows no difference among cationic DOTAP LNP, CTS LNP and CTS LNP made with 2-channel.

[0040] FIGs. 21A-21C. FIG. 21A. Schematic illustration of making standard LNP: LNP is formed by mixing of lipid mixture and DNA plasmid in aqueous buffer, followed by dialysis in PBS or PBS containing Mg2in correspondence to Mg2+concentration in the aqueous buffer, unless otherwise noted. FIGs. 21B-21C) Representative Cryo-EM of DNA LNPs in citrate (FIG. 21A) or optimized TBS buffers (FIG. 21B). Scale bar denotes 25 nm.

[0041] FIGs. 22A-22D. FIG. 22A. Size of each formulated CTS LNPs. FIG. 22B. PDI of each LNPs. FIG. 22C. DNA entrapment efficiency of each LNP formulation. FIG. 22D. DNA expression in HeLa cells, demonstrated as % of lipofectamine positive control. Condition ‘CTS LNP’ is made using dilution cartridges with 3 channels in NanoAssemblr™ Ignite™ nanoparticle formulation systems.

[0042] FIGs. 23A-23B show circular Dichroism (CD) Spectroscopy. Testing how the buffer used during DNA LNP synthesis can change the fraction of DNA in the B- form. FIG. 23 A. DNA was dissolved in either the most common buffer for mRNA LNP synthesis, citrate (pH 4), or TBS buffers (pH 6) with 4mM Mg2+, then subjected to CD spectroscopy. The ellipticity signal at 245nm and 275 nm correlates with B-form DNA, and highest for the TBS buffers containing Mg2+. and lowest for citrate. FIG. 23B. CD spectrum shows highest 27 nm signal in TBS pH 6 buffer with 4mM Mg2+.

[0043] FIGs. 24A-24H shows improving transfection by optimizing the buffer used during LNP synthesis to ensure DNA stability'. FIGs. 24A-24B. Effect of Mg2+in LNP formulation on cell transfection. A macrophage-like cell line (RAW 264.7) was transfected with DNA LNPs encoding luciferase, synthesized and dialyzed with the listed buffers. By far the highest expression was seen with TAE buffer containing Mg2+and dialyzed against Mg2+containing PBS. FIGs. 24C-24E. Effect of pH and buffer in LNP formulation on macrophage transfection. Comparing DNA expression in macrophages using 4 buffering molecules at pH 5 (FIG. 24C) and pH 6 (FIG. 24D) and with added Mg2+(FIG. 24E), showing TBS with Mg2+at pH 6 is the optimal buffer. FIGs. 24F-24H. Effect of pH and buffer in LNP formulation on epithelial cell transfection. Using an epithelial cell line (HeLa) produces the same general trends as seen in macrophages, with the exception that in the presence of Mg2+, TAE outperforms TBS.

[0044] FIG. 25 demonstrates increasing Mg2+concentration in citrate buffer reduces DNA expression.

[0045] FIGs. 26A-26F show synchrotron Size-Exclusion Chromatography in-line with Small-Angle X-ray Scattering (SEC-SAXS). FIG. 26A. Plot of mean X-ray intensity (in cm1) as a function of frame number from in-line size-exclusion analysis of standard LNPs (Std) using SAXS, shown as a black line. In FIGs. 26A and 26C, an asterisk (*) denotes the peak fraction used for subsequent SAXS analysis, and the shaded region denotes the region of data analyzed using singular value decomposition (SVD) analysis. FIG. 26B. SVD analysis of SEC-SAXS peak region. Only two significant values (SVs) are identified, providing a relative measure of monodispersity within the peak fraction. FIG. 26C. Plot of mean X-ray intensity (in cm'1) as a function of frame number from in- line size-exclusion analysis of CTS particles (CTS) using SAXS. FIG. 26D. SVD analysis of SEC-SAXS peak for CTS particles, where only two SVs are identified. FIG. 26E. Shape Distribution Function Analysis. FIG. 26F. DENSS Analysis. Ab initio electron density is shown in orthogonal views. Reconstructions were visualized using PyMOL 2.5.2 Molecular Graphics System (Schrodinger, LLC, New Your, NY, USA) with five contour levels of density rendered: 15o, lOo, 5o, 2.5o, and -0.7o. The sigma (o) level denotes the standard deviation above the average electron density value of the generated model.

[0046] FIG. 27 shows CTS LNP with different ratios of DOTAP:DOPE in RAW macrophages and HeLa cells.

[0047] FIGs. 28A and 28B show the effect of varying cores in CTS LNP. FIG. 28A. Characterization of 2-channel LNP with one channel of all the lipid components + one channel of DNA, and CTS LNP with one channel of standard LNP lipid mixture (ionizable lipid, cholesterol, helper lipid and PEG lipid), one channel of core lipid (DOTAP / DOPE or GMO), and one channel of DNA. FIG. 28B. To evaluate the importance of our mixing strategy, we compared LNPs prepared using conventional bulk mixing versus our multi-stage microfluidic approach. The standard method involved simultaneous mixing of DNA with both core and shell lipid components, while the multistage process sequentially formed core lipoplexes before encapsulation with shell lipids. Transfection studies in RAW macrophages and HeLa cells across multiple core lipid formulations demonstrated that the multi-stage mixing protocol and the resulting coreshell architecture significantly enhanced transfection efficiency.

[0048] FIG. 29A shows a schematic of loading drug into CTS LNP. FIG. 29B shows representative UPLC peaks of small molecule drug only, empty LNP without drug, LNP loaded with drug, showing overlapping peak at retention time of the drug in just drug only and drug-loaded LNP.

[0049] FIG. 30 is a table showing entrapment efficiency and drug-to-lipid ratio for the various tested drugs. FIG. 31 demonstrates that BX-795 can only be effectively loaded in CTS LNP, but not in regular LNP formulation. BX-795 was loaded into CTS LNP using a lipid concentration of 25mM. Drug / Lipid (D / L): 0.013. Loading of BX-795 improves cell viability, expression, and reduces IFN-beta release.

[0050] FIG. 32 demonstrates that C-178 can only be effectively loaded in CTS LNP and in regular LNP formulation, but significantly better in CTS LNP. C-178 was loaded into CTS LNP using a lipid concentration of 25mM, D / L: 0.025. Loading of C-178 improves cell viability, expression, animal survival and reduces IFN-beta release.

[0051] FIG. 33 demonstrates that JSH-23 can only be effectively loaded in CTS LNP, but not in regular LNP formulation. JSH-23 was loaded into CTS LNP using a lipid concentration of 25mM, D / L: 0.025. Loading of JSH-23 improves cell viability, expression, and reduces IFN-beta release.

[0052] FIG. 34 demonstrates that loading of GW4869 improves expression in RAW cells.

[0053] FIG. 35 shows that T-cell factor 1 increases expression in CTS-DNA-LNP. EL4 cells at le6 cells / well, were treated with CD5-CTS LNP loaded with TCF1. Luciferase expression was measured after 24 hours.

[0054] Detailed Description of the Invention

[0055] Described herein is a new way to synthesize cargo-loaded lipid nanoparticles (LNPs that we have termed Multi-Stage Mixing (MSM). Provided herein are nextgeneration LNPs generated using this “core-then-shell” method, achieved by a multichannel system.

[0056] Compared to mRNA-LNPs, there are five major hurdles that DNA-LNPs face for achieving protein expression (Fig 8A): 1) The synthesis conditions of LNPs have been optimized for mRNA, not DNA, and this may lead to deleterious alterations to DNA’s structure (PMID: 34548658, PMID: 33178668). Next, after the LNPs are delivered to cells and the LNPs achieve endosomal escape, DNA cargo face intracellular hurdles: 2) Intracellular DNA sensors, such as cGAS-STING and AIM2, repress protein translation upon sensing cytosolic DNA (PMID: 33927185); 3) Intracellular DNases rapidly degrade DNA if it is found outside the nucleus (PMID: 29054961); 4) slow cytosolic trafficking of DNA towards nucleus in the viscous cytoplasm (PMID: 29054961); and 5) nuclear entry of pDNA is highly inefficient, in large part because the nuclear pore complex does not permit cargo > 39 nm. (PMID: 20630994)

[0057] To overcome all five hurdles at once, here we have reimagined the LNP synthesis process, with one initial goal: compactify the DNA. If we could loosely pack the DNA into a sphere (a ‘'core’’), it would be protected during synthesis (problem #1), would not have free and flexible linear segments that would bind to intracellular DNA sensors (#2) or DNases (#3), and it would increase the mobility of large pDNA in the cytoplasm (#4) and fit more easily through the nuclear pore complex (#5). To accomplish the compactification of DNA inside LNPs. we innovated the first practical multi-stage mixing (MSM) of LNPs. In standard LNPs, there is a single mixing step: the nucleic acid (in aqueous buffer) is mixed with the lipids (in organic solvent) via microfluidics or confined impingement jet mixing (Fig 8C). Here we describe the first version of MSM that we used to create “core-then-shell” (CTS) DNA-LNPs, by first forming a “core” of compactified DNA, and then building around that a “shell” containing the standard LNP lipids.

[0058] In certain embodiments of MSM production of CTS-DNA-LNPs, there are two stages of mixing (Fig 10A). At first, DNA is combined with the cationic lipid DOTAP and the zwitterionic lipid DOPE. Literature shows that certain ratios of DNA, DOTAP, and DOPE can form lipoplexes containing inverse hexagonal phase, a lipid structural motif show n to enhance endosomal escape by LNPs (PMID: 20578750, 37364130). In addition, compactifying DNA at a N / P ratio of less than 2.0 is reported to form relatively loose compacts and enhance nuclear delivery efficiency, while a high N / P ratio would result in tightly-condensed rigid-body-like particles. Since the nuclear pore complex can only facilitate a < 39 nm rigid body passing through, too tight condensation of DNA within nanoparticles w ould impose a negative effect on the nuclear entry (PMID: 26066769). Thus, the ratio of DOTAP is kept low to avoid forming nanoparticle-size rigid bodies and achieve loose condensation of pDNA. However, these lipoplex cores are not thermodynamically stable, aggregating to >2000nm, making them unfeasible for loading into LNPs or injecting in vivo. The MSM approach encapsulates these lipoplexes in a lipid wrapper with the standard LNP lipids before they have a chance to grow, forming a shell around the core.

[0059] Using a variety of physical characterization methods, we show that MSM produces DNA-LNPs with a radically different structure than standard LNPs, with the structure matching the hypothesized core and shell. Further, we show CTS-LNPs improve in vitro DNA transfection by several orders of magnitude and even in classically “hard-to-transfect” primary cells like cortical neurons and mouse embryonic fibroblasts. Finally, we show that co-loading CTS-DNA-LNPs with a STING inhibitor C178 improves expression further, even beating the decades-old gold standard of lipofectamine in vitro, and drives in vivo protein at levels that are multiple orders of magnitude higher than standard DNA-LNPs, and now comparable to mRNA-LNPs at their peak.

[0060] In addition, we show that this methodology is useful for generating LNP carrying cargo other than DNA, including siRNA, mRNA, peptides, proteins, protein-DNA complexes, peptide-DNA complexes. lipid-DNA complexes, proteolysis targeting chimeras (PROTAC), or small molecules.

[0061] Described herein is the first use of a multi-channel system to make LNP. It has not been previously attempted to make an unstable core composed of DNA / mRNA / siRNA and lipid and / or other bioactive components (e.g. peptides, small molecule drugs, lipids), and load into an outer shell of lipid to form a stable LNP. We are also the first to develop the multi-channel system that allows the incorporation of bioactive components and DNA / mRNA / siRNA into LNP at the same time. This technology not only provides the next generation of LNP with controllable and safer profile, but also a platform for loading various bioactive components that broadens the application of LNPs.

[0062] To control the supramolecular structure (spatial arrangement of molecules) of LNPs using MSM, in this study we start with the simplest possible arrangement of molecules within an LNP: two layers, with each layer composed of different molecular species. As LNPs are spheres, a two- layer arrangement equates to a “core” and a “shell.” Thus, the simplest MSM protocol is a synthesis that forms a core and then a shell (“core- then-shell” synthesis [CTS]).

[0063] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0064] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.

[0065] As used herein, the term “about” refers to a variant of ±10% from the reference integer and values therebetween. For example, “about” 40 base pairs, includes ±4 (i.e., 36 - 44. which includes the integers 36. 37. 38. 39. 40. 41. 42, 43, 44). For other values, particularly when reference is to a percentage (e g., 90% identity, about 10% variance, or about 36% mismatches), the term “about” is inclusive of all values within the range including both the integer and fractions.

[0066] As used throughout this specification and the claims, the terms “comprising”, “containing”, “including”, and its variants are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like.

[0067] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly know n one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine. T represents thymine, U represents uracil.

[0068] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The term “lipid” refers to a group of organic compounds that are derivatives of fatty7acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0069] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary' skill in the art. values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0070] As used herein, the term “administered in combination” or “combined administration” means that two or more agents (e.g., LNPs) are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g.. a synergistic) effect is achieved. In some embodiments, the administration in combination can be concurrent (i.e., all the LNPs are administered as part of a single formulation, or different LNPs in different formulation are administered simultaneous), or consecutive (e.g., several LNPs in several formulations are administered consecutively).

[0071] In the context of the present disclosure, substitutions (even when they are referred to as amino acid substitution) are conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.

[0072] When used with respect to two or more moieties, the terms “associated with.” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association'’ need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization-based connectivity sufficiently stable such that the “associated” entities remain physically associated.

[0073] As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats a tumor, an effective amount of an agent is, for example, an amount sufficient to reduce or decrease a size of a tumor or to inhibit a tumor grow th, as compared to the response obtained without administration of the agent. The term “effective amount” can be used interchangeably with “effective dose,” “therapeutically effective amount.” or “therapeutically effective dose.”

[0074] As used herein, the term “helper lipid” refers to a compound or molecule that includes a lipidic moiety (for insertion into a lipid layer, e.g., lipid bilayer) and a polar moiety (for interaction with physiologic solution at the surface of the lipid layer). Typically, the helper lipid is a phospholipid. A function of the helper lipid is to “complement” the amino lipid and increase the fusogenicity of the bilayer and / or to help facilitate endosomal escape, e.g., of nucleic acid delivered to cells. Helper lipids are also believed to be a key structural component to the surface of the LNP.

[0075] The term “ionizable amino lipid” includes those lipids having one, two, three, or more fatty acid or fatty alkyl chains and a pH -titratable amino head group (e.g., an alkylamino or dialkylamino head group). An ionizable amino lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the amino head group and is substantially not charged at a pH above the pKa. In certain embodiments, the ionizable lipid is partially charged, due to the presence of amide bonds. In certain embodiments, the ionizable lipid is positively charged at pH 6 - 7.4, which is the pH range these LNPs face during storage, when in blood, etc. Being positively charged at these pHs allows these ionizable lipids to condense DNA compactly.

[0076] The terms “nucleic acid sequence.” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.

[0077] The phrase “nucleotide sequence encoding” refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence which encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and poly adenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include sequences that encode signal peptides.

[0078] As used herein, the terms “coding region” and “region encoding” and variants thereof, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.

[0079] As used herein, the term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of DNA or mRNA molecule that encodes a polypeptide. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[0080] The term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes poly deoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g.. peptide nucleic acids ‘"PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one unnatural nucleobase. In some aspects, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase. e.g., 5- methoxyuridine). In some aspects, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and U in the case of a synthetic DNA, or A, C. T, and U in the case of a synthetic RNA.

[0081] The skilled artisan will appreciate that the T bases in the codon maps disclosed herein are present in DNA, whereas the T bases would be replaced by U bases in corresponding RNAs. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or an in-vitro translation (IVT) template, would have its T bases transcribed as U based in its corresponding transcribed mRNA. In this respect, both codon-optimized DNA sequences (comprising T) and their corresponding RNA sequences (comprising U) are considered codon-optimized nucleotide sequence of the present disclosure. A skilled artisan would also understand that equivalent codon-maps can be generated by replaced one or more bases with non-natural bases. Thus, e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn would correspond to a codon (RNA map in which U has been replaced with pseudouridine).

[0082] Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2, of cytidine and the C2-NH2, N' — H and C6-oxy, respectively, of guanosine. Thus, for example, guanosine (2-amino-6-oxy-9-P-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-P-D-ribofuranosyl-purine). Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (l-P-D-ribofuranosyl-2-oxy-4-amino- pyrimidine) to form isocytosine (l-P-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine (U.S. Pat. No. 5,681.702 to Collins et al.). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described by Switzer et al. (1993) Biochemistry 32: 10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides can be prepared using the method described by Switzer et al., 1993, supra, and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 to Collins et al. Other nonnatural base pairs can be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37, for the synthesis of 2,6-diaminopyrimidine and its complement (1-methy Ipyrazolo- [4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotide units which form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.

[0083] As used herein, the term “polypeptide7’ refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.

[0084] “Fragments’" of proteins or peptides in the context of the present invention may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence (or its encoded nucleic acid molecule), N-terminally and / or C-terminally truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid molecule). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide.

[0085] A fragment of a protein may ty pically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein.

[0086] Fragments of proteins or peptides may furthermore comprise a sequence of a protein or peptide as defined herein, which has a length of for example at least 5 amino acids, preferably a length of at least 6 amino acids, preferably at least 7 amino acids, more preferably at least 8 amino acids, even more preferably at least 9 amino acids; even more preferably at least 10 amino acids; even more preferably at least 11 amino acids; even more preferably at least 12 amino acids; even more preferably at least 13 amino acids; even more preferably at least 14 amino acids; even more preferably at least 15 amino acids: even more preferably at least 16 amino acids; even more preferably at least 17 amino acids; even more preferably at least 18 amino acids; even more preferably at least 19 amino acids; even more preferably at least 20 amino acids; even more preferably at least 25 amino acids; even more preferably at least 30 amino acids; even more preferably at least 35 amino acids; even more preferably at least 50 amino acids; or most preferably at least 100 amino acids. For example such fragment may have a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8. 9, or 10, (or even 6, 7, 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. Fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides. Furthermore, domains of a protein, like the extracellular domain, the intracellular domain or the transmembrane domain and shortened or truncated versions of a protein may be understood to comprise a fragment of a protein.

[0087] "V ariants’' of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and / or deleted amino acid(s). Preferably, these fragments and / or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property’. "Variants" of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three- dimensional structure or do not affect the binding region. Modifications to a three- dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption. Circular Dichroism and ORD of Polypeptides, in: Modem Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).

[0088] A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10. 20. 30, 50, 75 or 100 amino acids of such protein or peptide.

[0089] Furthermore, variants of proteins or peptides as defined herein, which may be encoded by a nucleic acid molecule, may also comprise those sequences, wherein nucleotides of the encoding nucleic acid sequence are exchanged according to the degeneration of the genetic code, without leading to an alteration of the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.

[0090] Provided herein are methods of generating lipid nanoparticles (LNP) that encapsulate a cargo using a multi-channel fluidic mixer. These methods are termed multi-stage mixing (“MSM”) or core-then-shell (“CTS”).

[0091] In standard RNA-LNP formation, the RNA is dissolved in an aqueous buffer (usually citrate buffer, pH 4), while the lipids are dissolved in an organic solvent (usually ethanol). The aqueous and organic solvents are then rapidly mixed in a single step, using the “vortex method” (pipetting one solvent phase into another phase while it is being vortexed), microfluidics, confined impingement jet mixing, etc. This method mixing of two phases has been used in all the early DNA-LNP papers as well.

[0092] Here, we developed a new method, called multi-stage mixing (MSM). In this method, we first condense the DNA or other cargo with a condensing molecule (such as those above), forming condensed “cores.” These are then immediately mixed with the remaining LNP components, to form the “shell” that surrounds the core.

[0093] In certain embodiments, the method utilizes a fluidic mixer. A multi-channel fluidic mixer is a device used in microfluidics to mix different fluid streams in controlled environments. A mixer has multiple inlet “channels”, allowing different fluids (e.g., reactants, samples, or reagents) to be introduced simultaneously. This setup enables precise mixing of different fluid streams. The flow rates of each channel can be precisely controlled, often through micro-pumps or pressure adjustments. This allows for finetuning of the mixing process, ensuring consistent concentrations and reactions.

[0094] Components of Compositions and Methods

[0095] Provided herein are new methods of generating lipid nanoparticles (LNPs) that encapsulate a cargo, and compositions generated according to those methods.

[0096] The term "lipid nanoparticle'’, also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids (e.g., cationic lipids, non- cationic lipids, and PEG-modified lipids). In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). The cargo, or a portion thereof, is encapsulated in a “core” within the LNP, and is surrounded by a lipid “shell”. See, FIG. 10A. This method is sometimes referred to as core-then-shell, multi-stage-mixing, or layer-by-layer protocol.

[0097] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 250 nm. from about 50 nm to about 200nm, from about 110 nm to 250 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 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, or 250 nm and are substantially non-toxic. In certain embodiments, the mRNA. when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease. As used herein, the mean diameter may be represented by the z-average as determined by dynamic light scattering. In certain embodiments, the size, PDI. zeta potential (surface charge), and / or DNA / RNA loading are measured to confirm the physicochemical property and stability. In certain embodiments, the CTS LNP can be stable for at least 4 months with unchanged size, PDI, DNA entrapment efficiency and transfection. See, e.g., Cho EJ, Holback H, Liu KC, Abouelmagd SA, Park J, Yeo Y. Nanoparticle characterization: state of the art, challenges, and emerging technologies. Mol Pharm. 2013 Jun 3; 10(6):2093-l 10. doi: 10.1021 / mp300697h. Epub 2013 Mar 21. PMID: 23461379; PMCID: PMC3672343, which is incorporated herein by reference.

[0098] In certain embodiments, the LNPs have a poly dispersity' index (PDI) from 0.05 to 0.2. Closely related to the size, polydispersity index or PDI, characterizes the width or spread of the nanoparticle size distribution profile. PDI is defined by the square of the standard deviation of the nanoparticle population over the nanoparticle mean diameter.

[0099] Core

[0100] The LNP described herein comprise a compact "core” that comprises the cargo and a condensing agent or agents.

[0101] Condensing Agents

[0102] In certain embodiments, the compositions provided herein incorporate one or more molecules capable of condensing and / or compacting DNA (or condensing agent), when the cargo comprises DNA. These condensing agents are also useful with other cargo, optionally in combination with other agents, such as a negatively charged molecule.

[0103] The condensing agent is any agent that is capable of compactifying the DNA, other nucleic acid, or other cargo to enhance cytosolic trafficking and nuclear entry. The condensing agent may be a lipid or lipid mixture, including a cationic lipid, ionizable lipid, or neutral lipid, a peptide, a protein, a polymer, or other agent. In certain embodiments, the condensing agent is a protein (e.g.. histone), peptide (especially those that are cationic), ionizable lipid, or charged lipid. In certain embodiments, the condensing agent is more than one agent, individually selected from those described herein.

[0104] The condensing agent is contained in the ‘‘core'’ of the LNP, as described herein. In nature, there are many molecules which complex with DNA to provide such protection and sometimes compactification. Examples include the compaction of DNA into sperm, which is accomplished by complexation with poly amines (spermine & spermidine) and cationic proteins, such as protamine.

[0105] In certain embodiments, the condensing agent is a cationic lipid. Cationic lipids are amphiphilic molecules, which consist of a hydrophilic and a hydrophobic region connected by a linker structure

[0033] , DOTMA and DOTAP (l,2-dioleoyl-3- trimethylammonium propane) are two most commonly investigated cationic lipids for transfection. In another embodiment, the cationic lipid is MVL-5. In another embodiment, the cationic lipid is SM-102. In certain embodiments, the core does not contain a cationic lipid.

[0106] A number of commercial preparations of cationic lipids are available which can be used to condense / compact the DNA or other cargo. These include, for example, LIPOFECTIN ® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N- (l-(2,3dioleyloxy)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, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA). In certain embodiments, the cationic lipid is a spermine or spermidine-conjugated lipid.

[0107] In another embodiment, the condensing agent is a neutral lipid. In other embodiments, the condensing agent is a glyceryl monooleate. In another embodiment, the condensing agent is DOPE. In certain embodiments, the core does not contain a neutral lipid.

[0108] Polyamines are also useful in condensing / compacting DNA or other cargo. Poly amines include spermine, spermidine, cadaverine, 1,3-diaminopropane, and putrescine. See, e.g., van Dam L, Korolev N, Nordenskiold L. Polyamine-nucleic acid interactions and the effects on structure in oriented DNA fibers. Nucleic Acids Res. 2002 Jan 15;30(2):419-28. doi: 10.1093 / nar / 30.2.419. PMID: 11788703; PMCID: PMC99836, which is incorporated herein by reference. Other useful condensing agents include polyethylenimine. chitosan and poly(P-amino)esters.

[0109] Cationic peptides and proteins from natural or non-natural sources are also useful in condensing / compacting DNA or other cargo. Cationic peptides and other basic polymers are positively charged and interact with the negatively charged phosphate backbone of DNA through electrostatic interactions. Cationic proteins useful herein include Poly-L-lysines and polylysine-containing peptides, histones, protamine, Brdt (sperm protein), RVG-9R, R15, p, Pep V, Tat49-57 derivatives, POLYTAT, L-Arg, D- Arg, and RPC. See, e.g., Tecle M, Preuss M, Miller AD. Kinetic study of DNA condensation by cationic peptides used in nonviral gene therapy: analogy’ of DNA condensation to protein folding. Biochemistry. 2003 Sep 9;42(35): 10343-7. doi: 10.1021 / bi034325e. PMID: 12950160, and Saccardo P, Villaverde A, Gonzalez- Montalban N. Peptide-mediated DNA condensation for non-viral gene therapy. Biotechnol Adv. 2009 Jul-Aug;27(4):432-8. doi: 10.1016 / j. biotechadv.2009.03.004. Epub 2009 Mar 31. PMID: 19341789, which are incorporated herein by reference.

[0110] In certain embodiments, the core does not contain any lipids.

[0111] Cargo

[0112] The LNP described herein encapsulate a cargo. The cargo may be DNA, RNA, peptide, protein, protein-DNA complex, peptide-DNA complex, lipid-DNA complex, proteolysis targeting chimera (PROTAC), or small molecule.

[0113] In certain embodiments, the cargo is DNA. The DNA cargo comprises an expression cassette that includes open reading frame encoding a protein of interest under control of regulators’ sequences that direct expression of the ORF in the target cell.

[0114] The DNA may be any ty pe of DNA that is useful to deliver the gene of interest. In certain embodiments, the DNA is circular DNA (e.g., plasmid DNA). In other embodiments, the DNA is circular single stranded DNA (cssDNA). In other embodiments, the DNA is linear DNA. In other embodiments, the DNA is single stranded DNA.

[0115] The regulatory sequences can include any of those conventionally used, such as a promoter, appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; TATA sequences; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequence); introns; sequences that enhance protein stability, etc. The expression cassette or vector may contain none, one or more of any of the elements described herein. In certain embodiments, the expression cassette further includes viral LTRs or ITRs.

[0116] The promoter may be selected based on the desired expression of the DNA cargo. In certain embodiments, the promoter is cell-specific, such as for delivery to the target cell. The target cell may, in certain embodiments, include any mammalian cell type, such as neurons, glia, hepatocytes, endothelial cells, epithelial cells, fibroblasts, muscle cells, adipocytes, chondrocytes, osteocytes, keratinocytes, melanocytes, t lymphocytes, b lymphocytes, macrophages, dendritic cells, granulocytes. NK cells, mast cells, hematopoietic stem cells, pancreatic islet cells, spermatocytes, oocytes, thyroid follicular cells, pituitary cells, astrocytes, microglia, Schwann cells, IPSCs, primary neurons, ocular cells, etc.

[0117] In another embodiment, the promoter is a ubiquitous or constitutive promoter. An example of a suitable promoter is a hybrid chicken p-actin (CBA) promoter with cytomegalovirus (CMV) enhancer elements. In another embodiment, the promoter is the CB7 promoter. Other suitable promoters include the human P-actin promoter, the human elongation factor- la promoter, the cytomegalovirus (CMV) promoter, the simian vims 40 promoter, and the herpes simplex virus thymidine kinase promoter. See, e.g., Damdindorj et al, (August 2014) A Comparative Analysis of Constitutive Promoters Located in Adeno- Associated Viral Vectors. PLoS ONE 9(8): el06472. Still other suitable promoters include viral promoters, constitutive promoters, regulatable promoters [see, e g., WO 2011 / 126808 and WO 2013 / 04943], In another embodiment, the promoter is an inducible promoter. The inducible promoter may be selected from known promoters including the rapamycin / rapalog promoter, the ecdysone promoter, the estrogenresponsive promoter, and the tetracycline-responsive promoter, or heterodimeric repressor switch.

[0118] In certain embodiments, the cargo is RNA. The RNA cargo comprises an expression cassette that includes open reading frame encoding a protein of interest under control of regulatory' sequences that direct expression of the ORF in the target cell. In certain embodiments, the RNA is messenger RNA (mRNA).

[0119] An mRNA may include a 5' untranslated region, a 3' untranslated region, an ORF and / or a poly A sequence. An mRNA may be a naturally or non-naturally occurring mRNA. An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA in the compositions comprise at least one modification which confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. An mRNA may include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified. For example, all cytosine in an mRNA may be 5-methylcytosine. In certain embodiments, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the mRNA. As used herein, the terms “stable” and “stability” as such terms relate to the nucleic acids of the present invention, and particularly with respect to the mRNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i. e.. endonucleases or exonucleases) which are normally capable of degrading such mRNA. Increased stability can include, for example, less sensitivity' to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such mRNA in the target cell, tissue, subject and / or cytoplasm. Stabilized mRNA molecules demonstrate longer half-lives relative to their naturally occurring, unmodified counterparts (e.g. the wild-type version of the mRNA). In some embodiments, the mRNA exhibits increased stability including resistance to nucleases, thermal stability, and / or increased stabilization of secondary structure. In some embodiments, increased stability exhibited by the mRNA is measured by determining the half-life of the mRNA (e g., in a plasma, cell, or tissue sample) and / or determining the area under the curve (AUC) of the protein expression by the mRNA over time (e g., in vitro or in vivo). An mRNA is identified as having increased stability if the half-life and / or the AUC is greater than the half-life and / or the AUC of a corresponding wild-type mRNA under the same conditions.

[0120] Also contemplated by the terms “modification” and “modified” as such terms are related to mRNA are alterations which improve or enhance translation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozak consensus sequence).

[0121] In some embodiments, the mRNA described herein have undergone a chemical or biological modification to render them more stable. Exemplary modifications to an mRNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring mRNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).

[0122] In some embodiments, the number of C and / or U residues in an mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acids of the present invention also include the incorporation of pseudouridine ty / ) or 5- methylcytosine (m5C). Substitutions and modifications to the mRNA of the present invention may be performed by methods readily known to one or ordinary skill in the art. In certain embodiments, the mRNA includes a 5’ cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal. A 5 -CAP is an entity, typically a modified nucleotide entity, which generally “caps” the 5 ’-end of a mature mRNA. A 5 ’-CAP may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. Preferably, the 5 ’-CAP is linked to the 5’-terminus via a 5 ’-5 ’-triphosphate linkage. A 5’-CAP may be methylated, e.g., m7GpppN, wherein N is the terminal 5’ nucleotide of the nucleic acid carrying the 5’- CAP, typically the 5’-end of an mRNA. m7GpppN is the 5’-CAP structure, which naturally occurs in mRNA transcribed by polymerase II. Accordingly, a mRNA sequence as described herein may comprise a m7GpppN as 5’-cap.

[0123] Further examples of 5'-CAP structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, 1 -(beta-D-erythrofuranosyl) nucleotide, 4'- thio nucleotide, carbocyclic nucleotide. 1,5-anhydrohexitol nucleotide. L-nucleotides. alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3.4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate. 3'- phosphoramidate, hexylphosphate, aminohexyl phosphate, 3 '-phosphate, 3'phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety.

[0124] Additional modified 5 ’-cap structures are capl (methylation of the ribose of the adjacent nucleotide of m7G), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7G), cap3 (additional methylation of the ribose of the 3rd nucleotide dow nstream of the m7G), cap4 (methylation of the ribose of the 4th nucleotide downstream of the m7G), ARC A (anti-reverse CAP analogue, modified ARCA (e.g. phosphothioate modified ARC A), inosine. Nl-methyl-guanosine. 2'-fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. The mRNA may instead or additionally include a chain terminating nucleoside. In certain embodiments, the mRNA includes a stem loop, such as a histone stem loop. A stem loop may include 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in. before, or after an untranslated region (a 5?untranslated region or a 3?untranslated region), a coding region, or a polyA sequence or tail.

[0125] In certain embodiments, the mRNA includes a polyA sequence. According to a further preferred embodiment, the mRNA compound comprising an mRNA sequence of the present invention may contain a poly -A tail on the 3'-terminus of typically about 10 to 200 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 40 to 80 adenosine nucleotides, or about 50 to 70 adenosine nucleotides.

[0126] In certain embodiments, the poly(A) sequence in the mRNA is derived from a DNA template by RNA in vitro transcription. Alternatively, the poly(A) sequence may also be obtained in vitro by common methods of chemical-synthesis without being necessarily transcribed from a DNA-progenitor. Moreover, poly(A) sequences, or poly(A) tails may be generated by enzymatic polyadenylation of the RNA according to the present invention using commercially available polyadenylation kits and corresponding protocols known in the art.

[0127] Alternatively, the mRNA as described herein optionally comprises a polyadenylation signal, which is defined herein as a signal, which conveys poly adenylation to a (transcribed) RNA by specific protein factors (e.g., cleavage and poly adenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II (CF I and CF II), poly(A) polymerase (PAP)). In this context, a consensus polyadenylation signal is preferred comprising the NN(U / T)ANA consensus sequence. In a particularly preferred aspect, the polyadenylation signal comprises one of the following sequences: AA(U / T)AAA or A(U / T)(U / T)AAA (wherein uridine is usually present in RNA and thymidine is usually present in DNA).

[0128] In some embodiments, the mRNA sequence comprises at least one 5'- or 3'-UTR element. In this context, an UTR element includes a nucleic acid sequence, which is derived from the 5'- or 3'-UTR of any naturally occurring gene or which is derived from a fragment, a homolog or a variant of the 5'- or 3'-UTR of a gene. Preferably, the 5'- or 3'-UTR element used according to the present invention is heterologous to the at least one coding region of the mRNA sequence of the invention. Even if 5'- or 3'-UTR elements derived from naturally occurring genes are preferred, also synthetically engineered UTR elements may be used. The term “3'-UTR element” typically refers to a nucleic acid sequence, which comprises or consists of a nucleic acid sequence that is derived from a 3'-UTR or from a variant of a 3'-UTR. A 3'-UTR element may represent the 3'-UTR of an RNA, preferably an mRNA. Thus, as used herein, a 3'-UTR element may be the 3'-UTR of an RNA, e.g.. of an mRNA, or it may be the transcription template for a 3'-UTR of an RNA. Thus, a 3'- UTR element preferably is a nucleic acid sequence which corresponds to the 3'-UTR of an RNA, preferably to the 3'-UTR of an mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, the 3'-UTR element fulfils the function of a 3'-UTR or encodes a sequence which fulfils the function of a 3'-UTR. The cargo can be any that is desirable to deliver to the subject. The cargo includes therapeutic proteins, nucleases (e.g., CRISPR-CAS DNA), diagnostic proteins, antibodies, etc, and DNA and RNA encoding the same. A non-limiting list of applications and cargoes is below. Where reference is made to cargo in the list below, the cargo may comprise the polypeptide / protein, or a nucleic acid encoding the polypeptide / protein.

[0129] ■ Atherosclerosis

[0130] • Cargo IL- 10, inhibitors of inflammatory cytokines (IL-6, IL- 1 beta), LXR modulators

[0131] ■ Heart attack:

[0132] • Cargo: proteins that prevent “in-stent thrombosis” such as antithrombotic proteins (thrombomodulin); proteins that control “re-endothelialization” of the stent (VEGF modulators); proteins that promote regeneration of myocardium (modulators of VEGF, FGF, G-CSF, HDF, SDF, IGF, neuregulin, periostin)

[0133] ■ Stroke (ischemic):

[0134] • Cargo: immunomodulators (IL-10), modulators of NGF, BDNF, and other neuronal grow th factors ■ Hypertension:

[0135] • Cargo: modulators of nitric oxide synthase, components of the ACE and neprolysin pathways, etc

[0136] ■ Sepsis: > Cargo, immunomodulators like IL- 10

[0137] ■ Local expression of peptide antibiotics / antimicrobials, antibodies, anti-inflammatory proteins, etc.

[0138] ■ Chronic infections:

[0139] ■ Cargo: Peptide antibiotics include LL37, anti-inflammatory proteins include IL- 10, and neutralizing antibodies exist against many bacteria

[0140] ■ Antibodies during pandemics: One-time delivery of neutralizing antibodies prevents infection for 6 months.

[0141] ■ Vaccines: antigens, polysaccharides, or toxoids to common pathogenic organisms (e.g..

[0142] SARS-CoV-2, Polio (IPV), Hepatitis A, Rabies, Pertussis, Measles, Mumps, and Rubella (MMR), Varicella (Chickenpox), Rotavirus, Hepatitis A, B, C, HPV, Tetanus, Haemophilus influenzae ty pe b (Hib) polysaccharide, Pneumococcal polysaccharides (PCV13. PPSV23), Meningococcal polysaccharides, Diphtheria)

[0143] ■ Pain: LNPs encoding proteins that quiet pain nerves.

[0144] • Cargo', modulators of various ion channels (eg., TRPV-1, sodium channels, etc), modulators of GABA receptors, modulators of inflammation (IL- 10).

[0145] ■ Wound healing:

[0146] ■ Cargo: modulators of growth factors such as EGF, FGFs, PDGF, TGF-a, and TGF-p.

[0147] ■ Bum wound healing: Cargo skin stem cells programmed via DNA-episomes

[0148] ■ Traumatic brain injury': " Cargo: Pro-regenerative proteins (e.g., BDNF, NGF, etc)

[0149] ■ Broken bones: can take months to heal, but we can speed that up with growth factors and anti -infl ammatori es .

[0150] ■ Cargo: BMPs, FGF, IGF, PDGF, TGF-beta, VEGF ■ Chronic toxin accumulation syndromes can be fixed by using DNA-LNPs to express proteins that clear the toxins:

[0151] • Cargo: Wilson’s disease (we prevent copper accumulation by expressing copperchelating proteins); hemochromatosis (express iron-chelating proteins); lysosomal storage diseases (lysosomal enzyme activator, lysosomal membrane proteins, or non- lysosomal proteins)

[0152] ■ Autoimmune: Express the mAbs and other therapeutic proteins that are common for diseases like rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease (IBD), etc. ■ Cargo: etanercept, antibodies like anti-TNF, IL-4, -IL-5, DNAse for lupus, etc.

[0153] ■ Deliver to immune cells proteins that prevent their activation.

[0154] ■ Cargo. IkB and mutations thereof, etc.

[0155] ■ Diabetes: Cargo: DNA-LNPs encoding for GLP-1 agonists

[0156] ■ NASH / MASH: - Cargo: FGFs, MOTS-c, TGF-beta superfamily members such as GDF15, etc.

[0157] ■ Inborn errors of metabolism:

[0158] ■ Cargo: enzymes to combat phenylketonuria, etc

[0159] ■ Surgical procedures

[0160] • Surgical wounds: express pro-healing and anti-microbial proteins » Implants (ex: hip replacement, defibrillators): similar to wounds, but also anti-fibrotic proteins will help with neural implants. Cargo DNA: See wound healing above, and antimicrobial proteins.

[0161] • Transplant: Cargo: Express tolerizing cargo (shRNA against MHC proteins; express PD1, PDL-1, CTLA-4 and other proteins that inhibit T-cell mediated cytolysis), anti- thrombotics (thrombomodulin, etc), pro-healing at the anastomosis (FGF, VEGF, etc.)

[0162] • Intra-abdominal surgery: deliver LNPs that secrete proteins that prevent intra-abdominal adhesions. Cargo: tPA, TGFbeta inhibitors such as the TGFbeta decoy receptor or antibody, antibodies targeting VEGF, IL-1, and TNF-alpha.

[0163] ■ Catheter-based procedures: • Intra-arterial catheters will allow for periodic delivery (every 6 months) to an organ that is hard to target by nanoparticle formulations alone. Usually intra-arterial catheters would be unacceptable for chronic diseases, but q6 months is reasonable for a severe disease. Cargo: same proteins as above for atherosclerosis. « Fibrotic diseases: includes IPF (idiopathic pulmonary fibrosis), retroperitoneal fibrosis, etc. Antifibrotic proteins are well known, but have off-target side effects, so local delivery is essential, but requires long-term delivery'. Cargo: Antibodies and decoy receptors to inhibit key cytokines and soluble factors: TGFbeta, CTGF, CCN2, PDGF, Oncostatin M, CCL2, CCL3, CXCL12. " CAR-T cells (and CAR macrophages, NK cells, etc):

[0164] • Cargo: any CAR

[0165] ■ DNA-vaccines for cancer:

[0166] • Cargo. Include typical cancer vaccine proteins, including tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), including antigens such as mesothelin, CEA, PSA, and many more.

[0167] ■ Chemo-related side effects:

[0168] ■ Cargo’. Chemo-induced esophagitis is treated with a peptide therapeutic, palifermin

[0169] ■ Anti-cancer antibodies: Both immunotherapies (e.g., anti-PDl) and direct anti-tumor antibodies - ADCs (antibody-drug-conjugates)

[0170] • Cargo’, antibodies targeting cancer antigens (e.g.. HER2) genetically fused to a toxic protein, such as tetanus or diphtheria toxin, or to a small protein binder such as neutravidin, that binds to biotin that would be covalently conjugated to a toxic small molecule. " Regenerative medicine in general: For example, after tissue injury, such as ARDS or IPF, there is a need to deliver grow th factors such as Wnts & PDGF, that regulate regeneration of the alveoli. Similar exist for the heart muscle (e.g., VEGF after MI), liver, skin, etc.

[0171] • Cargo’. In lung: Wnt, VEGF; for MI: VEGF, FGF, G-CSF, HDF, SDF, IGF, neuregulin, periostin ■ Alzheimer’s: Numerous genes have been implicated, but we have trouble delivering them to the brain. We can deliver DNA-LNPs via intrathecally (lumbar puncture or intra- cistema magna) injection every 6 months:

[0172] • Cargo’, antibodies that disrupt beta amyloid plaques such as those already tested clinically; if the LNPs reach the neurons, cargo can encode shRNA against beta-amyloid and tau, or encode protein variants of presenilin and other products in the beta-amyloid processing pathway.

[0173] ■ Parkinson’s:

[0174] • Cargo’, antibodies or other proteins that bind and disaggregate a-synuclein aggregation; shRNA to knockdown a-synuclein; antioxidant proteins such as catalase and SOD; antiinflammatories such as IL- 10, IL- Ira.

[0175] ■ PICS: Post-intensive care syndrome. After ICU admissions, patients have immunoparalysis, a persistent catabolic state, and neuromuscular weakness. All can be addressed by DLNPs - Osteoporosis: Less frequent dosing of the current antibodies (e.g., denosumab), and local delivery into the bones at highest risk (e.g., hips).

[0176] • Cargo’, antibodies such as denosumab (or other RANKL antibodies).

[0177] ■ Chronic low back pain: The problem is local inflammation and pain, and we can deliver via local injections anti-inflammatory' proteins and anesthetic proteins. » Cargo’, modulators of various ion channels (eg., TRPV-1, sodium channels, etc), modulators of GABA receptors, modulators of inflammation (IL- 10).

[0178] ■ Osteoarthritis: Similar to chronic low back pain, but delivery into joints, this time with pro-regenerative proteins, many of which have been shown preclinically, but have bad PK. » Cargo’. Wnt proteins such as Wntl6; cartilage delivery of anti-NFkB therapies (IKB variants such as IKB-super-repressor); anti-cytokine therapy such as local antibodies or decoy receptors of TNF-alpha.

[0179] • Grow th hormone replacement therapy for short stature:

[0180] ■ Cargo’, growth hormone " Thyroid diseases: • Cargo, proteins that encode the synthetic enzymes producing T3 & T4

[0181] ■ Spinal cord

[0182] • Spinal cord injury: o Cargo: IL- 10; neurotrophic factors such as BDNF . ALS: o Cargo'. shRNA against SOD

[0183] . SMA o Cargo: SMN1, SMN2

[0184] ■ Peripheral nervous system: » Regeneration after trauma o Cargo: NGF, BDNF, etc

[0185] ■ AMD (age-related macular degeneration): less frequent injections of anti-VEGF, and then later, express intracellular proteins that are therapeutic

[0186] • Cargo: anti-VEGF antibodies - Intracochlear injection of proteins that promote regeneration of hair cells

[0187] • Cargo: Myc, Notch 1, Wnt

[0188] ■ Sinusitis: delivery of anti-microbials and anti-inflammatories

[0189] • Cargo: IL- 10, anti-microbial peptides such as LL37

[0190] ■ COPD: Will deliver to airways anti-inflammatories, and to alveoli, pro-regenerative proteins.

[0191] • Cargo: IL- 10. pro-regenerative proteins as above, anti-HERl to reduce mucus hypersecretion; modulating antibodies or proteins of VEGF, FGF, TGF-[3, TNF-a, CXCL1, CXCL8, CCL2, mitogen-activated protein kinase p38, NFkB

[0192] ■ Asthma: Cargo: dupilumab " Pulmonary arterial hypertension:

[0193] • Cargo: modulators of BMPR2; shRNA against tryptophan hydroxylase- 1; modulators of the activin and inhibin family

[0194] ■ Heart failure: Delivery to the heart is difficult IV, but we can use intra-arterial (IA) catheter delivery every' 6 months • Cargo, modulators in the heart of pathways related to: AT-1 receptor, reactive oxygen species, TNF-alpha, NFkB, IL-6, STAT3, LIF, VEGF, and anti-apoptotic pathways

[0195] ■ Arrhythmias

[0196] • Cargo: inhibitors of tissue fibrosis (anti-TGF-beta. CTGF, Angll, PDGF). reactive oxygen species (SOD, catalase); expression of connexons and their modulators; modulators of NF At

[0197] ■ Alimentary tract:

[0198] • GERD: inject into LES (lower esophageal sphincter) DNA-LNPs encoding for proteins that regenerate the smooth muscle o Cargo: FGF, PDGF

[0199] ■ Liver

[0200] • Cirrhosis: Delivery of anti-fibrotics or pro-regenerative proteins o Cargo: anti-fibrotics as above, such as anti-TGFbeta, and pro-regenerative include HGF, FGF, KGF » Gallstone disease: delivery proteins that prevent gallstone formation

[0201] ■ Glomerular diseases are a leading cause of kidney failure, and we can deliver DNA- LNPs to the kidney

[0202] • Cargo: inhibitors of STING including shRNA, inhibitors of APOL1

[0203] ■ Muscular dystrophies: » Cargo: dystrophin

[0204] ■ Muscle regeneration: For sarcopenia of the elderly, post-hospitalization, post injury.

[0205] Many muscle growth factors are known, and we can do local injections, since muscle is a syncitium.

[0206] • Cargo: modulators of mTORCl, MEF2, SRF, PGC-la4, and YAP > Male pattern baldness: express growth factors for hair follicles

[0207] • Cargo: VEGF, shRNA against the DHT receptor

[0208] ■ Blood thinners for atrial fibrillation or post-pulmonary embolism:

[0209] • Cargo: proteins that inhibit thrombin or Factor Xa (thrombomodulin, anti-thrombin, etc)

[0210] Other DNA / RNA cargoes include those encoding anti-cancer therapies. Non- limiting examples of anti-cancer molecules include immune checkpoint inhibitors (e.g., CTLA-4 antibodies, PD-1 antibodies, PDL-1 antibodies), cytotoxic agents (e.g., Cly A, FASL, TRAIL, TNF-alpha), immunostimulatory cytokines and co-stimulatory molecules (e.g., 0X40, CD28, ICOS, CCL21, IL-2, IL-18, IL-15, IL-12, IFN-gamma, IL-21, TNFs, GM-CSF). antigens and antibodies (e.g., tumor antigens, neoantigens, CtxB-PSA fusion protein, CPV-OmpA fusion protein, NY-ESO-1 tumor antigen, RAFI, antibodies against immune suppressor molecules, anti-VEGF, Anti-CXR4 / CXCL12, anti-GLPl, anti- GLP2, anti-galectinl, anti-galectin3, anti-Tie2, anti-CD47, antibodies against immune checkpoints, antibodies against immunosuppressive cytokines and chemokines). DNA transfer vectors (e.g., endostatin, thrombospondin- 1, TRAIL, SMAC, Stat3, Bcl2, FLT3L, GM-CSF, IL-12, AFP, VEGFR2), and enzymes (e.g., E. coll CD, HSV-TK).

[0211] In one embodiment, the cargo encodes a neutralizing antibody against a viral pathogen. Such anti-viral antibodies may include anti-influenza antibodies directed against one or more of Influenza A. Influenza B, and Influenza C. The 20 type A viruses are the most virulent human pathogens. The serotypes of influenza A which have been associated with pandemics include, H1N1, which caused Spanish Flu in 1918, and Swine Flu in 2009; H2N2, which caused Asian Flu in 1957; H3N2, which caused Hong Kong Flu in 1968; H5N1, which caused Bird Flu in 2004; H7N7; H1N2; H9N2; H7N2; H7N3; and H10N7. Other target pathogenic viruses include, without limitation, arenaviruses (including funin, 25 machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picomoviridae (including rhinoviruses, echovirus), coronaviruses, paramyxovirus, morbillivirus. respiratory synctial virus, togavirus, coxsackievirus, JC virus, parvovirus Bl 9, parainfluenza, adenoviruses, reoviruses, variola (Variola major (Smallpox)) and Vaccinia (Cowpox) from the poxvirus family, and varicella-zoster (pseudorabies). Viral hemorrhagic 30 fevers are caused by members of the arenavirus family (Lassa fever) (which family is also associated with Lymphocytic choriomeningitis (LCM)). filovirus (ebola virus), and hantavirus (puremala). The members of picomavirus (a subfamily of rhinoviruses), are associated with the common cold in humans. The coronavirus family, which includes a number of non-human viruses such as infectious bronchitis virus (poultry), porcine transmissible gastroenteric virus (pig), porcine hemagglutinatin encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), canine coronavirus (dog). The human respiratory coronaviruses have been putatively associated 5 with the common cold, non-A, B or C hepatitis, and sudden acute respiratory syndrome (SARS). The paramyxovirus family includes parainfluenza Virus Type 1, parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3, rubulavirus (mumps virus, parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastle disease vims (chickens), rinderpest, morbillivirus, which includes measles and canine distemper, and pneumovirus, which 10 includes respiratory' syncytial virus (RSV). The parvovirus family includes feline parvovirus (feline enteritis), feline panleucopeniavirus, canine parvovirus, and porcine parvovirus. The adenovirus family includes viruses (EX, AD7, ARD, O.B.) which cause respiratory disease. Thus, in certain embodiments, an antibody may include an anti-ebola antibody, e.g., 2G4, 4G7, 13C6, an anti-influenza antibody, e.g., FI6, 15 CR8033, and anti-RSV antibody, e g, palivizumab, motavizumab.

[0212] A neutralizing antibody construct against a bacterial pathogen may also be selected for use. In one embodiment, the neutralizing antibody construct is directed against the bacteria itself. In another embodiment, the neutralizing antibody construct is directed against a toxin produced by the bacteria. Examples of airborne bacterial pathogens include, e.g., Neisseria meningitidis (meningitis), Klebsiella pneumonia (pneumonia), Pseudomonas aeruginosa (pneumonia), Pseudomonas pseudomallei (pneumonia), Pseudomonas mallei (pneumonia), Acinetobacter (pneumonia), Moraxella catarrhalis, Moraxella lacunata, Alkaligenes, Cardiobacterium. Haemophilus influenzae (flu), Haemophilus parainfluenzae, Bordetella pertussis (whooping cough), Francisella tularensis (pneumonia / fever), 25 Legionella pneumonia (Legionnaires disease), Chlamydia psittaci (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis (tuberculosis (TB)), Mycobacterium kansasii (TB), Mycobacterium avium (pneumonia), Nocardia asteroides (pneumonia), Bacillus anthracis (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever). Streptococcus pneumoniae (pneumonia), Corynebacteria diphtheria 30 (diphtheria), Mycoplasma pneumoniae (pneumonia). Bacillius anthracis. Antibodies against infectious diseases caused by parasites or by fungi may be encoded, including, e.g., Aspergillus species, Absidia corymbifera, Rhixpus stolonifer, Mucor plumbeaus, Cryptococcus neoformans, Histoplasm capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Penicillium species, Micropolyspora faeni, Thermoactinomyces vulgaris, Altemaria alternate, Cladosporium species, Helminthosporium, and Stachybotrys species.

[0213] In another embodiment, the cargo encodes an antibody against pathogenic factors of diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Rheumatoid arthritis (RA), Irritable bowel syndrome (IBS), chronic obstructive pulmonary disease (COPD), cancers, tumors, systemic sclerosis, asthma and other diseases. Such antibodies may be., without limitation, e.g., alpha-synuclein, anti-20 - vascular endothelial growth factor (VEGF) (anti-VEGF), anti-VEGFA, anti-PD-1, antiPDLl, anti-CTLA-4, anti- TNF-alpha, anti-IL-17, anti-IL-23, anti-IL-21, anti-IL-6, anti-IL-6 receptor, anti-IL-5, anti-IL-7, anti-Factor XII, anti-IL-2, anti-HIV, anti-IgE, anti-tumour necrosis factor receptor-1 (TNFR1), anti-notch 2 / 3, anti-notch 1. anti-OX40, anti-erb-b2 receptor tyrosine kinase 3 (ErbB3), anti-ErbB2, anti-beta cell maturation antigen, anti-B 25 lymphocyte stimulator, anti-CD20, anti-HER2, anti-granulocyte macrophage colonystimulating factor, anti-oncostatin M (OSM), anti-lymphocyte activation gene 3 (LAG3) protein, anti-CCL20, anti-serum amyloid P component (SAP), anti-prolyl hydroxylase inhibitor, anti-CD38, anti -glycoprotein Ilb / IIIa, anti-CD52, anti-CD30, anti -IL-1 beta, antiepidermal grow th factor receptor, anti-CD25, anti-RANK ligand, anti-complement system 30 protein C5, anti-CDl la, anti-CD3 receptor, anti-alpha-4 (a4) integrin, anti- RSV F protein, and anti-integrin a4p7. Still other pathogens and diseases will be apparent to one of skill in the art. Other suitable antibodies may include those useful for treating Alzheimer’s Disease, such as, e.g., anti-beta-amyloid (e.g., crenezumab, solanezumab, aducanumab), anti-betaamyloid fibril, anti-beta-amyloid plaques, anti-tau, a bapineuzamab, among others.

[0214] In certain embodiments, the cargo encodes a CRISPR-Cas enzyme, e.g., Cas9.

[0215] In some embodiments, the nucleic acids described herein have undergone a chemical or biological modification to render them more stable, e.g., nucleic acids containing one or more non-naturally occurring or modified. The modified nucleotide analog may be located for example at the 5'-end and / or the 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase- modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza- adenosine; 0- and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2'-OH- group may be replaced by a group selected from H. OR, R. halo, SH, SR, NH.sub.2, NHR, N.sub.2 or CN, wherein R is C.sub.l-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxy prolinol linkage as described in Krutzfeldt et al, Nature (Oct. 30, 2005). Soutschek et al, Nature 432: 173-178 (2004), and U.S. Patent Publication No. 20050107325. which are incorporated herein by reference in their entireties. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005, which is incorporated herein by reference in its entirety. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In some embodiments, the expressible nucleic acid sequence is in the form of DNA. In some embodiments, the expressible nucleic acid is in the form of RNA with a sequence that encodes the polypeptide sequences disclosed herein and. in some embodiments, the expressible nucleic acid sequence is an RNA / DNA hybrid molecule that encodes any one or plurality of polypeptide sequences disclosed herein.

[0216] It is shown herein that the CTS method is able to encapsulate cargoes other than nucleic acids, including small molecules, polysaccharides, and proteins. In certain embodiments, the cargo is any small molecule that is suitable for loading into a lipid nanoparticle. Small molecule drugs include, without limitation analgesics / anti- inflammatory agents, including aspirin, ibuprofen, acetaminophen, diclofenac, naproxen, indomethacin, celecoxib; antibiotics, including ciprofloxacin, amoxicillin, doxycycline, vancomycin, rifampicin, tetracycline, metronidazole, azithromycin; antivirals, including remdesivir, acyclovir, favipiravir, lamivudine, oseltamivir, ribavirin, tenofovir, zidovudine; anticancer agents, including doxorubicin, paclitaxel, docetaxel, camptothecin, gemcitabine, sorafenib, erlotinib, tamoxifen, methotrexate, cisplatin; cardiovascular drugs, including atorvastatin, simvastatin, amlodipine, losartan, enalapril, carvedilol, metoprolol, propranolol, clopidogrel; antifungals, including fluconazole, itraconazole, voriconazole, ketoconazole, amphotericin B, clotrimazole, terbinafine; antidiabetics, including metformin, glipizide, glyburide, sitagliptin, pioglitazone, saxagliptin, dapaghflozin. canagliflozin; antiulcer / Antireflux agents, including omeprazole, esomeprazole, pantoprazole, lansoprazole, ranitidine, cimetidine, famotidine; antimalarials, including chloroquine, hydroxychloroquine, artesunate, artemether, mefloquine, lumefantrine, quinine; antipsychotics, including risperidone, olanzapine, aripiprazole, haloperidol, clozapine, quetiapine, ziprasidone; antidepressants, including fluoxetine, sertraline, paroxetine, citalopram, venlafaxine, duloxetine, amitriptyline; anticonvulsants, including carbamazepine, phenytoin, lamotrigine, valproate, gabapentin, levetiracetam, topiramate; beta-adrenergic blockers, including atenolol, bisoprolol. labetalol. carvedilol, propranolol; antihistamines, including diphenhydramine, cetirizine, loratadine, fexofenadine, chlorpheniramine; bronchodilators, including albuterol, salbutamol, terbutaline, theophylline, montelukast, ipratropium; hormonal agents, including dexamethasone, hydrocortisone, prednisolone, levothyroxine, estradiol, testosterone.

[0217] In certain embodiments, the cargo is Trametinib, U0126, SP600125, minocycline hydrochloride, Lysophosphatidic acid. Arachidonic Acid, GW4869, Catalpalactone, Baricitinib, H-151, C-176, RU.521, G150, Astin C, Schaftoside, Raffinose, Resatorvid, Celastrol, Thiodigalactoside, PF-06928215. flurocein, FITC dextran 3k, anionic. FITC dextran 10k, anionic, FITC dextran 40k, anionic or neutral, FITC dextran 70k, anionic or neutral, FITC dextran 500k, anionic, or FITC dextran 2000k, anionic.

[0218] In certain embodiments, the cargo is BX-795, C-178, JSH-23, 4-phenylbutyric acid. FITC-dextran (lOkDa), dexamethasone-2 IP, dexamethasone, betamethasone, or dexamethasone palmitate.

[0219] In certain embodiments, a drug-to-lipid ratio of 0.0001 to 0.99 is utilized. In other embodiments, a drug-to-lipid ratio of 0.0001 to 0.750 is utilized. In other embodiments, a drug-to-lipid ratio of 0.0001 to 0.750 is utilized. In other embodiments, a drug-to-lipid ratio of 0.001 to 0.500 is utilized. In other embodiments, a drug-to-lipid ratio of about 0.0001, 0.0025, 0.013, 0.021, 0.059, 0.086, 0.118, 0.164, or 0.73 is utilized.

[0220] In certain embodiments, the cargo includes a nucleic acid and a small molecule drug. FIG. 29A provides an exemplary schematic for loading of either hydrophilic or hydrophobic drugs into the core of the LNP. For example, a hydrophilic drug is mixed with a nucleic acid in an aqueous solution and mixed with a second solution comprising an organic solvent and one or more lipids (e.g., a cationic lipid and a helper lipid) to form a core. The core is then rapidly mixed with a third solution comprising an ionizable lipid, a helper lipid, cholesterol, and / or a pegylated lipid to form the shell.

[0221] In certain embodiments, the cargo includes T cell factor 1.

[0222] In certain embodiments, the cargo is not a limitation of this invention. Shell

[0223] The LNP described herein comprise one or more “shell” layers that surround the core. The shell layer comprises one or more lipids. In one embodiment, the shell comprises one or more ionizable lipids as described herein, cholesterol, a helper phospholipid, and a polyethylene glycol-modified lipid. It is noted that it is permissible that one or more lipids that are included in the core are also included in the shell.

[0224] In certain embodiments, the shell comprises a lipid mixture (primarily cholesterol (or other sterol), phospholipids, PEGylated lipid). If included, ionizable lipid can be loaded in either / both shell or / and core phase. The shell can also include one or more antibody or antibody fragment, peptides, polymers, helper lipids (charged, specific structure), lipids with functionalized chemical groups. These components in the shell can serve as a cell penetrating reagent, tissue targeting reagent, etc.

[0225] As mentioned, in certain embodiments, the LNP comprises an ionizable cationic lipid. The cationic lipid is preferably ionizable, i.e.. it becomes 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. When positively charged, 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.

[0226] In some embodiments, the ionizable cationic lipid is present in a molar percentage of the lipid nanoparticle composition from about 0% to about 80%. In other embodiments, the ionizable cationic lipid is present in a molar percentage of the lipid nanoparticle composition from about 5% to about 30%. In some embodiments, the molar percentage of the ionizable cationic lipid is present from about 7.5% to about 20%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 11.9%. In some embodiments, the ionizable cationic lipid is present in a molar percentage of the lipid nanoparticle composition from about 15% to about 30%. In some embodiments, the molar percentage of the ionizable cationic lipid is present from about 15% to about 25%.

[0227] In certain embodiments, the LNP may comprise any further cationic or ionizable lipid, i.e., 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-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l- (2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxy spermine (DOGS), 1,2- dioleoyl-3-dimethylammonium propane (DODAP), N.N-dimethyl-2.3- dioleoyloxy)propylamine (DODMA), and N-(l,2dimyristyloxyprop-3-yl)-N,N-dimethyl- N-hydroxy ethyl ammonium bromide (DMRIE).

[0228] Other useful lipids include, without limitation, 98N12-5, C 12-200, PLGA, PEG, PEG-DMG, PEGylated lipids, amino alcohol lipids, and KL22. In certain embodiments the cationic lipid is DOTAP, DOPSA, DOTMA, DMRIE, or N-Tetamine-pLys40.

[0229] Additionally, 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- 3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l- (2,3dioleyloxy)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, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1.2-dilinolenyloxy-N.N-dimethylaminopropane (DLenDMA).

[0230] In one embodiment, the further cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in W02012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to. l,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2- dilinoleyoxy-3morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2- linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3- trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)-butanoic acid, ( 1 OZ, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -yl- 10, 13 -nonadecadi en- 1 -y 1 ester (DLin-MC3-DMA), N, N-dimethyl-2,2-di-(9Z,12Z)-9,l 2-octadecadien- 1-yl- 1,3- dioxolane-4-ethanamine (DLin-KC2-DMA). See also, e.g., WO2014 / 089486, US 2018 / 0353616A1, and US 8,853,377B2, which are incorporated by reference. In some embodiments, LNP comprises a cationic lipid (i.e. N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), or l,2-dioleoyl-3- trimethylammonium-propane (DOTAP)) with helper lipid DOPE. In some embodiments, LNP comprises an ionizable lipid Dlin-MC3-DMA ionizable lipids, or diketopiperazine- based ionizable lipids (cKK-E12). In some embodiments, polymer comprises a polyethyleneimine (PEI), or a poly(P-amino)esters (PBAEs). In some embodiments, the LNP comprises C14-4 / DOPE / Chol / PEG-lipid. See. Rybakova Y., Kowalski P. S., Huang Y., Gonzalez J. T., Heartlein M. W., DeRosa F., et al. . (2019). mRNA delivery for therapeutic anti-HER2 antibody expression in vivo. Mol. Ther. 27, 1415-1423 which is incorporated by reference. In other embodiments, the LNP comprises L319 / DSPC / Chol / PEG-DMG. See, Thran M., Mukherjee J., Pbnisch M., Fiedler K.. Thess A., Mui B. L. (2017). mRNA mediates passive vaccination against infectious agents, toxins, and tumors. EMBO Mol. Med. 9, 1434-1447 which is incorporated by reference. See also, e.g., WO2014 / 089486, US 2018 / 0353616A1, US2013 / 0037977A1, W02015 / 074085A1, US9670152B2, and US 8,853,377B2, which are incorporated by reference.

[0231] In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation. Phospholipids are particularly useful helper lipids. Exemplary helper 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- Icarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE). distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearioyl-2- oleoylphosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2- distearoyl-sn-glycero-3phosphocholine (DSPC). In some embodiments, the phospholipid is present in a molar range of about 20% to 100%. In some embodiments, the phospholipid is present in a molar percentage of the lipid nanoparticle composition from about 8% to about 20%. In some embodiments, the molar percentage of the phospholipid is present from about 10% to about 14%. In some embodiments, the molar percentage of the phospholipid is about 11.9%. In other embodiments, the phospholipid is present in a molar percentage of the lipid nanoparticle composition from about 20% to about 23%. In some embodiments, the molar percentage of the phospholipid is present from about 20% to about 21%.

[0232] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, GMO, and SM120. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 100: 1 to 1:99. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1. In other embodiments, the shell comprises no cationic lipid.

[0233] In various embodiments, the LNPs further comprise a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of total lipids to cholesterol ranges from about 100:0 to 0. 1:99.9. In some embodiments, the steroid is present in a molar percentage of the lipid nanoparticle composition from about 39% to about 46%. In some embodiments, the molar percentage of the steroid is present from about 40% to about 43%. In some embodiments, the molar percentage of the steroid is about 40.5%. In other embodiments, the steroid is present in a molar percentage of the lipid nanoparticle composition from about 15% to about 39%. In some embodiments, the molar percentage of the steroid is present from about 20% to about 27.5%.

[0234] The term “anionic lipid’' refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, Ndodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, Nglutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. In certain embodiments, the LNP comprises a glycolipid (e.g., monosialoganglioside GM1).

[0235] In some embodiments, the LNPs comprise a polymer conjugated lipid or other stealth molecule. 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. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1 -(monomethoxy-polyethylenegly col)-2,3-dimyristoylgly cerol (PEG-s-DMG) and the like. In certain embodiments, PEG can be replaced with other forms of lipid that also enhance serum stability and reduce clearance, aka, stealth molecule. These alternatives include lipids with poly(2-ethyl-2-oxazoline) (PEOZ) chains, poly glycerol fatty acid esters (PGFEs), and poly(carboxybetaine) (PCB) structures, polysarcosine (pSar) lipids, Poly(oxazoline) lipids, polyamides, and RAFT lipopolymers, among others. Wherever the term pegylated lipid is used herein, an alternative embodiment exists that utilizes another stealth molecule.

[0236] 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 glycollipids 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]-l,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 -poly ethyleneglycol)-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-l-O-(®-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3di(tetradeca noxy)propyl)carba mate or 2,3- di(tetradecanoxy )propyl-N-(w-methoxy(poly ethoxy )ethyl)carbamate. In various embodiments, the molar ratio of the ionizable lipid to the pegylated lipid ranges from about 100:1 to about 25: 1. In certain embodiments, the pegylated lipid comprises 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8. 0.9, 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20. 21. 22. 23. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. 36. 37. 38. 39. 40, 41,

[0237] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

[0238] 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

[0239] 90, 91. 92, 93, 94, 95, 96, 97, 98, 99, or 100 molar % of the shell.

[0240] In the context of the present invention, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated according to the methods described herein. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle.

[0241] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 200 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 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, and are substantially non-toxic. In certain embodiments, the DNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease. As used herein, the mean diameter may be represented by the z-average as determined by dynamic light scattering.

[0242] In certain embodiments, a shell layer comprises helper lipid, cholesterol, and pegylated lipid. In another embodiment, a shell layer comprises only pegylated lipid(s).

[0243] In certain embodiments, one or more shell layers comprises one or more proteins to facilitate interaction with the cell membrane. Such proteins include cell-surface proteins. In certain embodiments, the protein is a CD45 antibody, a CD47 antibody, or cell-penetrating peptide. Multi-Stage Mixing (MSM)

[0244] As described herein, we introduce a new way to synthesize cargo-loaded lipid nanoparticles (LNPs): Multi-Stage Mixing (MSM). LNP synthesis has for 20+ years involved single-step mixing, in which an organic solution (such as ethanol, containing dissolved lipids) and an aqueous solution (containing dissolved nucleic acids) are mixed in a single step by shooting the two streams of solvents into a single mixer, such as a herringbone mixer in microfluidics, or confined impingement jet mixers. In MSM, we mix two solutions, then a very short time later mix that with a third solution, and potentially add on additional mixing steps. MSM allows us to control the location inside the LNP of each of the different components (particular lipids, nucleic acids, or even peptides, proteins and small molecules).

[0245] The method includes utilizing one or more channels to generate the "core" of the LNP. The core includes a first solution that includes one or more condensing agents and a second solution that includes a cargo. These components are mixed in a controlled way that allows for condensation of the DNA or other cargo, while not forming too big a complex.

[0246] A first channel of the microfluidic mixer, comprising one or more condensing agents is provided. In certain embodiments, the first solution includes an ionizable lipid that is positively charged in a specific buffer (e.g., the buffer in which the cargo is present). In certain embodiments, the first solution includes a cationic lipid. In certain embodiments, the first solution includes an ionizable lipid and a helper lipid, optionally DOPE. In certain embodiments, the first solution includes DOTAP. In certain embodiments, the first solution includes DOTAP, DOPSA, DOTMA, DMRIE, or N- Tetamine-pLys40. In certain embodiments, the first solution includes cationic lipid + helper lipid, e.g. DOTAP + DOPE.

[0247] In certain embodiments, the first solution further includes an organic solvent or buffer. Various solvents and buffers are known in the art and are useful herein. These include, without limitation, magnesium acetate, Tris-acetate-EDTA (TAE) , Trisbuffered Saline(TBS), HEPES, Tris-borate (TB), Tris-borate-EDTA (TBE), Tris-EDTA (TE) buffer, phosphate-buffered-saline (PBS), sodium acetate, sodium citrate, HEPES- buffered saline (HBS), Dulbecco's Phosphate Buffered Saline (DPBS), citrate buffer, TAE buffer with Mg2+, saline, ethanol, acetone, isopropanol, methanol, chloroform, cyclohexane, toluene, dichloromethane, polyethylene glycol 400, propylene glycol, glycerin, ethylacetate, ethyl formate, methylethyl ketone, methyl acetate, ethyl acetate, isopropyl acetate, benzyd alcohol, butyl lactate, distilled water, and glycerol. Other buffers include Tris-acetate-EDTA (TAE), Tris-buffered Saline (TBS), I4EPES, Trisborate (TB), Tris-borate-EDTA (TBE), Tris-EDTA (TE) buffer. In certain embodiments, the buffer used is not a limitation of this invention. In other embodiments, the LNP are formed using multivalent ions such as Mg2+, Hexaamminecobalt(III) chloride, or Ca2+.

[0248] In certain embodiments, the condensing agent is a lipid mixture, optionally DOTAP and DOPE. In certain embodiments, the DOPE:DOTAP is provided in a molar ratio of 1 : 100 - 99: 1. In certain embodiments, the first solution comprises DOTAP at a lipid molar % of 3.6% and DOPE at a lipid molar % of 10.8%. In certain embodiments, the first solution comprises DOTAP at a lipid molar % of 1.96% and DOPE at a lipid molar % of 6.4%.

[0249] In certain embodiments, the second solution includes cargo provided in an aqueous buffer in a second channel of the microfluidic mixer. The cargo may be any of those described herein, including, without limitation, DNA, mRNA, siRNA, peptide, protein, protein-DNA complex, peptide-DNA complex, lipid-DNA complex, proteolysis targeting chimera (PROTAC), or small molecule.

[0250] In certain embodiments, the cargo: condensing agent is provided in a molar ratio of 1 : 100 - 99: 1.

[0251] The first solution and second solution are mixed at a flow rate ratio of 1 : 30 to 30: 1. The shell to core flow rate ratio is 1:30 to 30: 1. In one embodiment, the flow rate for mixing the core components is of O.lml / min to 20ml / min, optionally 12 ml / min. In one embodiment, the flow rate for mixing the core components is of 0. 1 ml / min to 30ml / min, optionally 12 ml / min.

[0252] The delay interval, or time after core forms before being mixed with shell components, is from 0.1ms to 300ms. In certain embodiments, the delay interval is 0.1ms. 0.2ms, 0.2ms, 0.4ms, 0.5ms. 0.6ms, 0.7ms, 0.8ms, or 0.9ms. In certain embodiments, the delay interval is about 1ms, 2ms, 3ms, 4ms, 5ms, 6ms, 7ms, 8ms, 9ms, 10ms, 11ms, 12ms, 13ms, 14ms, 15ms, 16ms, 17ms, 18ms, 19ms, 20ms, 21ms, 22ms, 23ms, 24ms, 25ms, 26ms, 27ms, 28ms, 29ms, 30ms, 31ms, 32ms, 33ms. 34ms, 35ms, 36ms, 37ms, 38ms, 39ms. 40ms, 41ms. 42ms, 43ms, 44ms, 45ms, 46ms. 47ms, 48ms. 49ms, 50ms, 51ms, 52ms, 53ms, 54ms, 55ms, 56ms, 57ms, 58ms, 59ms, 60ms, 61ms, 62ms, 63ms, 64ms, 65ms, 66ms, 67ms, 68ms, 69ms, 70ms, 71ms, 72ms, 73ms, 74ms, 75ms, 76ms, 77ms, 78ms, 79ms, 80ms, 81ms, 82ms, 83ms, 84ms, 85ms. 86ms, 87ms, 88ms, 89ms, 90ms, 91ms. 92ms, 93ms. 94ms, 95ms, 96ms, 97ms, 98ms. 99ms, 100ms, 101ms, 102ms, 103ms, 104ms, 105ms, 106ms, 107ms, 108ms, 109ms, 110ms, 111ms, 112ms, 113ms, 114ms, 115ms, 116ms, 117ms, 118ms, 119ms, 120ms, 121ms, 122ms, 123ms, 124ms, 125ms, 126ms, 127ms, 128ms, 129ms, 130ms, 131ms, 132ms, 133ms, 134ms, 135ms, 136ms, 137ms, 138ms, 139ms, 140ms, 141ms, 142ms. 143ms. 144ms, 145ms, 146ms, 147ms, 148ms, 149ms, 150ms. 151ms. 152ms. 153ms. 154ms. 155ms, 156ms, 157ms, 158ms, 159ms, 160ms, 161ms, 162ms, 163ms, 164ms, 165ms, 166ms, 167ms, 168ms, 169ms, 170ms, 171ms, 172ms, 173ms, 174ms, 175ms, 176ms, 177ms, 178ms, 179ms, 180ms, 181ms, 182ms, 183ms, 184ms, 185ms, 186ms, 187ms. 188ms, 189ms, 190ms, 191ms, 192ms, 193ms, 194ms, 195ms. 196ms. 197ms. 198ms. 199ms, 200ms, 201ms, 202ms, 203ms, 204ms, 205ms, 206ms, 207ms, 208ms, 209ms, 210ms, 211ms, 212ms, 213ms, 214ms, 215ms, 216ms, 217ms, 218ms, 219ms, 220ms, 221ms, 222ms, 223ms, 224ms, 225ms, 226ms, 227ms, 228ms, 229ms, 230ms, 231ms. 232ms, 233ms, 234ms, 235ms, 236ms, 237ms, 238ms, 239ms, 240ms, 241ms. 242ms. 243ms, 244ms, 245ms, 246ms, 247ms, 248ms, 249ms, 250ms, 251ms, 252ms, 253ms, 254ms, 255ms, 256ms, 257ms, 258ms, 259ms, 260ms, 261ms, 262ms, 263ms, 264ms, 265ms, 266ms, 267ms, 268ms, 269ms, 270ms, 271ms, 272ms, 273ms, 274ms, 275ms, 276ms, 277ms, 278ms, 279ms, 280ms, 281ms, 282ms, 283ms, 284ms, 285ms, 286ms. 287ms, 288ms, 289ms, 290ms, 291ms, 292ms, 293ms, 294ms. 295ms. 296ms. 297ms. 298ms, 299ms, or 300ms. In certain embodiments, the delay interval is from about 0.5ms to about 100ms. In certain embodiments, the delay interval is from about 1ms to about 75ms. In certain embodiments, the delay interval is from about 1ms to about 60ms. In certain embodiments, the delay interval is less than 80ms. In certain embodiments, the delay interval is less than 30ms, 31ms, 32ms, 33ms, 34ms, 35ms, 36ms, 37ms, 38ms, 39ms, 40ms, 41ms, 42ms, 43ms, 44ms, 45ms, 46ms, 47ms, 48ms, 49ms, 50ms, 51ms,

[0253] 52ms, 53ms, 54ms, 55ms, 56ms, 57ms, 58ms, 59ms, 60ms, 61ms, 62ms. 63ms, 64ms,

[0254] 65ms, 66ms, 67ms, 68ms. 69ms, 70ms. 71ms, 72ms, 73ms, 74ms, 75ms. 76ms, 77ms.

[0255] 78ms, 79ms, 80ms, 81ms, 82ms, 83ms, 84ms, 85ms, 86ms, 87ms, 88ms, 89ms, 90ms,

[0256] 91ms, 92ms, 93ms, 94ms, 95ms, 96ms, 97ms, 98ms, 99ms, or 100ms.

[0257] A third solution is provided that includes one or more lipid components that comprise the shell of the LNP. These components may be comprised in one. two, or more solutions which are then added simultaneously to the pre-formed core layer. In certain embodiments, this is accomplished via a 3-channel microfluidic mixer as described below for the condensing molecule DOTAP. In certain embodiments, more than 3 channels are utilized. However, the core components are always mixed and the core formed prior to mixing with the shell layer. In certain embodiments, the LNP comprises more than one shell layer.

[0258] In certain embodiments, a shell layer comprises helper lipid, cholesterol, and pegylated lipid. In another embodiment, a shell layer comprises only pegylated lipid(s).

[0259] In other embodiments, the DNA-LNP are formed using an inline dilution cartridge for making LNPs with DOTAP and DOPE. A mixture of DOTAP and DOPE is diluted in TBS buffer (solution 1), pDNA is also diluted in TBS buffer (solution 2). Mixture of cholesterol, ionizable lipid (e.g.. SM102), DMG-PEG and NOA (STING- inhibitory lipid) is diluted with 100% ethanol (solution 3). Solution 1 and 2 are inserted into lipid and DNA channels, respectively, while solution 3 is inserted into the dilution channel (channel labels are those on a standard NanoAssemblr Ignite). In other embodiments, a bespoke microfluidics chip is used to generate the CTS LNP, such as those show n in FIG. 4B.

[0260] In other embodiments, the DNA integrity is protected during LNP synthesis. LNP synthesis was developed for and optimized with RNA delivery in mind. Therefore, many of the conditions used to synthesize LNPs are suboptimal for DNA loading. In particular, DNA is more stable when complexed with specific ions (e.g., Mg2+). Additionally, we have found that particular buffers improve DNA-LNPs ability to express encoded proteins. Particular buffers and solutes might prevent DNA-damage, improve DNA condensation and thus transit through the nucleus, and allow improve opening of DNA in the nucleus.

[0261] In certain embodiments of CTS synthesis, in Mixing Stage 1. nucleic acid (in aqueous solution) is mixed with a cationic species, leading to electrostatic condensation to form a ‘"core” (Fig. 16A). In one example of CTS, the cationic species is the lipid DOTAP, and we additionally add a common LNP component, the zwitterionic lipid DOPE. We have subsequently accomplished CTS synthesis with many different cationic species, including ionizable cationic lipids like those typical of LNPs. Notably, these cores are not thermodynamically stable - they rapidly grow to sizes exceeding 2000 nm, making them unsuitable for both LNP incorporation and in vivo administration. To overcome this, in Mixing Stage 2. our CTS synthesis encapsulates these nascent cores with standard LNP lipids before they can expand, forming a stabilizing "shell" that arrests core growth.

[0262] In another embodiment, an example of MSM is the three-channel core-then-shell method of DOTAP / DOPE LNP. DOTAP is a positively charged lipid and DOPE is a neutral helper lipid. DOTAP + DOPE can together form a hexagonal structure that enables cargo nucleic acids in LNPs to escape from cellular endosomes more quickly and safely. If we mix DOTAP / DOPE with DNA first, it will form a >2000nm complex, and cannot be loaded into LNP by the traditional two-channel method. If we mix DOTAP / DOPE into the lipid phase first, DOTAP will be exposed on the surface of LNPs, which will lead to heterogeneity of surface charge on the LNP, and will cause coagulation after entering the blood. With our multi-channel system (MSM), we allow DNA and DOTAP / DOPE to mix briefly and quickly, and before they form a large complex, we introduce the lipid phase through the third channel, which rapidly wraps the DOTAP / DOPE-DNA / RNA inside the rest of the LNP components (e.g.. ionizable lipid. DSPC, cholesterol, DMG-PEG), thus forming a stable DOTAP / DOPE in the core.

[0263] In certain embodiments, the MSM system provides enormous benefits. In one embodiment, we are able to identify the unique structure of LNPs using Cryo-EM. In another embodiment, we are able to incorporate non-DNA nucleic acid (siRNA. mRNA) using MSM method and obtain more homogenous LNP. Previously we have disclosed DNA LNP using core-then-shell method, now we demonstrate its ability' to RNA LNP. RNA LNP has been widely used for many applications, including COVID- 19 vaccines. However, RNA LNPs on the market have major drawbacks. LNPs can induce endosome escape, cause endosomal membrane damage and inflammatory’ cascade. Empty LNPs without nucleic acid pay load induce order of magnitude higher toxicity' than those loaded with nucleic acid. With the novel MSM, we provide a new-generation of LNP that have better functionality and safer profile.

[0264] In another embodiment, we are able to modify the core component in MSM system and further improve LNP functionality. In another embodiment, we are able to load non-nucleic acid drugs (PROTAC, small molecule cargo drugs, peptides, proteins, etc.) using MSM. Many small molecule drugs cannot be loaded into liposomes or LNPs using traditional methods, unless they have special pKa or LogP. Using the MSM / core- then-shell method, we were able to load the drugs without specific chemical properties into the core-then-shell LNP in their original forms and maintain their bioactivity'.

[0265] In another embodiment, we are able to load histone inside the core with DNA, using MSM. Loading histone cannot be achieved by traditional method because it forms large aggregates. Loading of histone into LNP accelerates endosome escape of DNA, protects DNA from degradation in cytosol, enables nuclei localization, therefore significantly improves DNA expression.

[0266] In another embodiment, we are able to bury dangerous lipids in the core but maintain their function. Some lipids are dangerous on the surface (e.g., DOTAP is positively charged and causes clotting) but benign when buried inside, where they' can provide other benefits.

[0267] In another embodiment, we can increase the density of the LNPs’ brush border by adding PEGylated lipids (or other anti-fouling brush polymers) in a later step of MSM mixing.

[0268] Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided which contains the LNP described herein and a pharmaceutically acceptable carrier. In certain embodiments, the composition is formulated for in vivo delivery.

[0269] As used herein, ‘'carrier’' includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0270] In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

[0271] Methods and agents well known in the art for making formulations are described, for example, in ‘‘Remington's Pharmaceutical Sciences,” Mack Publishing Company, Easton, Pa. Formulations may, for example, contain excipients, carriers, stabilizers, or diluents such as sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes, preservatives (such as octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyd parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins, chelating agents such as EDTA. sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0272] A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of poly oxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), poly oxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter ‘ P’’ (for poloxamer) followed by three digits: the first tw o digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

[0273] These above compositions may be administered in a variety of volumes of carrier, excipient or buffer formulation, ranging from about 25 to about 1000 microliters, or higher volumes, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method.

[0274] Any suitable route of administration may be selected. Accordingly, pharmaceutical compositions may be formulated for any appropriate route of administration, for example, in the form of liquid solutions or suspensions (as. for example, for intratumoral administration, intravenous administration, for oral administration, etc.). Alternatively, pharmaceutical compositions may be in solid form (e.g, in the form of tablets or capsules, for example for oral administration). In some embodiments, pharmaceutical compositions may be in the form of powders, drops, aerosols, etc.

[0275] EXAMPLES

[0276] Example 1 : Materials and methods

[0277] Materials

[0278] SM-102, ALC-0315 and ALC-0519 were purchased from Echelon Biosciences (Ontario, Canada). 18:0 PC (DSPC. l,2-distearoyl-sn-glycero-3-phosphocholine), DMG- PEG 2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), 18: 1 (A9- Cis) PE (DOPE), 18: 1 TAP (DOTAP), 18:0 PE-DTPA and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL). RAW 264.7, HeLa cells, and mouse embryonic fibroblasts (MEFs) were purchased from American Type Culture Collection (Manassas, VA). Reporter lysis buffer and luciferin assay solution were purchased from Promega (Madison, WI). Plasmid DNA (pDNA) was purchased from Aldevron (<0.1 EU / pl endotoxin level). 5moU nucleoside-modified firefly luciferase mRNA was purchased from TriLink BioTechnologies. Indium-111 chloride (In-111) was purchased from BWXT Medical (Ottawa, Canada). Sodium iodine (1-125) was purchased from PerkinElmer (Waltham, MA). All other chemicals and reagents were purchased from Fisher Scientific (Hampton, NH), unless otherwise noted.

[0279] Animals

[0280] All animal studies were carried out in accordance with the Guide for Care and Use of Laboratory Animals under the protocol approved by the University of Pennsylvania Institutional Animal Care and Use Committee, and conformed to all relevant regulatory standards. Naive C57BL / 6 mice (male, aged 6-8 weeks, 23-25g), naive BALB / c mice (female, aged 6-8 weeks. 23-25g) were procured from the Jackson Laboratory (Bar Harbor, ME). The animals were housed in a controlled environment maintained at temperatures between 22-26 °C, with a 12 / 12-hour light / dark cycle, and provided with access to food and water. Cell culture

[0281] RAW264.7 mouse macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin (PS). HeLa cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and 1% PS. Mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% FBS, 2 mM L- GlutaMAX, IX non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 100 U / mL penicillin, 100 mg / mL streptomycin. All cells were incubated with 5% CO2 at 37°C.

[0282] LNP Synthesis

[0283] LNPs were formulated using NanoAssemblr Ignite nanoparticle formulation systems (Precision Nanosystems. Vancouver. Canada), or using microfluidics devices designed in-house (Fig 13A). The aqueous phase was made in different buffers including 50 mM citrate buffer (pH 4.0), Tris-acetate-EDTA (TAE), Tris-buffered saline (TBS), and HEPES with pDNA. For standard LNPs, an organic phase containing a mixture of lipids dissolved in ethanol at a designate molar ratio (Fig 8C) was mixed with the aqueous phase containing DNA, at a flow rate ratio of 1 :3 and at a total lipid to nucleic acid weight: weight ratio of 40: 1. For CTS LNPs, channel 1 (core lipid phase) contains lipid mixture of DOPE and DOTAP; channel 2 (shell lipid phase) contains lipid mixture of ionizable lipid, DSPC, PEGylated lipid and cholesterol at a designated molar ratio (Fig 10A); channel 3 (core aqueous phase) is pDNA in aqueous buffer. 3 channels were mixed at a flow rate ratio of 1 : 1:3. For drug-loaded CTS LNPs, sting inhibitor C178 was added to channel 2.

[0284] LNPs were dialyzed against IX PBS or IX PBS containing MgChfor 2 hours. Mg2concentration in the dialysis buffer corresponds to its concentration in the DNA- containing aqueous buffer, unless otherwise stated. After dialysis, LNPs were sterilized using a 0.22 pm filter, and stored at 4 °C for later use.

[0285] In-House Microfluidics Device Fabrication A custom, dual-stage microfluidic mixer was fabricated in the Quattrone Nanofabrication Facility at the University' of Pennsylvania. Design of the device was completed in AutoCAD (Autodesk, San Rafael, CA). Briefly, using a mask aligner, each layer of the multilayer architecture was photolithographically patterned onto a 100mm silicon wafer (ID 775; University Wafer, South Boston, MA) spray coated with SI 805 photoresist (Dow, Midland, MI). After patterning and development, deep reactive ion etching (SPTS Rapier Si DRIE, Newport, UK) was performed. After the silicon was patterned and etched, the device was anodically bonded to a 100-mm Borofloat 33 glass wafer (ID 517; University Wafer) using an EVG 510 Wafer Bonding System (EVG Group, Oberosterreich, Austria).

[0286] Dual-valve pressure regulators (Alicat Scientific, Tucson, AZ) were used to pressurize 15mL vessels (Elveflow. Paris, France), with outputs mechanically coupled to the input ports of the microfluidic mixer. Separate inputs were provided for the ethanol and aqueous phase components, to allow for a two-step nanoprecipitation. The micromixer accommodated individual mixing of constituent components of the ethanol and aqueous phases to separately form the core and shell, which then combined and underwent further mixing. Pressures were chosen to allow the total flow rate through each input to combine to a total flow rate of 1.2mL / min. CTS LNP samples were collected directly into a dialysis cassette (Thermo Fisher, Waltham, MA), and dialyzed against IX PBS containing 4 mM MgCU for 2 hours.

[0287] LNP Characterization

[0288] Hydrodynamic nanoparticle size, poly dispersity7index (PDI), and bulk zeta potential of LNPs were measured by dynamic light scattering using Zetasizer Pro ZS (Malvern Panaly tical, Westborough, MA). Distribution of zeta potential of individual LNP was measured by ZetaView instrument by Particle Metrix (Inning am Ammersee, Germany).

[0289] Encapsulation efficiency and concentration of pDNA or mRNA were measured using a Quant-iT-PicoGreen, or Ribogreen, respectively. 50 pL of TE buffer and 50 pL of 2% Triton X-100 (in TE buffer) were added in duplicates to a black bottom 96-well plate. LNP formulations were diluted in the TE buffer, and 50 pL of each formulation was added to the TE buffer and 2% Triton X-100. After 10 minutes of incubation time, RiboGreen or PicoGreen reagent was diluted 1:100 or 1 :200, respectively, in the TE buffer and added to each well. Picogreen fluorescence was measured at excitation of 480 nm and emission at 520 nm, and RiboGreen signal was measure using an excitation 485 nm of and emission of 528 nm by a plate reader (Spectramax M2; Molecular Devices, San Jose, CA).

[0290] Drug encapsulation was measured by high-performance liquid chromatography, and drug entrapment efficiency was calculated as weight of loaded drug in LNPs over weight of total drug added into LNP formulation.

[0291] Cryo Electron Microscopy

[0292] To prepare samples for cryo-EM imaging, lacey carbon-coated 300 mesh copper grids (Electron Microscopy Sciences) were glow discharged at 15 mA for 30 s with the PELCO easiGLOW glow7discharge system (Ted Pella). Then, 8 pL of sample was applied to the grids. The grids w ere blotted with filter paper and plunge frozen in liquid ethane using Vitrobot Mark IV. under 4 °C and 100% humidity. The grids were kept in liquid nitrogen until imaging. Cryo-EM images were collected with Titan Krios Cryo- TEM (ThermoFisher).

[0293] Circular Dichroism Spectroscopy

[0294] All CD experiments were carried out using Aviv CD spectrometer. The following parameters were used: range 200-320 nm, data pitch = 1 nm, CD scale = 200 mdeg / 0. 1 dOD, FL scale = 200 mdeg / 0.1 dOD, Digital Integration Time = 1 sec, bandwidth = 1 nm. pDNA were measured at the concentration of 30 pg / mL. Buffer solutions alone were used as blanks to establish baselines for correction. Normalized raw CD data were used for plotting and analysis.

[0295] Differential Scanning Calorimetry Samples of LNPs were prepared for cal ori met ry measurements at particle concentrations of 3-5ell nanoparticles per mL in PBS. PBS was used as a reference solution for all differential scanning calorimetry measurements. Reference PBS was draw n from the same aliquot used to dilute nanoparticle samples. Lipid nanoparticle suspensions and reference solutions were heated from 4°C to 130°C at a target rate of 1°C per minute in a TA Instruments Nano DSC operating in scanning mode. Background measurements of reference PBS vs. reference PBS were obtained to confirm stability of the measurements and reference vs. reference thermograms were subtracted from all LNPs vs. PBS reference thermograms. Sigmoidal fits were applied to baselines of background-corrected thermograms in TA Instruments NanoAnalyze software. After baseline subtraction, thermogram data were normalized to instantaneous scan rate and molar concentration of nanoparticles as measured by nanoparticle tracking analysis. The resultant molar heat capacity thermograms were integrated to yield enthalpy data or normalized to temperature then integrated to yield entropy data. Enthalpy, entropy, and Gibbs energy of any phase transitions were extracted from this data.

[0296] Small Angle X-ray Scattering

[0297] Dynamic Light Scattering. A Nanobrook Omni particle sizer (Brookhaven Instruments Corporation, Holtsville, NY, USA) was used to record data at 25°C in polystyrene 1-cm cells using a standard diode laser at 640 nm, with scattering recorded at an angle of 90°. Three scans were recorded for each sample and hydrodynamic radii (Stokes radii) were calculated using the BIC Particle Solutions software v3.6.0.7122.

[0298] SEC-MALS. 100 pL of DNA-LNP at a particle concentration of -1011 particles were injected and eluted isocratically at 0.2 ml / min from a 7.8 mm TSKgel G6000PWxl- CP sizing column equilibrated in IX PBS at room temperature. Absolute molar mass of the DNA-LNPS was determined in-line using multi-angle light scattering. Light scattering from the column eluent was recorded at 18 different angles using a DAWN- HELEOSII MALS detector (Wyatt Technology' Corp.) operating at 658 nm. Eluent concentration using a mass averaged dn / dc value of 0.16 cm3 / g was determined using an in-line Optilab T-rEX Interferometric Refractometer (Wyatt Technology Corp.). The weight-averaged molar masses of species within defined chromatographic peaks were calculated using the ASTRA software version 8.2.2 (Wyatt Technology Corp.), by construction of Debye plots (KC / R0 versus. sin2[0 / 2]) at one second data intervals. The weight-averaged molar mass was then calculated at each point of the chromatographic trace from the Debye plot intercept and an overall average molar mass was calculated by weighted averaging across the peak. Normalization coefficients for diode counting efficiency were obtained using a BSA standard.

[0299] SEC-SAXS. Synchrotron Size-Exclusion Chromatography in line with Small Angle X- ray Scattering. Size-exclusion chromatography (SEC)-SAXS data were collected at beamline 16-ID (LiX) of the National Synchrotron Light Source II (Upton, NY). Data were collected at a wavelength of 1.0 A in a three-camera configuration, yielded accessible scattering angle where 0.006 < q < 3.0 A'1, where q is the momentum transfer, defined as q = 4a: sin(0) / X. where / . is the X-ray wavelength and 20 is the scattering angle; data to q<0.5 A-1were used in subsequent analyses. 100 pL of LNP at a particle concentration of -1011particles were injected and eluted isocratically at 0.2 ml / min from a 7.8 mm TSKgel G6000PWxl-CP sizing column equilibrated in IX PBS (Invitrogen), at room temperature. Eluent from the column flowed into a 1 mm capillary for subsequent X-ray exposures at 1-s intervals. Plots of intensity from the forward scatter closely correlated to in-line UV and refractive index (RI) measurements. SAXS Analysis. SVD- EFA analysis of the SEC-SAXS data sets were performed, as implemented in the program RAW. Buffer subtracted profiles were analyzed by singular value decomposition (SVD) and the ranges of overlapping peak data determined using evolving factor analysis (EFA) as implemented in REGALS. The determined peak windows were used to identify the basis vectors for each component and the corresponding SAXS profiles were calculated. When manually fitting the pair distribution function P(r)using the program DIFT, the maximum diameter of the particle (Dmax) was incrementally adjusted to optimize / 2figures, to minimize the discrepancy between the fit and the experimental data to a qmax :-o 8 / Rg. and to optimize the visual qualities of the distribution profile. Deconvolution of the primary Braggs peaks in SAXS were using multiple Lorentz with the built-in functions implemented in OriginPro 2024b (Northampton, MA, USA), as previously described: y = yo + (2*area / n) *(width / (4*(x - xc)2 width2)) where yo is an offset and was set to the X-ray baseline. xcis a center of function and corresponds to the center of the SAXS peak. In the fitting, the peak position was fixed and the area and width of the peaks were not restrained. Peaks were assigned to inverse hexagonal (H||) or lamellar (La) phases based on peaks corresponding to the first order Bragg peak and corresponding peaks at integral increments.

[0300] SASView (available at sasyiew.org) was used to fit experimental data to empirical models for core-shell sphere and ellipsoid models. In this fitting, scattering length densities (SLDs) were fixed to calculated values and the shell diameter to that of a lipid bilayer (54 A). The SLDs for the component parts of the DNA-LNP formulations used in this fitting were calculated using MULCh.

[0301] DENSS

[0042] was used to calculate the ab initio electron density map directly from the DIFT output. Twenty reconstructions of electron density were performed in the slow mode with default parameters and subsequently averaged and refined. Reconstructions were visualized using PyMOL 2.5.2 Molecular Graphics System (Schrodinger, LLC, New Your, NY, USA) with five contour levels of density rendered with these respective colors: 15o (red), lOo (green), 5o (cyan), 2.5o (blue), and -0.7o (blue). The sigma (o) level denotes the standard deviation above the average electron density value of the generated model.

[0302] In vitro LNP transfection

[0303] HeLa cells and RAW 264.7 macrophages were seeded in 96-well plates and incubated overnight (seeding density of 5xl03and 5xl04cells / well, respectively), and treated with 200 ng / mL or 500 ng / mL of luciferase-pDNA-LNP, respectively. Following 24 hour LNP treatment, 10% cell counting kit-8 (CCK-8, Enzo Life Sciences) reagent in complete medium was added to cells for 30 minutes incubation and run a colorimetric assay to measure cell viability, according to manufacturer’s protocol. To test the transfection efficiency of luciferase-pDNA-LNP, cells were lysed by cell culture lysis reagent and cell lysates were tested using luminometer upon adding luciferase assay substrate.

[0304] MEFs were seeded in ibidi p-slide 8 well glass bottom chamber (Fitchburg, WI) at the density of 2xl04cells / well. 200 ng / rnL of eGFP pDNA LNP or lipofectamine were added in complete medium and incubated for 24 hours. eGFP-positive cells were imaged using Leica Stellaris 5 confocal microscopy (Leica Microsystems).

[0305] For the primary neuronal culture, E16-E18 embryos were collected for neuronal culture. Single cells were obtained and seeded in 8 well glass bottom chamber at 6xl04cells / well. Neurobasal Plus medium (Gibco, A3582901) with B27 supplement (Gibco, #A3582801), 1 x GlutaMax and 1% P / S was used for neuronal maintenance. Replace half of the medium every 3 days. On day 3, 5 uM AraC was used for inhibiting proliferating cells. At day 11. neurons were treated with 200ng / mL of eGFP pDNA LNP or lipofectamine, and were imaged 24 hours later.

[0306] Cytokine measurements were carried out on cell culture medium from RAW macrophages after 4-hour treatment, with IFN- (■ ELISA kit (Abeam), according to the manufacturer’s instructions.

[0307] Radiolabeling Biodistribution

[0308] For biodistribution studies, LNPs or fibrinogen were traced with In-111 or 1-125, respectively. LNPs were formulated as described above with 0.1 mol% of 18:0 PE- DTPA using metal-free buffers. Trace metals were removed from the buffers using a Chelex 100 resin, per manufacturer’s instruction. In-111 was mixed with LNPs at a specific activity of 1 pCi of In-111 per 1 pmol of lipid. The mixture w as incubated at room temperature for 30 minutes. Fibrinogen was radiolabeled with 1-125 using the lodogen method. Glass tubes coated with lOOpg of lodogen reagent were incubated with fibrinogen (2 mg / mL) and 1-125 (115 pCi per pg protein) for 5 minutes on ice. Unincorporated In-111 or 1-125 w as removed with a Zeba 7kDa desalting spin column (ThermoFisher Scientific). Thin layer chromatography was used to confirm radiolabeling efficiency. All materials were confirmed to have >90% radiochemical purity prior to use. To trace distribution, fibrinogen was injected 2 minutes prior to LNP intravenous injection. 30 minutes later, blood and organs were harvested, and the radioactivity was quantified with a gamma counter (Wizard2, PerkinElmer). The gamma data and organ weights were used to calculate the tissue biodistribution injected dose per gram.

[0309] DNA uptake and nuclei colocalization

[0310] DNA was labeled with SYBR Green by mixing SYBR Green I Nucleic Acid Gel Stain (Invitrogen, Waltham) at 2000X with 1 mg / mL pDNA at room temperature for 30 minutes. Free dye was removed by Zeba desalting spin column. DNA concentration and fluorescent intensity' was confirmed by NanoDrop Microvolume Spectrophotometers (Thermo Scientific). SYBR Green labeled pDNAs were then used to either mix with lipofectamine 2000 to form lipoplexes or to make pDNA-LNPs following previously mentioned LNP synthesis procedure.

[0311] HeLa cells were seeded in ibidi p-slide 8 well glass bottom chamber at a density of 2x104cells / well and grew overnight. Hoechst 33258 (Cayman Chemical) was added 10 minutes prior to LNP treatment. Cells were treated with 2000 ng / mL of SYBR Green- labeled pDNA-LNPs or pDNA-lipofectamine 2000 in OPTI-MEM medium. Time-lapse imaging was acquired under 5% CO2 at 37°C to trace DNA uptake and nuclei transport. For colocalization analysis, Fiji ImageJ software is used to perform nuclei mask and raw integrated density of SYBR green signal within the nuclei mask were measured and calculated.

[0312] Nanoparticle Tracking Analysis mRNA and plasmid DNA was labeled using Label IT® Nucleic Acid Labeling Kit (MoBiTec GmbH. Goettingen, Germany) according to the manufacturer's protocol. Briefly, nucleic acid was mixed with Label IT® Reagent at the (w:v) ratio of 5:2, and reacted at 37 °C for 1 hour. Unreacted dye of DNA was purified using G50 Microspin Purification Columns. The labeled mRNA was purified by ethanol precipitation. The final concentration was determined using a NanoDrop One Spectrophotometer. Cy5 labeled mRNA and Cy3 labeled DNA were used to form LNPs to analyze nucleic acid payload in individual nanoparticles. Briefly, LNPs were diluted with deionized water and analyzed using nanoparticle tracking analysis through a Nanosight NS300 (Malvern Panalytical, Westborough, MA). Light scattering mode was used to determine total LNP concentration. Fluorescent mode was used to determine fluorescent nucleic acid loaded LNP concentration. The size of free fluorescent nucleic acid is below the instrument measurement limit, thus was not reported. Percentage of LNP loaded with nucleic acid was calculated as concentration of fluorescent LNP over that of total LNP.

[0313] Quantitative endosomal escape assessments by galectin-9 reporter cell

[0314] HeLa cells expressing mCherry-coupled galectin-9 (mCherr -GAL9) were obtained by transfection using plasmids encoding mCherry-galectin 9 (Addgene, Watertown, MA). The galectin-9 reporter HeLa cells were then cultured in ibidi p-slide 8 well glass bottom chamber at a density of 2xl04cells / well and grew overnight. Cells were dosed with 400 ng / mL of pDNA-LNPs for 24 hours. After fixation with 4% paraformaldehyde (PF A), cells were stained with DAPI and imaged using confocal microscopy. Within individual cell regions of interest, galectin-9 puncta was analyzed via measuring the integrated densify of mCherry signals using Fiji ImageJ software. p-STING imaging and quantification

[0315] Raw macrophages were seeded in ibidi p-slide 8 well glass bottom chamber at a densify of 2x105cells / well and grew overnight. 1000 ng / mL of pDNA-LNPs were used to treat cells for 4 hours. After treatment, cells were washed with PBS for three times and fixed with 4% paraformaldehyde. For phosphorylated STING staining, cells were permeabilized in 0.1% Triton for 10 minutes, and stained with primary antibody (Phospho-STING (Ser366). 1:200) in blocking buffer containing 10% bovine serum albumin at 4 °C overnight, follow ed by a secondary antibody (Alexa Fluor 594- conjugated goat-anti rabbit antibody, 1:500) at room temperature for 2 hours. Cell nuclei were labeled using DAPI. Images w ere acquired by confocal microscopy and mean fluorescent intensify of individual cells were measured by Fiji ImageJ software. In Vitro and In Vivo Cytokine measurement

[0316] Cytokine measurements were carried out on cell culture medium or plasma with a LegendPlex 13-plex Mouse Inflammation Panel (Biolegend), or IFN-b and IL-6 ELISA kit (Abeam), according to the manufacturer’s instructions. Blood was collected from mice in a EDTA pre-coated tube. Plasma was obtained by centrifugation of whole blood at 500 g for 5 minutes.

[0317] In Vivo Imaging System

[0318] LNPs were injected intravenously into the BALB / c mice. One dose of lOmg / kg baricitinib was injected intravenously 5 minutes prior to injection of C-178 loaded CTS DNA-LNP. At the time of imaging, mice were intraperitoneally injected with 100 uL of 30 mg / mL D-luciferin sodium salt under 3% isofl urane-induced anesthesia, then placed in an IVIS Spectrum machine (PerkinElmer) belly up and imaged for whole body chemiluminescence every70.2 minutes with automatically determined exposure time for 10-12 images, until the signal reached the peak intensity.

[0319] Statistics

[0320] All results are expressed as mean ± SEM unless specified otherwise. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software) * denotes p<0.05, ** denotes p<0.0I, *** denotes pO.OOL **** denotes pO.OOOl.

[0321] Example 2: Core-then-shell loading method

[0322] The core-then-shell method is developed to load protein-, peptide-, or lipid-DNA complexes, which are very hard to load or cannot achieve applicable size, into DNA LNP. Core-then-shell can stabilize the structure and enhance the DNA expression. In order to achieve stable DNA-LNP, the flow- rate, flow rate ratio and component ratio require thorough adjustment.

[0323] A mixture of DOTAP and DOPE is diluted in TBS buffer (pH=6, 4mM Mg2+) (i) or 100% ethanol (ii) at a lipid molar % of 3.6% and 10.8%, respectively (solution 1). pDNA is diluted in TBS buffer (solution 2). Mixture of cholesterol, ionizable lipid (e.g., SMI 02), DMG-PEG and NOA (STING-inhibitory lipid) is diluted with 100% ethanol at a lipid molar % of 38.7%, 45.3%, 1.5%, and 0. 18%, respectively (solution 3). Inline dilution cartridge used for making LNPs with DOTAP. Solution 1 and 2 are inserted into lipid and DNA channels, respectively, while solution 3 is inserted into the dilution channel (channel labels are those on a standard NanoAssemblr Ignite). Flow rate is 12ml / min. Flow rate ratio is 1 : 1 : 0.66 for formulation (i) and 3: 1: 1 for formulation (ii).

[0324] Multi-Stage Mixing (MSM)

[0325] Multi-Stage Mixing (MSM) is a new way to synthesize DNA- or RNA-loaded lipid nanoparticles (LNPs). We mix two solutions, then a very short time later we mix with a third solution. Additional mixing steps may be added.

[0326] Three-Channel Core-Then-Shell Method

[0327] One example of MSM is the “the three-channel core-then-shell method.” This method utilizes a multi-channel system, in which one channel contains DNA / RNA and / or other components in the aqueous phase (1), and one channel contains lipids or other components in either aqueous phase or organic phase (2). We mix phase (1) and (2) first and we then join the third phase containing lipids in organic phase (3) with the first two channels to wrap up the system to form stable LNP.

[0328] Three-Channel Core-Then-Shell Method of DOTAP / DOPE LNP.

[0329] If we mix DOTAP / DOPE into the lipid phase first, DOTAP will be exposed on the surface of LNPs, which will lead to heterogeneity of surface charge on the LNP, and will cause coagulation after entering the blood. With our multi-channel system (MSM), we allow DNA and DOTAP / DOPE to mix briefly and quickly, and before they form a large complex, we introduce the lipid phase through the third channel, which rapidly wraps the DOTAP / DOPE-DNA / RNA inside the rest of the LNP components (e.g., ionizable lipid, DSPC, cholesterol, DMG-PEG), thus forming a stable DOTAP / DOPE in the core.

[0330] The size and surface properties (NLSand zeta view) of the LNPs made by multichannel core-then-shell method is examined. Their size is around 150 nm, with a poly disperse index of less than 0.1, and can be stable for several weeks. Their surface charge is neutral and uniform.

[0331] Advantages

[0332] (1) We identify the unique structure of LNPs using Cryo-EM. Using MSM, the DNA and lipids from the first two solutions form a core, and are wrapped with a shell-like structure that contains lipids from the third solution.

[0333] (2) We incorporate non-DNA nucleic acid (siRNA, mRNA) using MSM method and obtain more homogenous LNP. We use the core-then-shell method to generate RNA LNP. Using traditional single-step mixing, LNPs with SM-102 ionizable lipid (used in Modema vaccine) has -95% empty LNPs; LNPs with ALC-0315 ionizable lipid (used in Pfizer vaccine) has -50% empty LNPs. Using MSM. we use the same formulation as the traditional method, but instead we incorporate SM-102 into the core together with mRNA and then wrap the mixture with the lipid mixture containing helper lipid and cholesterol. We obtain 60% LNPs with payload, increased by 12-fold compared to the traditional LNP synthesis method. In addition, it is known that ionizable lipid amount is a leading cause of cell toxicity. We reduce the SM-102 amount to half and obtain more than 50% LNPs with pay load. The expression of these two LNPs are similar to the LNPs made by traditional method. Further, we improve the core by introducing DOPE / DOTAP. This method increases the ratio of LNP with payload to 98%, with higher expression, and potentially lower toxicity. The results suggest that we have solved the long-standing problem of LNP heterogeneity using traditional method.

[0334] (3) We modify the core component in MSM system and further improve LNP functionality7. DOTAP / DOPE is not the only combination that can form the core of LNP and improves LNP homogeneity and expression. Replacing DOTAP with other cationic lipids (DOPSA. DOTMA, DMRIE, N-Tetamine-pLys40, etc), with proper ratio with DOPE, we can form different core structure-then-shell LNP. By changing the ratio of cationic lipid to DOPE, or changing the ratio of cationic lipid / DOPE to DNA, we can change LNP nanostructure, improve LNP payload and nucleic acid loading, hence improving expression and reducing toxicity. According to different ionizable lipid, we can modify the composition ratio in the core, and get better LNP profile (higher expression, less toxicity).

[0335] (4) We load non-nucleic acid drugs (PROTAC, small molecule cargo drugs, peptides, proteins, etc.) using MSM. For example, we load 2 small molecule drugs (BX- 795 and phenylboronic acid). Both drugs cannot be loaded into liposomes or LNPs using traditional method. BX-795 is a TBK.1 inhibitor. In in vitro and in vivo studies, loading of BX-795 significantly improves DNA-LNP expression, reduces cellular toxicity and improves survival rate of injected animals. Our results suggest that this novel MSM system can co-load small molecule drugs with nucleic acid, which helps to minimize the toxicity of LNPs and improve the functionality, thus broadening the applications of LNPs. By doing this, we can also control in which stage / phase we put in the drug, different from traditional method where the drug is randomly distributed in the LNPs.

[0336] (5) We bury dangerous lipids in the core but maintain their function. Some lipids are dangerous on the surface (e g., DOTAP is positively charged and causes clotting) but benign when buried inside, where they can provide other benefits. We compare LNPs with the same lipid composition but formulated either via MSM / core- then-shell or the traditional lipid mixture. We find that LNPs containing DOTAP formed via MSM have neutral and uniform surface charge, while LNPs containing DOTAP formed via traditional mixing have positive surface charge (which is known to induce clotting) with a bimodal distribution of surface charge among the population of particles.

[0337] (6) By introducing MSM, we increase the density of the LNPs’ brush border by adding PEGylated lipids (or other anti-fouling brush polymers) in a later step of MSM mixing.

[0338] Example 3: Optimizing the buffer used during LNPs synthesis ensures DNA stability, and thereby markedly improves DNA-based protein expression

[0339] The first hurdle in DNA-LNPs reaching maximal expression is that the standard synthesis conditions of LNPs may alter DNA’ s structure in deleterious ways. In particular, for the decades of mRNA-LNP synthesis, the nucleic acid has been first dissolved in a pH 4 buffer (typically citrate), to maximize the fraction of ionizable lipids’ terti ary amines that are protonated. However, this low pH may destabilize the helix and lower the thermal melting point of DNA, and cause DNA to adopt conformations other than the B-form, which is the classical right-handed double helix and the most common biologically active structure present in cells under normal physiological conditions. Other conformations can include A-form, Z-form, G-quadruplex, and others, as well as less defined conformations that might occur after partial acid hydrolysis of the DNA. (PMID: 11159454 and ncbi_nlm_nih_gov / books / NBK6545 / )

[0340] To assess the effect of buffer and pH on DNA conformations, we synthesized DNA-LNPs in different buffers and measured B-form DNA. We analyzed pDNA in various buffers by circular dichroism (CD) spectroscopy, which is able to measure B- form DNA via the relative magnitude of the ellipticity7signal in the range of 200 - 320 nm. A typical B-form DNA has a positive maximum around 290 nm, a negative maximum around 245 nm and a broad positive peak between 260 - 280 nm. We compared two different buffers: citrate pH 4 (classic mRNA-LNP synthesis buffer) vs TBS pH 6 with 4 mM Mg2+. Tris being the most common buffering agent used in most laboratory DNA reactions, and Mg2+stabilizes the B-form DNA helix via a variety7of mechanisms, mostly involving electrostatic interaction between Mg2' and DNA’s phosphates (PMID: 18512983). As shown in FIG. 8B, the CD spectroscopic signal for B- form DNA signal is significantly lower for LNPs made in citrate vs TBS buffer. Next, we titrated Mg2+to the TBS buffer. The addition of 4 mM Mg2+increased the B-form DNA signal in CD spectroscopy, compared to 0 and 12.5 mM Mg2+(FIG. 9A). Collectively, these results suggest that the traditional buffer used during LNP synthesis (citrate pH 4, no Mg2+) perturbs the DNA B-form conformation, and this defect can be rescued by switching to TBS pH 6, buffer containing at least 4 mM Mg2+.

[0341] Having shown that the synthesis buffer can greatly affect the DNA conformation, we next sought to determine if the buffer similarly affects DNA-LNPs’ ability to drive protein expression. We synthesized DNA-LNPs using a standard LNP formulation (FIG. 8C) and encapsulating plasmid DNA (pDNA) encoding luciferase. We synthesized LNPs in one of two buffers, citrate pH 4 or TAE pH 6, which is another Tris-based buffer containing EDTA. After synthesis, we performed the standard dialysis on LNPs. Instead of using solely the standard PBS, we compared the PBS dialysis buffer with and without Mg2. The structure of the buffer-optimized LNPs was analyzed by cryo-electronmicroscopy (CryoEM). We found that the optimal DNA-LNPs (TBS, pH 6, Mg2+) had a similar CryoEM appearance as standard LNPs (citrate. pH 4. no Mg2+).

[0342] In the in vitro transfection experiments, RAW 264.7 macrophages were treated with these DNA-LNPs and luciferase expression was measured at 24 and 48 hours (FIG. 8D-E). The Tris-based buffer led to much greater expression than the citrate buffer, fitting with Tris buffer preserving B-form DNA (FIG. 8B). Further, Mg2+in the dialysis buffer was essential for any expression at all, again fitting with the importance of Mg2+for stabilizing B-form DNA. It is worth noting that the addition of in Mg2+acidic buffer reduced the luciferase expression (FIG. 9B), possibly due to the weakened Mg2+-DNA interactions in the presence of protons failing to stabilize the DNA double helix. Additionally, Mg2+can act as a cofactor of DNA related enzymes such as DNase I and topoisomerase. (PMID: 19188255)

[0343] Next, we compared a broader range of synthesis buffers for their abi 1 i ty to produce DNA-LNPs that drive high-level protein expression in RAW macrophages (FIG. 8F-H). The luciferase expression results show that TBS is by far better than TAE and HEPES, with pH being optimal. And the addition of Mg2+dramatically improves performance. We also confirmed this general trend in a second cell line, HeLa epithelial cells (FIG. 8J-L), except that TAE outperforms TBS when Mg2+is present. These results confirm that across multiple cell types, Tris buffers are best, at pH 6. and Mg2+is essential.

[0344] Example 4: Multi-stage mixing (MSM) to create core-then-shell (CTS) LNPs improves the encapsulation of nucleic acids

[0345] While buffer optimization preserves DNA stability during LNP synthesis, that is only the first hurdle DNA-LNPs face compared to mRNA-LNPs (FIG. 8A), and the resulting expression is only ~5% of that by lipofectamine, the gold-standard in vitro transfection reagent (FIG. 9C-D). The remaining hurdles prevent effective DNA expression. These hurdles could be solved individually by various chemical and genetic methods, such as covalent modification of DNA, co-loading inhibitors, and helper RNA molecules. However, a more elegant approach would attack all 5 intracellular hurdles without requiring the risks of introducing new chemical and genetic entities. To do this, we sought a physical method that would solely change how we pack DNA into LNPs. In particular, a potential solution is to compactify DNA into a “core,” which will protect DNA from DNA sensors and DNases, and can more easily fit through the nuclear pore complex.

[0346] To produce LNPs with a “core” of DNA, we developed multi-stage mixing (MSM) (FIG. 10A). In Mixing Stage 1, the DNA (here plasmid DNA) is mixed with helper lipid DOPE and a cationic molecule, for which we used as a prototype the cationic lipid DOTAP with aN:P of DOTAP: DNA = 0.63, lower than the threshold of 2 for tight core, which limits effective nuclear entry (PMID: 26066769). Notably, DOTAP at a final molar % of total lipids is only 1.96%. This is far lower than the 10-50% range of DOTAP that has been used for organ tropism, which we previously showed induced clotting (PMID: 32251383,38394670). Indeed, we show' in FIG. 11A-D that this low' level of DOTAP does not induce accidental lung tropism or clotting. These cores then are immediately mixed with the standard LNP lipids, which form a “shell” around the “core.” Thus, we call these “core-then-shell” (CTS) DNA-LNPs.

[0347] This initial version of CTS produces LNPs that are somew'hat bigger than standard DNA-LNPs, but the poly dispersity index (PDI) is also lower. The zeta potential of the LNPs measured in bulk (a population measure) on a Malvern ZetaSizer shows that CTS-LNPs are less negatively charged than the standard DNA-LNPs (made via standard tw'O-channel mixing, shown in FIG. 8C), but the CTS-LNPs are still -5 mV. This negative zeta potential is important given the known toxicities of positively charged LNPs, which was theoretically possible here, given the presence of a small amount of a cationic DOTAP in the LNPs.

[0348] Next, we wanted to compare the nucleic-acid-loading properties of CTS-LNPs made via MSM, as compared to standard LNPs. This is particularly important in light of multiple recent studies show ing that within a standard LNP population, only a small fraction of the individual particles are actually loaded with detectable rnRNA, while the rest are "empty" (PMID: 38529613, 36151112).

[0349] Further, we recently showed that empty LNPs are much more inflammatory than LNPs containing nucleic acids (PMID: 38659905). To compare to those recent studies, we loaded fluorescently-labeled DNA, and traced individual nanoparticles via nanoparticle tracking analysis (NT A) on a Malvern Panalytical NanoSight. The NanoSight can trace all nanoparticles via light scattering (FIG. 10D, right panel), and detect whether each LNP contains detectable DNA via the fluorescent signal FIG. 10D, left panel). Comparing the distribution of standard LNPs (FIG. 10E) vs CTS-LNPs (FIG. 10F), it is evident that a higher fraction of LNPs have detectable DNA in them when formed via the CTS process than via standard LNP synthesis. This is quantified in FIG. 10G. which shows that 83% of CTS-LNPs have detectable DNA, while only 53% of standard LNPs do. We observed a similar trend in CTS-LNPs loaded with rnRNA (FIG. 10I-L). The quantification results show that more than 95% of CTS LNPs contain detectable rnRNA, while only 5% of standard LNP do. Of course, this loading fraction depends on the sensitivity of the assay (here NT A) and the particular lipid composition and the cargo. However, this data shows that at least for some LNP formulations, CTS provides nucleic acid loading that is much more uniform across the population of particles.

[0350] Example 5: Structural analysis reveals that core-then-shell (CTS) DNA LNPs possess a true core and shell that is highly distinct from traditional LNPs

[0351] From Figure 10, it is evident that the CTS protocol generates DNA-LNPs that, compared to standard LNP synthesis, load nucleic acids more uniformly. However, Figure 2’s data had not proven that the CTS protocol actually generated a structure that was distinct from the amorphous LNPs formulated by standard LNP synthesis. Therefore, we performed CryoEM, which revealed distinct morphological differences between standard and CTS-formulated LNPs (FIG. 12A). Standard LNPs displayed a uniform, homogeneous internal structure without clear compartmentalization. In contrast, CTS LNPs exhibited a striking core-shell architecture, characterized by a well-defined central region surrounded by a distinct outer layer. This structural organization suggests that the CTS protocol enables more controlled assembly of LNP components, potentially leading to better-defined spatial arrangement of the nucleic acid pay load and lipid components.

[0352] We then sought to determine whether it was the multi-stage mixing (MSM) that produced the different LNP properties, or whether it was the presence of a small amount of a cationic DOTAP. To test this, we measured zeta potentials of individual LNPs with a Particle Metrix ZetaView. We compared two batches of DNA-LNPs, which both contained the same molar ratio of lipids as each other, including 1.96% DOTAP. In one batch, we used MSM microfluidics as described in FIG. 1 OA. In the other batch, we used traditional, single-step microfluidic mixing (shown in FIG. 8C) that mixes all the lipids from both core and shell phase with DNA. The histogram of zeta potentials for the MSM LNPs had the same single, broad peak as standard LNPs that contained no DOTAP (blue and black curves in FIG. 12B). However, the formulation that used single-step mixing produced two sharp peaks, one at +5mV and one at -5mV. Thus, MSM produces radically different particles than single-step mixing. This proves that LNPs’ key characteristics are not just determined by the LNP lipid ratio, but also are highly dependent on the mixing protocol.

[0353] To further validate that this core-shell architecture represents the predominant structure across the CTS-LNP population, we employed bulk thermal analysis (FIG. 12C). Differential scanning calorimetry (DSC) reveals distinct phase transitions and structural ordering by measuring the energy required to heat samples, providing insights into their molecular organization. The standard LNPs exhibited a single broad, shallow peak centered around 80°C, suggesting a relatively amorphous structure with heterogeneous thermal transitions. In contrast, CTS LNPs displayed two sharp, well- defined endothermic peaks: a major transition at approximately 75°C and a smaller transition at 85°C. The presence of these discrete thermal transitions indicates that CTS LNPs possess a more ordered internal architecture with distinct structural domains, consistent with the core-shell morphology observed in cryo-EM imaging. The dramatically different thermal profiles between the two formulations provide strong evidence that the CTS protocol generates LNPs with fundamentally different structural organization compared to the standard synthesis method.

[0354] Next, we examined CTS-LNPs by another technique that measures physical properties averaged across an ensemble of particles: small angle x-ray scattering (SAXS). SAXS probes internal structure at the nanoscale by measuring how x-rays scatter from electrons in the sample, providing information about particle size, shape, and internal organization without requiring sample perturbation. Importantly, unlike cryo-EM which images individual particles, SAXS measurements represent ensemble averages across millions of particles in solution, offering statistical validation of structural features across the entire population. SAXS analysis provided additional quantitative evidence for the distinct structural organization of CTS LNPs. While both standard and CTS LNPs showed primary Bragg peaks at similar q-values (~0. 115-0. 118 A-1), their scattering profiles revealed fundamentally different internal architectures (FIG. 12D). The standard LNPs exhibited characteristics typical of traditional lipid nanoparticles, with well-defined shape distribution functions yielding a radius of gyration (Rg) of 156 A and maximum particle dimension (Dmax) of 400 A (FIG. 12E, Table 1).

[0355] Table 1

[0356] Table of Parameters Determined by SEC-SAXS Analysis

[0357] n.d. - Not Determined

[0358] ALorentz fitting was only performed on profiles derived with characteristic Braggs peak near q~0.05-0.25 A’1, using the program Origin.

[0359] BAll Inverse Fourier Transform (IFT) analyses were performed using DIFT

[0360] (PMID: 36249494) in conditions where Qmin — 2a / D max. In all experiments, qmin = 0.006 A-1.cThe number of singular values shown were determined using REGALS

[0361] (PMID: 33708400) analysis. Peak boundaries used for these analyses are shown in

[0362] Figure 12.

[0363] Din samples with 3 singular values, the third component w as a buffer artifact extracted from the pool, with no further analysis performed.

[0364] In contrast, CTS LNPs showed a markedly different scattering pattern that could not be analyzed by conventional shape distribution analysis, as evidenced by undeterminable Rg and Dmax values. Instead, their scattering profile required fitting with a core-shell sphere model with poly dispersity term (FIG. 12F), providing strong validation of the compartmentalized structure observed by crvo-EM.

[0365] The distinct structural organization of CTS LNPs was further supported by their intermediate scattering intensity (Ip = 0.46 cm ') compared to standard LNPs (Ip = 6.73 cm '), suggesting a more complex internal arrangement (Table 1 ). While standard LNPs showed characteristics of uniform lipid organization with a single dominant spacing (d = 53.2 A, FIG. 12G), CTS LNPs displayed scattering features consistent with multiple structural domains (FIG. 12H). This SAXS analysis aligns with both the core-shell morphology observed in cryo-EM and the discrete thermal transitions detected by DSC, collectively demonstrating that the CTS protocol generates LNPs with a truly distinct structural organization optimized for DNA delivery.

[0366] Example 6: CTS -LNPs enhance DNA transfection by orders of magnitude

[0367] After confirming the unique structure of CTS LNPs, we wanted to check if CTS- LNPs led to improved transfection, which was the original goal of developing MSM and the CTS design. In RAW macrophages, we compared luciferase expression from plasmid DNA delivered via CTS-LNPs vs standard DNA-LNPs. CTS-LNPs displayed > 1 Ox higher expression than standard LNPs, and levels comparable to mRNA-LNPs (FIG. 14A), and ~3-fold higher in transfection efficiency, quantified as % of cells that are transfected by plasmid encoding GFP (FIG. 14B). In HeLa epithelial cells, CTS-DNA- LNPs displayed ~150-fold higher expression than standard DNA-LNPs (FIG. 14D), and double the transfection efficiency (FIG. 14E). Thus, compared to standard LNP formulations and single-step mixing, using MSM to create CTS-LNPs improves protein expression from DNA cargo by 1-3 orders of magnitude. Throughout this manuscript, the CTS-LNPs are made using dilution cartridges with 3 channels in NanoAssemblr™ Ignite™ nanoparticle formulation systems. CTS-LNPs can also be obtained by an inhouse fabricated microfluidic device (FIG. 13, Table 2).

[0368] Table 2

[0369] Lipid molar ratio and mass ratio of total lipid: DNA for each tested CTS LNP formulations

[0370]

[0371] We have screened a range of CTS-LNP formulations varying the weight weight ratio of shell lipids:pDNA, and have obtained LNP products with comparable size, PDI, DNA entrapment efficiency' and expression, comparing to CTS-LNPs made by NanoAssemblr.

[0372] We had originally hypothesized that the CTS process would primarily work via the benefits of compactification of DNA, and enhancing cytoplasmic trafficking and nuclear entry. However, another possibility is that CTS-LNPs might affect LNPs' endosomal escape, which is ty pically the rate limiter for LNPs' transfection of mRNA. To test this, we applied the same MSM and CTS design on mRNA and tested the protein expression level of CTS-mRNA-LNPs in both RAW and HeLa cells, figuring out that the augmentation of expression of CTS-mRNA-LNPs is 2-4 fold, compared to standard mRNA LNP (FIG. 14C and FIG. 14F). The enhanced mRNA expression indicates that CTS design alleviates the hurdles imposed by endo / lysosomes including inefficient endosomal escape, oligonucleotide sensors and enzymes. However, the expression improvement in the mRNA system is much less than the multiple orders of magnitude improvement for DNA. This could give real mechanistic insight: the bulk of CTS's benefits for DNA-LNPs’ improvement is something that is DNA-specific. This lowers the probability that CTS’s dominant effects on improving pDNA transfection is solely via endosomal escape.

[0373] To quantify the endosome escape event, we transfected HeLa cells with a Gal9- GFP plasmid and monitored LNP -mediated endosomal disruption (FIG. 14G). Under baseline conditions, cells showed diffuse cytoplasmic Gal9-GFP distribution (PMID: 33594247). Upon treatment with different LNP formulations, we observed distinct Gal9- GFP puncta formation, indicating sites of endosomal membrane disruption (FIG. 14H). Quantification revealed that CTS-LNPs induced significantly higher Gal9-GFP puncta formation compared to standard LNPs (FIG. 141). However, this modest enhancement in endosomal escape cannot fully account for the 1-3 orders of magnitude improvement in transfection efficiency observed with the CTS formulation.

[0374] We next examined nuclear delivery kinetics using fluorescently-labeled DNA. Live-cell imaging in HeLa cells revealed that CTS-LNPs achieved nuclear accumulation rates comparable to lipofectamine over a 60-minute period (FIG. 14J). Quantitative analysis show ed nearly identical uptake profiles between both delivery systems (FIG. 14K), suggesting that while CTS-LNPs match the nuclear delivery efficiency of this gold-standard transfection reagent, additional mechanisms beyond endosomal escape and nuclear entry must contribute to its superior transfection capabilities.

[0375] Example 7: Co-loading CTS-DNA-LNPs with STING inhibitors further increases expression and enables in vivo expression peaks comparable to mRNA-LNPs

[0376] Before moving CTS-DNA-LNPs in vivo, it was essential to address whether their DNA cargo activates intracellular DNA-sensors, especially cGAS-STING. Previous w ork has shown that standard DNA-LNPs strongly activate STING in vivo, leading to significant toxicity upon intravenous administration (PMID: 38915627).

[0377] We had initially hypothesized that the core-then-shell structure w ould provide enhanced protection of DNA through compaction within the core, thereby shielding it from both cGAS-STING sensing and DNase degradation after endosomal escape (FIG. 8A). However, our findings presented an interesting paradox: while CTS-LNPs showed improved endosomal escape (FIG. 14G-I) and may provide better DNA protection from DNases (FIG. 8A), these very advantages could potentially increase cytosolic DNA accumulation over time. This raised the possibility that despite DNA compaction, the higher cytosolic DNA levels might actually enhance activation of DNA sensors like cGAS-STING. To test this, we incubated RAW macrophages with CTS-pDNA-LNPs, standard pDNA-LNPs, or lipofectamine with pDNA and measured STING activation using phospho-STING immunostaining. We found that CTS-LNPs indeed activated STING more strongly than standard LNPs, reaching levels comparable to lipofectamine DNA delivery (FIG. 15 A, 15B). Thus, the core-then-shell structure ultimately led to increased STING activation, probably by increasing the integrated amount of DNA exposed to the cytosol over time.

[0378] To mitigate STING activation, we incorporated C-178, a small molecule STING inhibitor that covalently binds to Cys91, into our CTS-DNA-LNPs (FIG. 15C). C-178 was efficiently incorporated during CTS formulation, with the drug entrapment efficiency of 97.5% and drug-to-lipid ratio of 0.025. The loading of C-178 did not affect DNA entrapment efficiency. CTS-LNP loaded with C-178 effectively suppressed STING-mediated immune responses, as evidenced by complete elimination of IFN-0 production in RAW macrophages (FIG. 15D). Importantly, C-178 co-loading enhanced transgene expression by approximately 4-fold in RAW macrophages and 20-fold in HeLa cells, compared to CTS LNP only (FIG. 15E-15F).

[0379] Having solved the STING activation issue, we next evaluated these optimized CTS-DNA-LNPs in challenging primary cell types. In mouse embryonic fibroblasts, C- 178-loaded CTS-DNA-LNPs significantly outperformed lipofectamine in transfection efficiency (FIG. 15G-15H). Similarly, in primary cortical neurons, traditionally considered highly resistant to transfection, our formulation achieved superior gene delivery compared to lipofectamine (FIG. 15I-15J).

[0380] In vivo studies demonstrated that while CTS-DNA-LNPs without C-178 showed enhanced 5-fold higher liver expression compared to standard formulations (FIG. 15K- 15L), they induced significant toxicity leading to mortality (FIG. 15M). Co-loading with C-178 completely eliminated this toxicity while maintaining high transgene expression levels. Most remarkably, CTS-DNA-LNPs co-loaded with C-178 achieved superior expression using only 5 pg of plasmid DNA compared to standard LNPs containing 25 pg of DNA - a 5-fold reduction in dose. This optimized formulation produced peak expression levels approaching those of mRNA-LNPs (FIG. 15N), but with the crucial advantage of sustained expression over several months (FIG. 150). The ability to achieve mRNA-like expression peaks with a reduced DNA dose, while maintaining long-term expression, makes CTS-DNA-LNPs particularly promising for treating chronic diseases requiring sustained protein production.

[0381] Example 8: Discussion

[0382] A long-standing goal in genetic medicine is to control gene expression in the diseases with the greatest burden on society: common chronic diseases. These diseases include the top killers, like atherosclerosis, heart failure, and chronic obstructive pulmonary disease (COPD), as well as the top causes of illness and disability, such as Alzheimers, chronic pain, and chronic kidney disease. (PMID: 38675196) Unfortunately, genetic medicine tools have thus far been best suited to either rare monogenic diseases (AAV and CRISPR) or vaccines (mRNA-LNPs). By contrast, DNA-LNPs offer unique advantages that make them particularly well-suited for treating common chronic diseases: they can express or knockdown (by expressing short hairpin RNA [shRNA]) even large cargo genes (unlike AAV) because there is no major cargo capacity constraint (unlike AAV’s rigid capsid); maintain expression for ~6 months per dose (unlike the hours of expression of mRNA-LNPs): achieve genetically-encoded cell-type specificity with inclusion of large promoter regions (also not possible with mRNA-LNPs); avoid permanent integrate or disrupt the genome (unlike AAV and CRISPR); minimize immunogenicity (unlike AAV); and leverage existing mRNA-LNP targeting technologies. Thus, DNA-LNPs can, with continued development, fill the greatest gap in genetic medicine, opening up therapy for dozens of common chronic diseases.

[0383] However, two major problems have historically limited DNA-LNPs' clinical translation. First, DNA-LNPs were noted for decades to be extremely toxic (PMID: 38915627). Second, standard DNA-LNPs express proteins orders of magnitude lower than mRNA-LNPs and AAV. In this current study, we remedy this poor expression by the invention of multi-stage mixing (MSM) to create core-then-shell (CTS) DNA-LNPs that express at 2-3 logs higher levels than standard DNA-LNPs. With co-loading of STING inhibitor into CTS DNA-LNPs. we demonstrated improvements in transfection in multiple cell lines, multiple primary cell types considered to be “difficult-to-transfect”, and in vivo in mice. Thus, CTS DNA-LNPs now express at levels comparable to the gold-standard mRNA-LNPs, but without mRNA's limitations of a half-life of hours and lack of promoter regions.

[0384] Importantly, the CTS LNPs developed here represent a fundamentally different approach from traditional core-shell nanoparticles created through layer-by-layer (LbL) techniques. While LbL methods typically involve sequential deposition of oppositely charged materials onto a preformed core (PMID: 39086513). our multi-stage mixing (MSM) approach employs a sophisticated microfluidic design that achieves two critical innovations simultaneously: (1) controlled DNA compactification using precisely titrated DNA-condensing agents that facilitate nuclear entry, and (2) rapid capture of thermodynamically metastable core matrices by lipid shells before aggregation can occur. The microfluidic architecture is crucial to this process. Traditional LbL requires multiple separation and purification steps between layer additions, which can lead to material loss and batch-to-batch variability. (PMID: 39086513) In contrast, our MSM system achieves precise spatiotemporal control over the mixing process in a continuous flow format. The first mixing stage creates DNA-lipid complexes with moderate compactification - critically maintaining a N / P ratio below 2.0 to avoid overcondensation that would inhibit nuclear entry (PMID: 26066769). The second stage rapidly envelopes these nascent cores with a lipid shell before they can grow beyond the optimal size range, effectively "freezing" the thermodynamically metastable state of the core. This process occurs in milliseconds, preventing the formation of large aggregates (>2000 nm) that typically plague attempts to create DNA-lipid cores through bulk mixing methods.

[0385] Our MSM approach offers distinct advantages over conventional core-shell particle synthesis through its precise control over the degree of DNA compactification, allowing optimization for nuclear transport. The system enables capture of metastable intermediates that would be impossible to achieve through equilibrium processes, while maintaining continuous flow production that enhances reproducibility and scalability. Compared to multi-step LbL processes, our method minimizes material loss and prevents aggregation through rapid shell formation. These capabilities stem directly from the careful optimization of microfluidic design parameters, including channel geometries, flow rates, and mixing ratios, which work in concert to achieve the delicate balance between DNA compactification and structural stability. This represents a significant advance in nanoparticle engineering, demonstrating how precise control over assembly kinetics through microfluidic design can access otherwise unattainable particle architectures.

[0386] While we have now shown the CTS structure massively improved transfection, there is great need for future papers deeply exploring the mechanism of CTS’s improved efficacy. We were guided in our design of CTS by the hypothesis that compactifying DNA into a core w ould protect it from intracellular DNases, and enable it to pass more easily through the viscous cytosol and the narrow nuclear pore complex (FIG. 8A). However, proving whether or not these are indeed the mechanisms of CTS’s improved efficacy will require many future studies. Thus far, we have a few glimpses at the mechanisms. First, we found that it is essential to maintain DNA in the B-form, by optimizing the buffer. Second, we found that CTS improves mRNA-LNPs’ transfection, but only by ~2-fold. which is much less than ~ 1,000-fold improvement by CTS of DNA- LNP’s transfection. Third, we found that CTS improves endosomal escape (measured by Gal-9-GFP puncta), but only by <2-fold. Fourth, we found that the time course of nuclear translocation of DNA is similar for CTS-DNA-LNPs as it is for lipofectamine. These four observations allow us to roughly estimate the relative contributions of different mechanisms to CTS’s improved transfection: maintenance of the B-form plays a moderate role; improved endosomal escape plays a modest role (based on Gal-9 and mRNA expression data); and CTS allow s rapid translocation to the nucleus.

[0387] While these tentative conclusions on mechanism are consistent with our initial hypothesis, we need more definitive mechanistic studies in the future. Such studies could include comparing CTS vs standard DNA LNP in the following ways: measuring degradation of cargo-DNA after it enters the cell, including whether the DNA is partially damaged (nicked, linearized, or its conformational changes); track the movement of cargo-DNA through the cytosol and nuclear pore complex, especially after plasma membrane permeabilization (to eliminate the effect on endosomal escape). Such mechanistic studies will not only elucidate key science in genetic medicine, but also guide further engineering of MSM and CTS-LNPs.

[0388] Indeed, MSM and CTS-LNPs may open up a vast design space of materials for genetic medicine. Customized design MSM may allow for multiple shell layers. Within just the CTS architecture, there is tremendous room for optimization, and may allow for loading into LNPs of chemical species that would normally partition out of LNPs. For example, we can change out the molecules used to condense the DNA and vary the microfluidic design to change the size and material properties of the LNPs. Finally, there is exciting work to be done to scale-up the manufacturing of MSM and CTS, which may be done with highly parallelized microfluidics (PMID: 34189917, 37556502), or perhaps with macro-scale mixers like the confined impinging jet mixers used for the COVID vaccines (PMID 39248526).

[0389] With continued innovation of MSM and CTS-LNPs, it is possible to foresee a time when DNA-LNPs achieve their promise of safe, long-term control of expression of any protein. Such DNA-LNPs would be able to treat a vast number of common chronic diseases. All of this can be made possible with materials science and engineering.

[0390] Example 9:

[0391] To control the supramolecular structure (spatial arrangement of molecules) of LNPs using MSM, in this study we start with the simplest possible arrangement of molecules within an LNP: two layers, with each layer composed of different molecular species. As LNPs are spheres, a two- layer arrangement equates to a “core” and a “shell.” Thus, the simplest MSM protocol is a synthesis that forms a core and then a shell (“core- then-shell” synthesis [CTS]). In CTS synthesis, in Mixing Stage 1, nucleic acid (in aqueous solution) is mixed with a cationic species, leading to electrostatic condensation to form a “core” (Fig. 16A). In our first example of CTS, the cationic species is the lipid DOTAP, and we additionally add a common LNP component, the zwitterionic lipid DOPE. We have subsequently accomplished CTS synthesis with many different cationic species, including ionizable cationic lipids like those typical of LNPs. Notably, these cores are not thermodynamically stable - they rapidly grow to sizes exceeding 2000 nm, making them unsuitable for both LNP incorporation and in vivo administration. To overcome this, in Mixing Stage 2. our CTS synthesis encapsulates these nascent cores with standard LNP lipids before they can expand, forming a stabilizing "shell" that arrests core growth. Based on lipoplex studies showing that an N:P of DOTAP:DNA ratios above 2 impede nuclear delivery of DNA, we selected a DOTAP:DNA N:P of 0.63

[0017] , Notably, this leaves DOTAP at a final molar % of total lipids of only 1.96%. This is far below the 10-50% range used for DOTAP LNPs with lung tropism, which we previously showed induced thrombosis [18, 19], Indeed, we show in Fig. 20 that this low level of DOTAP, buried in the core, does not induce accidental lung tropism or clotting.

[0392] Cryo-electron microscopy confirmed successful formulation of the hypothesized core-shell architecture (Fig. 16B). While standard LNPs exhibited a characteristic amorphous structure (Fig. 21 A), CTS LNPs displayed a distinctive core-shell architecture, marked by a well-defined central region surrounded by two distinct outer shells. This shows that MSM / CTS enable control of the supramolecular structure of LNPs.

[0393] These CTS LNPs can be manufactured using either commercial dilution cartridges with 3 channels in the NanoAssemblr™ Ignite™ machine, or our in-house fabricated microfluidic devices, which are orders of magnitude cheaper than the singleuse Ignite cartridges (Fig. 16C). For the studies presented in this manuscript, we primarily used the cartridge-based system, which produced CTS LNPs that are larger than standard DNA-LNPs but show a lower poly dispersity index (PDI) (Fig. 16D), indicating more uniform size distribution. While the ^-potential measured in bulk shows these CTS LNPs are less negatively charged than standard DNA-LNPs, they still maintain a slight negative charge of -5 mV (Fig. 16E). This is important given known toxicities of positively charged LNPs. The DNA entrapment efficiency remains high at -90% for both standard and CTS LNPs (Fig. 16F). Notably, our in-house microfluidic devices (Fig. 22, Table 2) produced CTS LNPs with comparable characteristics across all these parameters when screened across a range of shell lipid:DNA weight ratios, demonstrating that the multi-stage mixing (MSM) process is the key enabling factor for successful CTS LNP formation.

[0394] Having established the basic physical characteristics of CTS LNPs, we next examined the uniformity of nucleic acid loading across the particle population. Recent studies have shown that within a standard LNP population, only a small fraction of the individual particles is actually loaded with detectable mRNA, while the rest are "empty" (undetectable mRNA) [20, 21], We recently showed that empty LNPs are much more inflammatory than LNPs containing nucleic acids

[0010] ,

[0395] To assess loading uniformity at the single-particle level, we prepared LNPs with fluorescently labeled DNA and analyzed them via nanoparticle tracking analysis (NT A) on a Malvern Panalytical NanoSight. The NanoSight can trace all nanoparticles via light scattering (Fig. 16G, right panel), and detect whether each LNP contains detectable DNA via the fluorescent signal (Fig. 16G, left panel). Comparing standard LNPs (Fig. 16H) with CTS LNPs (Fig. 161), it is evident that a higher fraction of CTS LNPs have detectable DNA in them. This is quantified in Fig. 16J, which show s that 83% of CTS LNPs have detectable DNA, while only 53% of standard LNPs do. We observed a similar trend in CTS LNPs loaded with mRNA (Fig. 16K-N). The quantification results show that more than 95% of CTS LNPs contain detectable mRNA, while only 5% of standard LNPs do. This loading fraction depends on the RNA / DNA detection threshold of the assay and is likely sensitive to lipid composition and cargo identity. However, this data shows that at least for some LNP formulations, CTS provides nucleic acid loading that is more uniform across the population of particles.

[0396] Structural analysis reveals that core-then-shell (CTS) DNA-LNPs possess a true core and shell that is highly distinct from traditional LNPs

[0397] To directly show that the CTS protocol generates LNP structures that are distinct from standard LNPs, we characterized LNPs with single particle zeta potential measurements, differential scanning microcalorimetry (DSC), small angle X-ray scattering (SAXS), and multi angle light scattering (MALS). We measured particle a- potentials at the single-molecule level using an NTA device that performs microelectrophoresis. We compared two compositionally identical formulations of DNA- LNPs containing 1.96% DOTAP, prepared using different mixing methods: one with MSM microfluidics, as described in Fig. 16A, and the other using traditional single-step microfluidic mixing (shown in Fig. 21A). The o-potential distribution for MSM LNPs showed a broad range of moderate to large potentials like that of standard LNPs without DOTAP, ranging from 0 to -50 mV (blue and black curves in Fig. 17A). However, the DOTAP formulation produced with single-step mixing had two sharp peaks, one at +5mV and one at -5mV. Thus, MSM produces particles with significantly different surface charge properties compared to single-step mixing.

[0398] These findings indicate that physicochemical properties, and thus supramolecular arrangement of LNPs' molecules, are influenced not only by lipid composition but also by the mixing protocol. To further investigate the lipid organization and physical stability of CTS formulations, we performed a thermal analysis using differential scanning calorimetry (DSC) (Fig. 17B). This technique can identify phase transitions and structural ordering by measuring the energy required to heat samples, providing insights into molecular organization. Thermograms of standard LNPs displayed a single, broad, shallow exothermic peak centered around 80°C, suggesting a relatively amorphous structure with heterogeneous thermal transitions. In contrast, CTS LNPs yield two sharp, well-defined exothermic peaks: a major transition at approximately ~75°C and a minor transition at ~85°C. The presence of these distinct thermal transitions indicates that CTS LNPs possess a more ordered internal architecture with defined structural domains. The contrasting thermal profiles between the two formulations strongly suggest that the CTS protocol produces LNPs with a fundamentally different internal organization (supramolecular structure) compared to the standard formulation method.

[0399] We next applied different modalities of light scattering to quantitate the size and mass of these particles in solution. Using dynamic light scattering (DLS), we observe that CTS particles are almost two-fold larger in hydrodynamic radius (Rh) in solution when compared to the standard LNP formulation, with a lower poly dispersity index (PDI) (Fig. 17C). CTS particles had a determined Rh of 151 A ± 2.3 (PDI = 0.14) compared to 80.2 A ± 2.5 (PDI = 0.28) for the standard formulation. Consistent with these differences in size, the particles had very different retention times and weight-average mass profiles (Mw), as determined by size-exclusion chromatography in-line with multiangle light scattering (SEC-MALS) (Fig. 17D&E). CTS particles had a Mw = 23.7 MDa± 2.1% across the sizing peak, whereas standard particles were almost five-fold smaller in mass (Mw = 5.4 MDa ± 3. 1%). Owing to the larger size of the CTS particles, we were also able to determine the radius of gyration (Rg) for the CTS particles (38 nm ± 5.5%).

[0400] We next employed synchrotron small-angle X-ray scattering (SAXS) to further investigate the size and shape, and to better interrogate the internal structure of these particles. We employed in-line size-exclusion chromatography (SEC) to minimize and measure the possible effects of sample poly dispersity in our analyses by performing singular value decomposition (SVD) analysis (see Methods and Fig. 25). In this approach, very’ different scattering profiles were obtained for the CTS and standard particles (Fig. 17F), including well-defined primary' Bragg peaks at q-values between ~0. l-0.25 A-l, indicative of ahighly ordered internal structure (Fig. 17G&H). In this experimental configuration (where qmin*Dmax:-<j it), it yvas possible to determine the size and shape of the standard LNP using the inverse Fourier transform (Rg of 156 A, Dmaxof 400 A), and to calculate ab initio electron density at loyv resolution (Table 1 and Fig. 25). It rvas not possible to apply the same conventional approaches to the CTS particles owing to their considerably larger sizes. However, empirical models for core-shell spheres and ellipsoids could be readily fit to the experimental data through the low and middle-q regime with a poly dispersity' term included, using calculated scattering length densities (SLDs) for the lipid and DNA components in X-rays and a fixed lipid bilayer depth of 54 A. In the fitting, the resulting particle radii determined are largely consistent yvith the other measures reported herein (Fig. 171).

[0401] As seen in Fig. 17G&H. both samples show overlapping but distinguishable peaks. For the CTS particles, these are the peaks at q=0.10 A-1and 0.12 A-1, while for the standard particles these are the peaks at q=0.115 A-1and 0.125 A-1. Using the relationship d=2n / q. where d is the distance between the repeat lipid-DNA structures, the peaks indicated organized structure at d = 59.8 A and 52.3 A respectively for the CTS particle and 54.6 A and 52.3 A for the standard particles respectively (Table 1). The pattern of the Braggs peak in this data provides direct information on the structural arrangement through the reciprocal spacings between peaks. Inverse hexagonal phases (hexosomes) have peaks spaced in the pattern of l, 3, 4.. and so on, while bicontinuous particles (cubosomes) have different integral spacings [22, 23] ( l, 2, 3..). In the CTS data, the q=0.10 A-l primary peak is readily assigned to a l, ^2, ^3 spacing, indicative of cubosome phases (C), with corresponding peaks at q=0.15 A-l and 0.18 A-l. The overlapping primary peak at q=0.12 A-l is readily assigned to corresponding peaks spaced in the pattern of l, 2, x / 3, at q=0.18 A-l and 0.22 A-l. Several additional weak peak features could not be assigned. In addition to primary' peaks, a broader and less well-defined shoulder in the profile at q=0.06-0.09 A-l is observed which can be assigned to a higher order and more disordered phase. In contrast to the CTS structure, the standard LNP is better assigned with peak integrals consistent with an inverse hexagonal phase (H||), with the q=0. 115 A-l peak readily assigned corresponding peaks at q=0.20 A-1and 0.23 A-1. The overlapping peak at 0.125 A-l could be assigned as an additional cubosome phase with corresponding peaks at q=0.18 A-1and 0.22 A-1, and like with the CTS particle, a region of disorder between q=0.05- 0.1 A-l could also be assigned. These X-ray analyses indicate that the CTS particles feature a cubosome / cubic structure very distinct from the canonical inverse hexagonal phases that define the standard LNPs.

[0402] CTS LNPs enhance DNA transfection by orders of magnitude

[0403] DNA-LNPs face five major “hurdles” to achieving high protein expression (Fig. 18 A): 1) DNA structural perturbations during LNP synthesis, as conditions optimized for mRNA may destabilize DNA's structure; 2) Upon cell uptake and endosomal escape, intracellular DNA sensors such as cGAS-STING repress protein translation; 3) Cytosolic DNases rapidly degrade DNA outside the nucleus; 4) DNA must navigate through the pores of the cytosolic gel; and 5) DNA must traverse the nuclear pore complex, which restricts passage of cargo >39 nm. We hypothesized that we could address the first hurdle by optimizing synthesis conditions to preserve DNA structure. We further hypothesized that we could address the next four hurdles by compactifying DNA into small spheres (“cores”), which would protect the DNA from DNA sensors and DNases (hurdles 2 & 3) and enable easier transit of DNA through the narrow passages of the cytosolic gel and nuclear pore complex (hurdles 4 & 5). More specifically, we hypothesized that CTS could produce DNA cores that possessed such properties.

[0404] We began by focusing on hurdle #1 above. The standard synthesis conditions of LNPs may alter DNA's structure in deleterious ways. In mRNA-LNP synthesis, the nucleic acid is dissolved in pH 4 citrate buffer to maximize protonation of ionizable lipids [2], However, this low pH may destabilize the DNA helix and cause conformational changes from the biologically active B-form, the most common biologically active structure present in cells under normal physiological conditions. Other conformations can include A-form, Z-form, G-quadruplex, and others, as well as less defined conformations that might occur after partial acid hydrolysis of the DNA

[0024] .

[0405] To assess the effect of buffer and pH on DNA conformations, we analyzed DNA in different buffers by circular dichroism (CD) spectroscopy, which measures B-form DNA via relative magnitude of the ellipticity signal in the range of 200 - 320 nm. A typical B-form DNA has a positive maximum around 290 nm, a negative maximum around 245 nm and a broad positive peak between 260 - 280 nm. We compared pH 4 citrate (classic mRNA-LNP synthesis buffer) vs pH 6 TBS with 4 mM Mg2+. Tris is the most common buffer used in DNA reactions, and Mg2+ stabilizes the B-form DNA helix via a variety of mechanisms, mostly involving electrostatic interaction between Mg2+ and DNA’s phosphates

[0025] , As shown in Fig. 23 A, the CD spectroscopic signal for B- form DNA is significantly disrupted by citrate buffer vs TBS buffer.

[0406] Titrating Mg2+ in the TBS buffer, we found that 4 mM Mg2+ slightly improves B-form DNA signal in CD spectroscopy, compared to 0 and 12.5 mM Mg2+ (Fig. 23B). Having established the impact of buffer conditions on DNA conformation, we next evaluated how these conditions affect DNA-LNPs' transfection efficiency. We synthesized luciferase-encoding DNA-LNPs in different buffers, dialyze against buffers containing corresponding Mg2+ as in their DNA dissolving buffer, and tested their performance in RAW 264.7 macrophages and HeLa cells (Fig. 24). The Tris-based buffers (TAE) at pH 5 outperformed citrate buffer (pH 4). Critically, we discovered that Mg2+ in the dialysis buffer was essential for expression, likely due to its role in stabilizing DNA structure - though notably, this benefit was only observed at pH > 4, as Mg2+ showed no positive effects in acidic conditions (Fig. 25), possibly due to weakened Mg2+-DNA interactions in proton-rich environments

[0026] , A comprehensive screen of buffer conditions across both cell types revealed that Tris-based buffers at pH 6 with Mg2+ consistently produced the highest expression levels, with TBS showing particularly strong performance in macrophages and TAE in HeLa cells. Cryo-EM analysis confirmed that these buffer-optimized LNPs (TBS, pH 6, Mg2+) maintained similar morphology to standard LNPs while achieving significantly higher expression levels (Fig. 21 C).

[0407] With optimized buffer conditions maintaining DNA structure during synthesis, we next investigated how CTS architecture could overcome the remaining intracellular hurdles, all of which were hypothesized to benefit from the DNA compactification of CTS. We compared luciferase expression from plasmid DNA delivered via CTS LNPs vs standard DNA-LNPs. CTS LNPs yield >10x higher expression than standard LNPs in RAW macrophages, and ~150-fold higher in HeLa cells, with the levels comparable to mRNA-LNPs (Fig 18B-C). Comparing transfection efficiency, quantified as % of cells that are transfected by plasmid encoding GFP, CTS DNA-LNPs outperform standard LNPs by three-fold in RAW macrophages and two-fold in HeLa cells (Fig 18D-E). Thus, compared to standard LNP formulations and single-step mixing, using MSM to create CTS LNPs improves protein expression from DNA cargo by 1-3 orders of magnitude.

[0408] We had originally hypothesized that the CTS process would primarily work via benefits of compact DNA packaging, enhancing cytoplasmic trafficking and nuclear entry’. However, another possibility is that CTS LNPs might affect LNPs' endosomal escape, which is typically the rate limiter for LNPs' transfection of mRNA

[0027] , To test this, we applied the same MSM CTS design for mRNA delivery and tested the protein expression level of CTS-mRNA-LNPs in both RAW and HeLa cells, figuring out that the augmentation of expression of CTS-mRNA-LNPs is 2-4 fold, compared to standard mRNA-LNP (Fig. 18F-G). The enhanced mRNA expression indicates that CTS design alleviates the hurdles imposed by endo / lysosomes including inefficient endosomal escape. However, the expression improvement in the mRNA system is much less than the multiple orders of magnitude improvement for DNA. This could give real mechanistic insight: the bulk of CTS's benefits for DNA delivery’ is DNA-specific. This lowers the probability that CTS predominantly improves DNA transfection via effects on endosomal escape.

[0409] To quantify endosomal escape events, we transfected HeLa cells with a galectin (Gal)-9-GFP plasmid and monitored LNP -mediated endosomal disruption (Fig. 18H). Galectins are primarily expressed in the cytosol and can be recruited to endosomes when membrane are damaged. Under baseline conditions, cells showed diffuse cytoplasmic Gal-9-GFP distribution

[0028] , Upon treatment with different LNP formulations, we observed distinct Gal-9-GFP puncta formation, indicating sites of endosomal membrane disruption (Fig. 181). Quantification revealed that CTS LNPs induced higher Gal-9-GFP puncta formation than standard LNPs (Fig. 18J). How ever, this modest (though statistically significant) enhancement in endosomal escape cannot fully account for the 1- 3 orders of magnitude improvement in transfection observed with CTS LNPs.

[0410] We next examined nuclear delivery kinetics using fluorescently labeled DNA. Live-cell imaging in HeLa cells revealed that CTS LNPs achieved nuclear accumulation rates comparable to Lipofectamine over a 60-minute period (Fig. 18K). Quantitative analysis showed nearly identical uptake profiles for both delivery’ systems (Fig. 18L), suggesting that while CTS LNPs match the nuclear delivery efficiency of this gold- standard transfection reagent, additional mechanisms beyond endosomal escape and nuclear entry' must contribute to its superior transfection capabilities.

[0411] Co-loading CTS DNA-LNPs with STING inhibitors further increases expression and enables in vivo expression peaks comparable to mRNA-LNPs

[0412] Before moving CTS DNA-LNPs in vivo, it was essential to address whether their DNA cargo activates intracellular DNA-sensors, especially cGAS-STING. Our previous work has shown that standard DNA-LNPs strongly activate STING in vivo, leading to significant toxicity upon intravenous administration

[0013] , and represses translation

[0029] ,

[0413] We had initially hypothesized that the CTS structure would provide enhanced protection of DNA through compaction in the core structure, shielding it from both cGAS-STING sensing and DNase degradation after endosomal escape (Fig. 18 A). However, our findings indicate CTS LNPs’ improved endosomal escape (Fig. 18H-J) may actually increase cytosolic DNA accumulation over time, enhancing activation of DNA sensors like cGAS-STING. To test this, we incubated RAW macrophages with CTS DNA-LNPs, standard DNA-LNPs, or Lipofectamine with DNA and measured STING activation by immunostaining of phosphorylated STING

[0030] , We found that CTS LNPs indeed activated STING more strongly than standard LNPs, reaching levels comparable to Lipofectamine-based DNA delivery (Fig. 19A-B).

[0414] To mitigate STING activation, we loaded our CTS DNA-LNPs with C-178, a small molecule STING inhibitor that covalently binds to Cys91, which is the same mechanism employed by mammal’s endogenous STING inhibitors, nitrated lipids (Fig. 19C)

[0031] , C-178 was efficiently incorporated during CTS LNP synthesis, with a drug entrapment efficiency of 97.5% and drug-to-lipid molar ratio of 0.025. The loading of C- 178 did not affect DNA entrapment efficiency. CTS LNPs loaded with C-178 effectively suppressed STING-mediated immune responses, as evidenced by complete elimination of IFN-p production in RAW macrophages (Fig. 19D). Importantly, C-178 co-loading enhanced transgene expression by approximately 4-fold in RAW macrophages and 20- fold in HeLa cells, compared to CTS LNP only (Fig. 19E-F).

[0415] Having solved the STING activation issue, we next evaluated STING-inhibiting C-178-loaded CTS DNA-LNPs in challenging primary cell types. In mouse embryonic fibroblasts, C-178-loaded CTS DNA-LNPs significantly outperformed Lipofectamine in transfection efficiency (Fig. 19G-H). Similarly, in primary cortical neurons, traditionally considered highly resistant to transfection, our formulation achieved superior gene delivery compared to Lipofectamine and preserved neuron morphology (Fig. 191-J). In vivo studies demonstrate that CTS DNA-LNPs without C-178 enhance liver luciferase expression 5-fold over standard DNA-LNPs (Fig. 19K-L), but also induce significant toxicity leading to mortality (Fig. 19M). Loading CTS LNPs with C-178 eliminates this toxicity while maintaining high transgene expression. A 5 ug dose of plasmid DNA in CTS DNA-LNPs loaded with C-178 achieved orders of magnitude superior expression to a 25 ug dose in standard LNPs - despite the 5-fold reduction in dose. C-178-loaded CTS DNA-LNPs produced peak expression levels approaching those of mRNA-LNPs (Fig. 19N), but with the crucial advantage of sustained expression (Fig. 190). The ability to achieve mRNA-like expression peaks with a reduced DNA dose, while maintaining long-term expression, makes CTS DNA-LNPs very promising for treating chronic diseases.

[0416] DISCUSSION:

[0417] These studies show that LNPs’ supramolecular structure (spatial arrangement of molecules) has a major effect on their performance. Further, we show that supramolecular structure can be controlled not just by the constituent molecules’ identity, but also by the methodology of mixing. In particular, multi-stage mixing (MSM) allows for control of supramolecular structure, even enabling capture of favorable metastable states, while maintaining continuous flow production. MSM's simplest protocol, a core-then-shell (CTS) two- layer synthesis, improved numerous LNP performance metrics, including: lower poly dispersity index (PDI) (Fig. 16E); a much lower fraction of “empty” LNPs (undetectably low concentration of nucleic acid cargo) (Fig. 16N), which we previously showed are inflammatory; prevention of side effects (e.g., clotting) by burying particular lipids in the core (Fig. 20); improved protein expression by mRNA-LNPs (Fig. 18F-G); and most impressively, improvement in DNA- LNPs’ protein expression by 2-3 orders, bringing it to levels similar to the peak of mRNA-LNPs (Figs. 18 & 19).

[0418] This enormous improvement in DNA-LNPs’ expression may now allow DNA- LNPs to sen e a long-standing goal in genetic medicine: common chronic diseases. These diseases include the top causes of death like atherosclerosis, heart failure, and chronic obstructive pulmonary disease (COPD), as well as the top causes of illness and disability, such as Alzheimer’s disease, chronic pain, and chronic kidney disease

[0032] , Unfortunately, genetic medicine tools have thus far been best suited to either rare monogenic diseases (AAV and CRISPR) or vaccines (mRNA-LNPs). By contrast, DNA- LNPs offer unique advantages that make them particularly well-suited for treating common chronic diseases: they can express or knockdown (by expressing short hairpin RNA [shRNA]) even large cargo genes because they impose no major cargo capacity constraint (unlike AAV's rigid capsid)

[0033] ; maintain expression for ~6 months per dose (unlike the hours of expression with mRNA-LNPs)

[0032] ; achieve genetically-encoded cell-type specificity with inclusion of large promoter regions (not possible with mRNA- LNPs); avoid permanent integration or disruption of the genome (unlike AAV and CRISPR)

[0034] ; minimize immunogenicity (unlike AAV)

[0034] ; and leverage existing mRNA-LNP targeting technologies. Thus, DNA-LNPs can, with continued development, fill the greatest gap in genetic medicine, opening up therapy for dozens of common chronic diseases.

[0419] Importantly, the CTS LNPs developed here represent a fundamentally different approach from traditional core-shell nanoparticles created through layer-by-layer (LbL) techniques. While LbL methods typically involve sequential deposition of oppositely charged materials onto a preformed core [9], our multi-stage mixing (MSM) approach employs a sophisticated microfluidic design that achieves two critical innovations simultaneously: (1) controlled compact packaging of DNA using precisely titrated DNA- condensing agents that facilitate nuclear entry; (2) rapid capture of thermodynamically metastable core matrices by lipid shells before aggregation can occur. The microfluidic architecture is crucial to this process. Traditional LbL requires multiple separation and purification steps between layer additions, which can lead to material loss and batch-to- batch variability [9], In contrast, our MSM system achieves precise spatiotemporal control over the mixing process in a continuous flow format. The first mixing stage creates DNA-lipid complexes with moderate compacting - critically maintaining an N:P ratio below 2 to avoid over-condensation that would inhibit nuclear entry

[0017] , The second stage rapidly envelopes these nascent cores with a lipid shell before they can grow beyond the optimal size range. This process occurs in milliseconds, preventing the formation of large aggregates (>2000 nm) that typically plague attempts to create DNA- lipid cores through bulk mixing methods.

[0420] Our MSM approach offers distinct advantages over conventional core-shell particle synthesis through its precise control over the stoichiometry’ of the compact lipoplex DNA core, allowing optimization for nuclear transport. The system enables capture of metatable intermediates that would be impossible to achieve through equilibrium processes, while maintaining continuous flow production that enhances reproducibility and scalability. Compared to multi-step LbL processes, our method minimizes material loss and prevents aggregation through rapid shell formation. While we have shown the CTS structure massively improves transfection, there is great need for future work deeply exploring the mechanisms of CTS’s improved efficacy. We were guided in our design of CTS by the hypothesis that compactifying DNA into a core would protect it from intracellular DNases, and enable it to pass more easily through the viscous cytosol and the narrow nuclear pore complex (Fig. 18A). However, proving whether or not these are indeed the mechanisms of CTS’s improved efficacy will require additional studies. Thus far, we have a few glimpses at the mechanisms. First, we found that it is essential to maintain DNA in the B-form, by optimizing the buffers in which DNA is formulated into LNPs and into which LNPs are dialyzed following synthesis. Second, we found that CTS improves mRNA-LNPs’ transfection, but only by ~2-4-fold, which is much less than the ~1, 000-fold improvement by CTS for DNA-LNP’s transfection. Third, we found that CTS improves endosomal escape (measured by Gal-9- GFP puncta), but only by <2-fold. Fourth, we found that the time course of nuclear translocation of DNA is similar for CTS DNA-LNPs and Lipofectamine. These four observations allow us to roughly estimate the relative contributions of different mechanisms to CTS’s improved transfection: maintenance of the B-form plays a moderate role; improved endosomal escape plays a modest role (based on Gal-9 and mRNA expression data); and CTS allows rapid translocation to the nucleus.

[0421] While these tentative conclusions on mechanism are consistent with our initial hypothesis, we need more definitive mechanistic studies in the future. Such studies could include comparing CTS vs standard DNA-LNPs in the following ways: measuring degradation of cargo-DNA after it enters the cell, including whether the DNA is partially damaged (nicked, linearized, or conformational changes); tracking the movement of cargo-DNA through the cytosol and nuclear pore complex, especially after plasma membrane permeabilization (to eliminate the effect on endosomal escape): and modifying CTS to produce larger or smaller cores, and compactify the DNA with different cations or other species, to probe structure-function relationships. Such mechanistic studies will not only elucidate key science in genetic medicine and material science, but also guide further practical engineering of MSM and CTS LNPs.

[0422] Indeed, MSM and CTS LNPs may open up a vast design space of materials for genetic medicine. Customized MSM designs may allow for multiple shell layers. Within just the CTS architecture, there is tremendous room for optimization, possibly allowing loading of chemical species that would normally partition out of LNPs. For example, we can change out the molecules used to condense the DNA and vary the microfluidic design to change the size and material properties of the LNPs. Finally, there is exciting work to be done to scale-up the manufacturing of MSM and CTS, which may be done with highly parallelized microfluidics [35, 36], or perhaps with macro-scale mixers like the confined impinging jet mixers used for the COVID vaccines [7], With continued innovation of MSM and CTS LNPs, it is possible to foresee a time when DNA-LNPs achieve their promise of safe, long-term control of expression of any protein. Such DNA- LNPs would be able to treat a vast number of common chronic diseases. All of this can be made possible by applying novel materials engineering techniques like MSM to control the supramolecular structure of LNPs.

[0423] References Pardi, N. et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J Control Release 217, 345-351 (2015). Zhang, W. et al. The Expression Kinetics and Immunogenicity of Lipid Nanoparticles Delivering Plasmid DNA and mRNA in Mice. Vaccines (Basel) 11, 1580 (2023). Herweijer, H. et al. Time course of gene expression after plasmid DNA gene transfer to the liver. The Journal of Gene Medicine 3, 280-291 (2001). Yu, L. & Liu, P. Cytosolic DNA sensing by cGAS: regulation, function, and human diseases. Sig Transduct Target Ther 6, 1-15 (2021). Chauvin, S. D., Stinson, W. A., Platt, D. J., Poddar, S. & Miner, J. J. Regulation of cGAS and STING signaling during inflammation and infection. J Biol Chem 299. 104866 (2023). Laczko, D. et al. A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity 53, 724-732. e7 (2020). Chase, L. S., Zaleski, M. H., Morrell, L. J. & Brenner, J. S. Automated measurement of distance-walked as a “sixth vital sign” for detecting infusion reactions during preclinical testing. International Journal of Pharmaceutics 645, 123369 (2023). Type I interferons in infectious disease - PMC. https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7162685 / . Parhiz, H. et al. Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE). Journal of Controlled Release 344, 50-61 (2022).. Omo-Lamai, S. et al. Lipid Nanoparticle- Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects. bioRxiv 2024.04. 16.589801 (2024) doi: 10. 1101 / 2024.04. 16.589801. . Omo-Lamai, S. et al. Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions. bioRxiv (2023) doi: 10. 1101 / 2023.07.21.550080. . Cheng, Z. et al. The interactions between cGAS-STING pathway and pathogens. Sig Transduct Target Ther 5, 1-15 (2020). . Domizio. J. D. et al. The cGAS-STING pathway drives ty pe I IFN immunopathology in COVID-19. Nature 603, 145-151 (2022). . Wang, D., Zhao, H., Shen, Y. & Chen. Q. A Variety of Nucleic Acid Species Are Sensed by cGAS, Implications for Its Diverse Functions. Frontiers in Immunology' 13, (2022). . Zhu, Y. et al. Multi-step screening of DNA / lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression. Nat Commun 13, 4282 (2022). . Zhu, Y. et al. Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery. Biomaterials 308, 122559 (2024). . Melo. T., Montero-Bullon, J.-F., Domingues, P. & Domingues, M. R. Discovery of bioactive nitrated lipids and nitro-lipid-protein adducts using mass spectrometry-based approaches. Redox Biol 23. 101106 (2019). . Hansen, A. L. et al. Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling. Proc Natl Acad Sci U S A 115, E7768-E7775 (2018). . Kaminski, J. J. et al. Synthetic Ohgodeoxynucleotides (ODN) Containing Suppressive TTAGGG Motifs Inhibit AIM2 Inflammasome Activation. J Immunol 191,

[0424] 10.4049 / jimmunol. 1300530 (2013). . Chatterjee, S., Kon, E., Sharma, P. & Peer, D. Endosomal escape: A bottleneck for LNP-mediated therapeutics. Proceedings of the National Academy of Sciences 121, e2307800120 (2024). . Bai, H., Lester, G. M. S., Petishnok, L. C. & Dean, D. A. Cytoplasmic transport and nuclear import of plasmid DNA. Biosci Rep 37, BSR20160616 (2017). . Zanta, M. A., Belguise-Valladier, P. & Behr, J. P. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci U S A 96, 91-96 (1999). . Spennde. J. J., Choi. S. J. & Szoka, F. C. Phage display selection of a peptide DNase II inhibitor that enhances gene delivery. J Gene Med 3, 101-108 (2001). . Jacob, A. et al. Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells. Cell Stem Cell 21, 472-488. elO (2017). . Jacob, A. et al. Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells. Nat Protoc 14, 3303-3332 (2019). . Abo, K. M. et al. Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell-derived alveolar epithelium. JCI Insight 7, el55589.

[0425] All documents cited in this specification are incorporated herein by reference. In addition, US Provisional Patent Application Nos 63 / 653,163, 63 / 653,164, and 63 / 718,985 are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for generating lipid nanoparticles (LNP) encapsulating a cargo, the method comprising: mixing a first solution comprising one or more condensing agents with a second solution comprising the cargo, whereby the cargo is condensed, then mixing the condensed cargo with a third solution comprising one or more lipids in an organic solvent, thereby generating LNP having a core containing the cargo with a lipid shell.

2. A method for generating lipid nanoparticles (LNP) encapsulating a cargo, the method comprising: in a nanoparticle formulation system comprising a first channel, a second channel, a third channel, and a fourth channel,(a) providing a first solution comprising one or more condensing agents and an organic solvent or an aqueous buffer in the first channel;(b) providing a second solution comprising the cargo and a second aqueous buffer in a second channel;(c) providing a third solution comprising one or more lipids in a second organic solvent and a fourth solution comprising one or more polymers, proteins, or peptides in a third aqueous buffer, the third solution and fourth solution in the third channel;(d) mixing the first solution and the second solution;(e) introducing the mixture of (d) with the third solution and fourth solution to form the LNP.

3. A method for generating lipid nanoparticles (LNP) encapsulating a cargo, the method comprising: in a nanoparticle formulation system comprising a first channel, a second channel, and a third channel,(a) providing a first solution comprising one or more condensing agents and an organic solvent or an aqueous buffer in the first channel;(b) providing a second solution comprising the cargo and a second aqueous buffer in a second channel;(c) providing a third solution comprising one or more lipid, peptide, protein, or polymer in a second organic solvent in the third channel;(d) mixing the first solution and the second solution;(e) introducing the mixture of (d) with the third solution to form the LNP.

4. The method of any one of claims 1 to 3. wherein the first solution comprises a condensing agent that comprises one or more of a cationic lipid, helper lipid, ionizable lipid, amphiphilic lipid, protein, peptide, or polymer.

5. The method of any one of claims 1 to 4. wherein the first solution comprises a cationic lipid, optionally DOTAP, DOPSA. DOTMA, DMRIE, or N-Tetamine-pLys40. and a helper lipid, optionally DOPE.

6. The method of any one of claims 1 to 5. wherein the cargo comprises DNA, RNA, peptide, protein, protein-DNA complex, peptide-DNA complex. lipid-DNA complex, proteolysis targeting chimera (PROTAC), or small molecule.

7. The method of any one of claims 1 to 6, wherein the third solution comprises cholesterol, helper lipid, ionizable lipid, and / or pegylated lipid.

8. The method of any one of claims 1 to 7, wherein the first organic solvent and second organic solvent are the same.

9. The method of any one of claims 1 to 8. wherein the first, second, and third aqueous buffers are the same.

10. The method of any one of claims 1 to 9. wherein:I l lthe first solution comprises DOTAP at a lipid molar % of 3.6% and DOPE at a lipid molar % of 10.8%; and / or the third solution comprises cholesterol at a lipid molar % of 37.53%, ionizable lipid (e.g.. SM102) at a lipid molar % of 44.65%. DMG-PEG at a lipid molar % ofI.37%, DOPE at a lipid molar % of 6.4%, and DOTAP at a lipid molar % of 1.96% diluted with 100% ethanol.I I. The method of any one of claims 1 to 10, wherein the third solution comprises (a) a cationic lipid in an amount from about 0 to about 80% of total lipid present in the particle; (b) a non-cationic lipid in an amount from about 20% to about 99.9% of the total lipid present in the particle, wherein the non-cationic lipid comprises a mixture of a phospholipid and a cholesterol or derivative thereof; (c) a polyethylene glycol (PEG)- lipid conjugate in an amount from about 0. 1 to about 10% of the total lipid in the particle, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 5000 to about 20000 daltons.

12. The method of any one of claims 1-11, wherein the core or the shell further comprises nitro-oleic acid (NOA).

13. The method of any one of claims 1-12, wherein the flow rate ratio is from 0.1: 100: 100-100:0.1:0.1-100: 100:0.1.

14. The method of any one of claims 1 to 12, wherein the LNPs have a diameter of about 1 lOnm to about 300nmand / or a poly dispersity index (PDI) of about 0.1.

15. The method of any one of claims 1 to 14, wherein the LNPs have a neutral surface charge.

16. A LNP made by the method of any one of claims 1 to 15.

17. A LNP comprising a core and a shell, wherein the core comprises a cargo and a condensing agent, wherein the shell comprises an ionizable lipid, helper lipid, cholesterol, and / or PEG.

18. The LNP of claim 17, comprising more than one shell layer.

19. A method of delivering a cargo to a subject comprising administering the LNP of claim 16 to 18 to the subject.

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