RNA-LNP formulations delivered by HD-map

Abstract

The present invention relates to stable nucleic acid LNP vaccine formulations for administration via a microprojection array in which the microprojections are densely packed and in which the vaccine formulations are rapidly sprayed or layered on to the microprojections in relatively small amounts such that the formulations dry rapidly. The present invention relates in particular to stabilized RNA-LNP vaccines delivered by high density micro-array patches (HD-MAPs).

Description

C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 1 - RNA-LNP FORMULATIONS DELIVERED BY HD-MAP Cross-Reference to Related Applications

[0001] This application claims priority to US Provisional Patent Application No. 63 / 692,790 filed on 10 September 2024 and US Provisional Patent Application No.63 / 716,340 filed on 5 November 2024. Background of the Invention

[0002] The present invention relates to stable nucleic acid LNP vaccine formulations for administration via a microprojection array in which the microprojections are densely packed and in which the vaccine formulations are rapidly sprayed or layered on to the microprojections in relatively small amounts such that the formulations dry rapidly. The present invention relates in particular to stabilized RNA-LNP vaccines delivered by high density micro-array patches (HD-MAPs). Description of the Prior Art

[0003] In recent years, attempts have been made to devise new methods of delivering drugs and other bioactive materials, for vaccination and other purposes, which provide alternatives that are more convenient and / or enhanced in performance to the customary routes of administration such as intramuscular and intradermal injection. Limitations of intramuscular and intradermal injection include: cross-contamination through needle-stick injuries in health workers; injection phobia from a needle and syringe; and most importantly, as a result of its comparatively large scale and method of administration, the needle and syringe cannot target key cells in the outer skin layers. This is a serious limitation to many existing and emerging strategies for the prevention, treatment and monitoring of a range of untreatable diseases. There is also a need to reduce the amount of material delivered due to toxicity of the material or due to the need to conserve the material because it is difficult or expensive to produce.

[0004] In an effort to solve some of the issues referenced above microprojection arrays or microneedle arrays have been utilized to deliver various materials through the skin. Devices which comprise a plurality of projections which can penetrate the skin so as to deliver aC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 2 - bioactive material or stimulus to a predetermined site. The projections can be solid and the delivery end of the projection is designed such that it can be inserted into targeted cells or specific sites in the skin.

[0005] One of the challenges of using devices that contain microneedles and / or microprojections is the need to coat the projections. Rapid spray coating of microprojection / microneedle drug delivery and vaccine platforms allows allocation of the coating to the delivery platform, minimizing the inefficiencies associated with spray coating or dip coating that may overcoat or undercoat the microprojections. Dip coating or spray coating is less accurate than ink jet coating. Designing a stable nucleic acid vaccine that may be distributed on a surface such as a microneedle or microprojection and dried poses challenges. In addition, each component of the vaccine composition affects the viscosity, drop formation, dry time, adhesion and stability of the vaccine. Other challenges to delivering a vaccine via a microprojection / microneedle array include coating the microprojections / microneedles with enough vaccine to be efficacious when administered, formulating a vaccine such that the drop size is sufficiently small to permit penetration into the skin with each projection of the array. There is also a need to provide microprojection / microneedle arrays that enable coating of the microprojections / microneedles with compositions that have components that are incompatible with each other in solution.

[0006] Recently vaccines that contain nucleic acids have been commercialized, in particular the Covid-19 mRNA vaccines. Such vaccines contained mRNA but also utilize lipid nanoparticles (LNPs) as a delivery mechanism. The challenge is to compose a formulation which combines nucleic acids and LNP delivery that can be applied to microprojection or microneedles of a microprojection array such that the formulation remains stable over time and does not aggregate such that the microprojection array can be used to deliver the vaccine formulation to the skin or an animal or human.

[0007] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 3 - Summary of the Present Invention

[0008] In one broad form an aspect of the present invention relates to composition comprising an RNA molecule and a lipid nanoparticle (LNP) and an additive, wherein the RNA molecule is encapsulated in the LNP.

[0009] In one embodiment the present invention relates to compositions where the RNA molecule is a self-amplifying RNA (samRNA).

[0010] In one embodiment the present invention relates to compositions where the RNA molecule is a messenger RNA (mRNA), a small interfering RNA (siRNA), a short hairpin RNA or small hairpin RNA (shRNA), a microRNA (miRNA), a miRNA inhibitor (antagomirs / antimirs), or a messenger-RNA-interfering complementary RNA (micRNA).

[0011] In one embodiment the present invention relates to compositions where the RNA molecule encodes a polypeptide or a fragment thereof.

[0012] In one embodiment the present invention relates to compositions where the RNA molecule encodes a polypeptide antigen.

[0013] In one embodiment the present invention relates to compositions where the RNA molecule encodes a viral polypeptide.

[0014] In one embodiment the present invention relates to compositions where the viral polypeptide is or is derived from a spike protein of a virus.

[0015] In one embodiment the present invention relates to compositions where the virus is an influenza virus.

[0016] In one embodiment the present invention relates to compositions where the virus is a coronavirus.

[0017] In one embodiment the present invention relates to compositions where the coronavirus is SARS-CoV-2.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 4 -

[0018] In one embodiment the present invention relates to compositions where the RNA molecule is a vaccine against a virus comprising the viral polypeptide or fragment thereof to be administered to a subject in need thereof.

[0019] In one embodiment the present invention relates to compositions which further comprise a second RNA molecule encapsulated in the LNP.

[0020] In one embodiment the present invention relates to compositions where the additive comprises one or more of sucrose, glucose, galactose, fructose, trehalose, maltose, histidine, sodium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium sulfate, sodium succinate, Tris, human albumin, hydrolyzed gelatin, protamine sulfate, benzalkonium chloride, Brij 35, Polaxamer 188, Polysorbate 20, Polysorbate 80, sodium docusate, Triton X-100, glycerol, mannitol, sorbitol, calcium chloride, DTT, EDTA, magnesium chloride, arginine, aspartic acid, glutaminc acid, glycine, histidine, isoleucine, lactic acid, lysine, maleic acid, malic acid, methionine, proline, urea, arginine, glutamic acid, isoleucine, gamma-cyclodextrin, 2-OH propyl beta cyclodextrin (HPBCD), sulfobutyl ether beta-cyclodextrin (SBECD), carboxymethylcellulose, dextran sulfate, dextran 40, PEG-3350, sodium hyaluronate, betaine, firoin, ectoine, gamma-cyclodextrin, alpha-cyclodextrin, methyl beta cyclodextrin (MBCD), random methylated beta-cyclodextrin (RAMEB), 2-OH propyl gamma cyclodextrin (HPGCD) and carboxymethyl beta cyclodextrin (CMBCD), hydroxypropyl methylcellulose (HPMC), maltodextrin, polyvinylpyrrolidone, guar gum, methylcellulose, beta-cyclodextrin and combinations thereof.

[0021] In one embodiment the present invention relates to compositions where the additive is a cyclodextrin.

[0022] In one embodiment the present invention relates to compositions where the cyclodextrin is selected from the group consisting of sulfobutyl ether beta-cyclodextrin (SBECD), 2-OH propyl beta cyclodextrin (HPBCD), methyl beta cyclodextrin (MBCD), random methylated beta-cyclodextrin (RAMEB), 2-OH propyl gamma cyclodextrin (HPGCD) and carboxymethyl beta cyclodextrin (CMBCD) and combinations thereof.

[0023] In one embodiment the present invention relates to compositions where the RNA:cyclodextrin ratio is from 1:5 to 1:80.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 5 -

[0024] In one embodiment the present invention relates to compositions where the RNA:cyclodextrin ratio is from 1:20.

[0025] In one embodiment the present invention relates to compositions which further comprise a non-cyclodextrin additive.

[0026] In one embodiment the present invention relates to compositions where the non- cyclodextrin additive is carboxymethyl cellulose.

[0027] In one embodiment the present invention relates to compositions where the carboxymethyl cellulose is present at a concentration of between 0.2 and 0.6%.

[0028] In one embodiment the present invention relates to compositions where the carboxymethyl cellulose is present at a concentration of 0.4%.

[0029] It will be appreciated that the broad forms of the invention and their respective features can be used in conjunction, interchangeably and / or independently, and reference to separate broad forms is not intended to be limiting. Brief Description of the Drawings

[0030] Various examples and embodiments of the present invention will now be described with reference to the accompanying drawings, in which: -

[0031] Figure 1 is a plot of particle size (DLS) versus additives stored at 2-8°C for mRNA- LNP formulations including various additives dried on LCP discs.

[0032] Figure 2 is a plot of encapsulation efficiency versus additives stored at 2-8°C for mRNA-LNP formulations including various additives dried on LCP discs.

[0033] Figure 3 is a plot of particle size (DLS) versus additives stored at 40°C for mRNA-LNP formulations including various additives dried on LCP discs.

[0034] Figure 4 is a plot of encapsulation efficiency versus excipients stored at 40°C for mRNA-LNP formulations including various additives dried on LCP discs.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 6 -

[0035] Figure 5A is a plot of encapsulation efficiency for unformulated control, trehalose control, HPBCD control, Trehalose post-print and HPBCD post print; Figure 5B is a plot of encapsulation efficiency for unformulated / no excipient, trehalose, HPBCD for LNP dried HD- MAP printed.

[0036] Figure 6 is a plot of delivery efficiency for 2ug of GF-S1G-05 (green fluorescent protein mRNA in LNP) 1:20 HPBCD, 0.4% CMC and 2ug of GF-S1G-05 (green fluorescent protein mRNA in LNP) 1:40 HPBCD, 0.4% CMC.

[0037] Figure 7 is a plot of delivery efficiency for 1ug and 0.5ug of hemagglutinin mRNA in LNP.

[0038] Figure 8 is a plot of remaining, delivered and coated mRNA for green fluorescent protein mRNA in LNP for 1ug and 0.5ug. The coated mRNA is the total amount of mRNA of eGFP coated onto the micro-array patch (MAP). The delivered mRNA is the amount of mRNA of eGFP removed from the MAP when the MAP was projected into the skin. The remaining mRNA is the amount of mRNA of eGFP left on the MAP after the MAP was projected into the skin and removed.

[0039] Figure 9A is a plot of the Z average (particle size) at various timepoints for MAPs coated with mRNA-LNP with various additives and stored at 2-8°C; Figure 9B is a plot of the Z average (particle size) at various timepoints for MAPs coated with mRNA-LNP with various additives and stored at 25°C; Figure 9C is a plot of the Z average (particle size) at various timepoints for MAPs coated with mRNA-LNP with various additives and stored at 40°C.

[0040] Figure 10A is a plot of the encapsulation efficiency at various timepoints for MAPs coated with mRNA-LNP with various additives and stored at 2-8°C; Figure 10B is a plot of the encapsulation efficiency at various timepoints for MAPs coated with mRNA-LNP with various additives and stored at 25°C; Figure 10C is a plot of the encapsulation efficiency at various timepoints for MAPs coated with mRNA-LNP with various additives and stored at 40°C.

[0041] Figure 11A is a plot of the Z average (particle size) at various timepoints for MAPs coated with mRNA-LNP (Formulation B) stored at 2-8°C, 25°C and 40°C; Figure 11B is a plotC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 7 - of the encapsulation efficiency at various timepoints for MAPs coated with mRNA-LNP (Formulation B) stored at 2-8°C, 25°C and 40°C.

[0042] Figure 12A is a plot of Z-average (particle size) at different timepoints of a luciferase mRNA-LNP with and without carboxymethylcellulose stored 2-8°C; Figure 12B is a plot of Z-average (particle size) at different timepoints of a luciferase mRNA-LNP with and without carboxymethylcellulose stored 40°C.

[0043] Figure 13A is a plot of encapsulation efficiency at different timepoints of a luciferase mRNA-LNP with and without carboxymethylcellulose stored 2-8°C; Figure 13B is a plot of encapsulation efficiency at different timepoints of a luciferase mRNA-LNP with and without carboxymethylcellulose stored 40°C.

[0044] Figure 14A is a plot of encapsulation efficiency for mRNA-LNP formulations with varying ratios of mRNA to HBPCD (1:10, 1:20, 1:40 and 1:60 w / w) with and without carboxymethylcellulose for material coated onto MAPs and eluted; Figure 14B is a plot of encapsulation efficiency for mRNA-LNP formulations with varying ratios of mRNA to HBPCD(1:10, 1:20, 1:40 and 1:60) with and without carboxymethylcellulose for the coating solution which was coated into MAPs.

[0045] Figure 15 is a plot of encapsulation efficiency for mRNA-LNP formulations with varying ratios of mRNA to HBPCD (1:5, 1:10, 1:20 w / w) with and without carboxymethylcellulose for material coated onto MAPs.

[0046] Figure 16 is a plot of encapsulation efficiency of an mRNA-LNP formulation with various additive ratios (1:5 1:10 and 1:20 w / w) stored on ice and at room temperature for various time periods (1, 2, and 4 hours) post formulation.

[0047] Figure 17 is a plot of the mean encapsulation efficiency of an mRNA-LNP formulation with various additive ratios (1:5, 1:10 and 1:20 w / w) stored on ice and at room temperature for various time periods (1, 2, and 4 hours) post formulation.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 8 -

[0048] Figure 18 is a plot of the encapsulation efficiency of an mRNA-LNP formulation at various concentrations of HPGCD at ratios up to 1:80 (with a fixed concentration of 0.4% CMC).

[0049] Figure 19 is a plot of the encapsulation efficiency of an mRNA-LNP formulation at various amounts of mRNA in which HPGCD and 0.4% CMC were in the formulation. Detailed Description of the Preferred Embodiments

[0050] The present disclosure generally relates to compositions and formulations of nucleic acid molecules, for example, DNA and RNA, with improved stability, and methods of making and using the same. The compositions and formulations can comprise lipid nanoparticles (LNPs). For example, some embodiments relate to compositions and formulations of RNA molecules that can be used to generate an immune response or for any other therapeutic purpose. Such compositions and formulations can be stable for periods of time at room temperature.

[0051] The present invention relates to devices and methods for coating microprojections with nucleic acid formulations on microneedle arrays with various substances. These substances may be liquid or non-liquid and may be applied to the microprojection array by various techniques including MJet spraying (US Patent No.11,254,126).

[0052] Microprojection and microneedle arrays can be in the form of MAPs having projections extending from a surface of a base. The projections and base may be formed from any suitable material, including but not limited to silicon and various polymers including liquid crystal polymers. The projections may be solid, non-porous and non-hollow.

[0053] In an array the MAP has a width W and a breadth B with the projections being separated by spacing. The projections may be provided in an array that is defined by a regular iteration of microprojections along a square or rectangular arrangement, but other arrangements of projections such as circular arrangement of the projections that are compatible with rotational spray coating may also be used. In order to further improve or enhance the targeting accuracy, the substrate may be designed such that the features to be coated are located on radial lines from the center point of the rotation or located on concentric circles or on a continuous spiral.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 9 - The substrate may be designed such that the feature spacing on each arc is designed to match an integer number of steps of the motor for a given radius. Each projection may include one or more support sections and an end section for penetrating tissue of the biological subject and projections will typically have a profile which tapers from the base to the tip.

[0054] The MAP is applied to the biological subject by positioning the MAP against a surface of a subject or by positioning the MAP near the subject if an applicator that can propel the MAP toward the skin is utilized. The ends of the projections penetrate the surface of the skin and may penetrate tissue beneath the surface of the skin to a given depth as the MAP is applied. The MAP may be used to deliver material to the tissues of a patient. The MAP may be delivered such that the projections pierce the Stratum Corneum (SC) and penetrate through the Viable Epidermis VE to penetrate the Dermis (DE) by a dermal penetration depth. The MAP can be provided in a variety of different configurations to suit different material or stimulus delivery requirements. Accordingly, the specific configuration of the MAP can be selected to allow the delivery of material particular tissues, at a specific depth, to induce a desired response.

[0055] The microprojection arrays may be divided into areas such that a different vaccine antigen or other substance such as an additive including excipients may be coated in each area. For example, the microprojection array may be divided into multiple quadrants usually of equal size such as half. quarter. 1 / 16th or 1 / 32nd where the same or different vaccine antigens or other substances such as excipients may be applied. These areas may have equal numbers of microprojections or unequal numbers of microprojections. In other embodiments some of the microprojections may be uncoated.

[0056] The microprojection array can be varied in size depending on its use. The area of the MAP will have an impact on the ability to penetrate the subject, but this must be balanced by the need to induce cell damage over a sufficiently large area to induce a response. The MAP may be of any shape including but not limited to square, rectangular or circular. The MAPs may have dimensions of between 0.5 x 0.5 mm and 20 x 20 mm, between 0.5 x 0.5 mm and 15 x 15 mm including 14mm x 14mm or 16mm x 16mm.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 10 -

[0057] The microprojection arrays may have a density of projections of between 1,000 to 10,000 per cm2or from 1,000 to 5,000 per cm2, or from 1,000 to 2,500 or from 1,000 to 2,000 per cm2or from 500 to 10,000 per cm2or from 500 to 5,000 per cm2, or from 500 to 2,500 or from 500 to 2,000 per cm2or from 500 to 1000 per cm2. The applicators of the present invention are often utilized to project high density microprojection arrays into the skin. Such high- density arrays are microprojection arrays of sufficient size and density such that forces that can be applied manually will be insufficient to overcome the elasticity of the skin. The projections are typically separated by between 10 µm and 200 ^m, between 30 µm and 150 ^m, between 50 µm and 120 ^m and more typically between 70 µm and 100 ^m.

[0058] The length of the projections may be from 200µm to 700µm or from 200µm to 600µm or from 200µm to 550µm or from 200µm to 500µm or from 200µm to 450µm or from 200µm to 400µm or from 200µm to 300µm or from 250µm to 700µm or from 250µm to 600µm or from 250µm to 550µm or from 250µm to 500µm or from 250µm to 450µm or from 300µm to 700µm or from 300µm to 600µm or from 300µm to 550µm or from 300µm to 500µm or from 300µm to 450µm or from 350µm to 700µm or from 350µm to 600µm or from 350µm to 550µm or from 350µm to 500µm or from 350µm to 400µm. In one embodiment of the HD-MAP the length of the microprojections is between 525µm to 575µm.

[0059] It may be desirable to incorporate a discontinuity into the effective profile of the projections and this can have benefits either in combination with the convex effective profile, or in isolation. A projection having a stepped effective profile parameters may control the depth of penetration of the projection in order to obtain a desired performance or to provide sufficient material to induce a desired response within the subject. In one embodiment of the MAPs of the present invention there are three sections to the microprojection: base, middle and end. In one embodiment of the three section, two step MAP projection the base may be approximately 300µm long and tapers along its length from approximately 130µm at the base to 110µm. The middle is approximately 152µm long and tapers from about 60µm to 40µm. The end section is about 100µm in length and tapers from about 20µm to 10µm at the top of the microprojection. Other embodiments include projections with a single discontinuity with the projection having two sections: an upper section and a lower section. In one embodiment the lengths of the two sections are equal and approximately 300µm in length in which the firstC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 11 - section tapers from approximately 130µm at the base to 110µm. There is a step or discontinuity between the first and second section and the second section tapers from about 60µm to 40µm.

[0060] The step can assist in ensuring more consistent depth of penetration in different biological subjects, despite variations in the tissue properties from subject to subject. In particular, during insertion of the projection, the step can impact on the dermal tissues, which typically present an increased resistance to penetration compared to tissues in outer layers of the skin (such as the viable epidermis, for example), thereby limiting further penetration of the projection. By providing projections with a suitably configured stepped profile, and using controlled application parameters, it is therefore possible to ensure that the tips of the projections extend into the dermis by a predetermined distance.

[0061] An advantage of using a stepped effective profile as discussed above is that the support section may be configured to effectively provide mechanical reinforcement for the projection, without impacting on the profile of the penetrating end section. This mechanical reinforcement may be provided by merely increasing the diameter of the projections along desired portions of the projection, but may also be provided in other ways, such as by providing buttress features radiating from the base of the projections, to even further strengthen the projections.

[0062] The microprojection array may be made of any suitable materials including but not limited to silicon, polymers, and plastic. In silicon embodiments the base thickness is about 60 um or silicon with a thin (1mm) polymer backing. The overall mass of some embodiments of the microprojection array is from about 0.2 grams to 2.0 grams. The microprojection array may have bevelled edges to reduce peak stresses on the edge of the array. The MAP can be quartered or subdivided by other ratios to reduce the stress load on the MAP and mitigate MAP breakage. Polymer embodiments may have reduced mass. The microprojection array may also have an overall weakly convex shape of the MAP to improve the mechanical engagement with skin and mitigate the effect of high speed rippling application: a ‘high velocity / low mass’ system. The microprojection array may have a mass of less than 2.0 grams or less than 1.8 grams or less than 1.7 grams or less than 1.6 grams or less than 1.5 grams or less than 1.4 grams or less than 1.3 grams or less than 1.2 grams or less than 1.1 gram or less than 1.0 grams, or less than 0.9 grams or less than 0.8 grams or less than 0.7 grams, or less than 0.6 grams or less than 0.5 grams. The microprojection array may have a mass of from about 0.4 grams to about 1.2 gramsC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 12 - or from about 0.4 grams to about 1.1 grams, or from about 0.4 grams to about 1.0 grams or from about 0.4 grams to about 0.9 grams, or from about 0.4 grams to about 1.8 grams or from about 0.4 grams to about 1.7 or from about 0.4 grams to about 0.6 grams or from about 0.4 grams to about 0.5 grams, or from about 0.5 grams to about 1.2 grams or from about 0.5 grams to about 1.1 grams, or from about 0.5 grams to about 1.0 grams or from about 0.5 grams to about 0.9 or from about 0.5 to about 0.8 grams or from about 0.5 grams to about 0.7 grams or from about 0.5 grams to about 0.6 grams . In one embodiment of the applicator / microprojection system the mass of the array is about 0.5 to about 0.7 grams, the array is projected at a velocity of about 20-26 m / s by the applicator.

[0063] The projection spacing is selected so that material from the projections is able to at least partially, and optionally at least completely, diffuse between the projections. Accordingly, the projections are typically separated by between 10 µm and 200 ^m or between 10 µm and 190 ^m or between 10 µm and 180 ^m or between 10 µm and 170 ^m or between 10 µm and 160 ^m or between 10 µm and 150 or between 10 µm and 140 ^m or between 10 µm and 130 ^m or between 10 µm and 120 ^m or between 10 µm and 110 ^m or between 10 µm and 100 ^m or between 10 µm and 90 ^m or between 10 µm and 80 ^m or between 10 µm and 70 ^m or between 10 µm and 60 ^m or between 10 µm and 50 µm or between 10 µm and 40 ^m or between 10 µm and 30 ^m or between 10 µm and 20 ^m

[0064] In some embodiments, more than one coating may be applied to the same projection. For instance, different coatings may be applied in one or more layers to provide the same or different materials for delivery to the tissues within the subject at the same time or different times if the layers dissolve in sequence. A first coating may be applied to modify surface properties of the projection and improve the ability of the second coating to coat the projection in a desirable manner. Multiple layers of the same coating formulation may be used with drying between each layer to allow a progressive build-up of coating to achieve a specific thickness and thus modify the effective cross section of the projection even further.

[0065] The devices and methods of the present invention relate to the delivery of nucleic acids, in particular RNA using a microprojection array. Delivering RNA to cells is complicated by RNase enzymes which degrade the RNA. The RNA must be taken into the cell such that it isC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 13 - translated into protein. Avoiding RNA degradation and ensuring cell delivery may be accomplished by combining the RNA with lipid nanoparticles such as those utilized by Pfizer and Moderna in Covid-19 vaccines. The LNPs of the Pfizer / BNT162b2 and Moderna mRNA- 1273 COVID mRNA vaccines contain four components: cholesterol, DSPC, a PEGylated lipid such as PEG2000-DMG and an ionizable lipid. Lipid nanoparticle or “LNP” refers to particles having at least one dimension on the order of nanometers which include lipids or other specified cationic lipids. Lipid nanoparticles typically comprise lipids and one or more compounds including neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the nucleic acid may be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle. Typically the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm. Lipid encapsulated may include full encapsulation or partial encapsulation of the nucleic acid.

[0066] There are a variety of ionizable lipids which may change the characteristics of the LNP. Other LNP functions include targeting, stability, reduced toxicity which may require other compounds. The lipid nanoparticles can be used to deliver any nucleic acid molecules, including but not limited to mRNA, antisense oligonucleotide, small interfering RNA (siRNA), self-amplifying RNA (samRNA), short hairpin RNAs or small hairpin RNA (shRNA), plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger-RNA- interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), In some embodiments, the nucleic acid comprises mRNA, samRNA, siRNA or shRNA.

[0067] Various lipids may be used to form LNPs described herein, and may include lipid compounds disclosed in US Patent Nos. 11,040,112, 10,723,692, 10,221,127, 10,166,298, 10,106,490, 9,738,593, 9,737,619 (e.g., in Table 1 of these patents), 9,675,668, 9,447,164, 9,301,993, 9,295,689, and 8,754,062, each of which is incorporated by reference herein to its entirety.

[0068] The nucleic acids delivered by the LNPs can encode polypeptides, including those that induce a host immune response. These polypeptides may originate from a pathogen (bacterial, fungal or viral protein). Prophylactic vaccines may be prepared using the nucleic acid-LNPC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 14 - delivery system described herein targeting COVID-19, respiratory syncytial viruses, influenza virus, etc., in which a pathogenic protein is encoded in the nucleic acid-LNP vaccine.

[0069] Nucleic acids carriers other than LNPs may be utilized by the devices and methods of the present invention. Liposome and lipidots may be used as carriers to deliver nucleic acids.

[0070] Microprojections may be coated with a vaccine compound that contains a single composition or multiple compositions either to the same pathogen or to different pathogens. In another embodiment the substance may be a vaccine composition having one or more additives and one or more antigens. In another embodiment the substance may be a vaccine composition having an adjuvant and one or more antigens. As described above vaccine compositions may be delivered by the MAP such that different antigens are located on different microprojections either independent one from another or in sections located on the MAP.

[0071] Coatings may be liquid or non-liquid. Liquid coating materials may be aqueous, however other coating solutions are possible, and the surface properties of the projection may need to be modified to accommodate a range of coating solutions. For an aqueous coating solution, the microprojections may be modified to be more "hydrophobic" in nature. A hydrophilic surface will cause an aqueous solution to completely wet it (assuming low viscosity). This would result in a large fraction of the liquid coating material being wicked onto the base of the projection array, which would impede its delivery to the skin. Increasing the solution viscosity slows down the wicking (or surface wetting) process. If a dry coating process is accomplished rapidly in comparison to the surface wetting, a larger fraction of the liquid coating material can be localized to the projections. By changing the contact angle of the projection surface (by chemically modifying it), the liquid coating solution wetting properties may also be altered. In making the surface more "hydrophobic", an aqueous coating solution will be inhibited from wetting the projection surface down to the base. Furthermore, a surfactant can be added to an aqueous coating solution which is placed on a "hydrophobic" projection. The surfactant may assist in wetting the hydrophobic surface by orienting the polar and non-polar groups of the surfactant at the surface, thus facilitating the wetting. If appropriate drying conditions (either with or without surfactant) are achieved, the result is that a significant portion of the coating material is retained near the projection tips. Striking a balance between the surface wetting properties (i.e. contact angle), solution viscosity, and the presence orC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 15 - absence of a surfactant or other additives (among other solution properties) can change the degree and uniformity with which the coating solution is localized to the projection ends. In a further embodiment, the microprojection surface may be altered such that the tips are hydrophilic and the lower portion of the shaft and base are hydrophobic. This can be accomplished using bulk lithographic processes. In this embodiment, the hydrophilic tip surface is easily wet, while the lower portion of the projection inhibits liquid travel towards the base due to its hydrophobic nature. Other methods of coating the microprojections include but are not limited to differential coatings using plasma polymers, spin coating, ink jet printing, mjet application, microimprinting and dip coating. Selection of the type of application of the composition onto the microprojections will depend on which method is most efficient while maintaining appropriate stability of the composition on the microprojections. While the initial material to be coated onto the microprojections may be liquid in many embodiments the material will be dried onto the microprojection such that an amount of water is removed from the composition. In some embodiments, a composition is dried so as to remove about or at least about 50%, 60%, 70%, 80%, 90%, 95% or more (and all sub-values and sub-ranges there between, including endpoints) of a starting amount of water.

[0072] The amount of nucleic acid used in the devices and methods of the present invention include amounts necessary to provide an immune response. A dose may include the use of more than one MAP if the dose on a single MAP is insufficient to provide an appropriate immune response. At least one dose selected from the group consisting of a 1µg dose, 2µg dose, 3µg dose, 4µg dose, 5µg dose, 6µg dose, 7µg dose, 8µg dose, 9µg dose, 10µg dose, 15µg dose, 20µg dose, 25µg dose, 30µg dose, 40µg dose, 50µg dose, 60µg dose, 70µg dose and a 80µg dose or more may be sufficient to induce an immune response in humans. The dose of the antigen may be administered to the human within a range of doses including from about 1μg to about 80μg, from about 1μg to about 80μg, from about 1μg to about 70μg, from about 1μg to about 70μg, from about 1μg to about 60μg or from about 1μg to about 50μg, from about 1μg to about 40μg, or from about 1μg to about 30μg, from about 1μg to about 25μg, from about 1μg to about 20μg, from about 1μg to about 15μg, from about 1μg to about 10μg, from about 2μg to about 10μg, from about 2μg to about 8μg, from about 3μg to about 10μg, from about 3μg to about 8μg, from about 3μg to about 5μg, from about 4μg to about 10μg, from about 4μgC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 16 - to about 8μg, from about 5μg to about 10μg, from about 5μg to about 9μg, and from about 5μg to about 8μg.

[0073] The present invention also relates to devices, formulations and methods for increasing the stability of vaccine formulations by including additives which include but are not limited to cyclodextrins, amino acids, reducing agents, carbohydrates and proteins and combinations thereof. Additives include but are not limited to Histidine, Sodium acetate, Sodium chloride, Sodium citrate, Sodium phosphate, Sodium sulfate, Sodium succinate, Gelatin, Hydrolysed Gelatin, Protamine sulfate, Arginine, Aspartic acid (sodium salt), Glutamic acid, Glycine, Isoleucine, Lactic acid, Lysine, Maleic acid, Malic acid (sodium salt), Methionine, Urea, EDTA, Magnesium chloride, Benzalkonium chloride, Brij 35, Poloxamer 188 (Pluronic F-68), Polysorbate 20, Polysorbate 80, Sodium docusate, Triton X-100, Lactose, Sucrose, Trehalose, Glycerol, Mannitol, Sorbitol, Gamma-Cyclodextrin, 2-OH propyl b-CD, Sulfobutyl ether beta- cyclodextrin, Carboxymethyl cellulose, Dextran sulfate, Dextran 40, PEG-3350, Sodium Hyaluronate, Sodium thioglycolate, Cysteine, Glutathione, gamma-cyclodextrin, alpha- cyclodextrin, methyl beta cyclodextrin (MBCD), random methylated beta-cyclodextrin (RAMEB), 2-OH propyl gamma cyclodextrin (HPGCD) and carboxymethyl beta cyclodextrin (CBC), hydroxypropyl methylcellulose (HPMC), maltodextrin, polyvinylpyrrolidone, guar gum, methylcellulose, beta-cyclodextrin and combinations thereof.

[0074] In certain embodiments of the nucleic acid / LNP formulations of the present invention the additives are cyclodextrins alone or in combination with other additives including other cyclodextrins. In some embodiments the additive may be carboxymethyl beta cyclodextrin (CMBCD) or 2-OH propyl gamma cyclodextrin (HPGCD) or 2-OH propyl beta cyclodextrin (HPBCD) or sulfobutyl ether beta-cyclodextrin (SBECD) alone or in combinations with other cyclodextrins and / or other additives. Combinations of additives include but are not limited to a combinations of carboxymethyl beta cyclodextrin (CMBCD) and 2-OH propyl beta cyclodextrin (HPBCD), or 2-OH propyl gamma cyclodextrin (HPGCD) and 2-OH propyl beta cyclodextrin (HPBCD).

[0075] The ratio of nucleic acid:additive may be 1:2 or 1:2.5 or 1:3 or 1:4 or 1:5 or 1:10 or 1:15 or 1:20 or 1:25 or 1:30 or 1:35 or 1:40 or 1:45 or 1:50 or 1:55 or 1:60 or 1:65 or 1:70 or 1:75 or 1:80 or 1:85 or 1:90 or 1:95 or 1:100. The ratio of nucleic acid:additive may be fromC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 17 - 1:2 to 1:100 or from 1:2 to 1:1:90 or from 1:2 to 1:80 or from 1:2 to 1:70 or from 1:2 to 1:60 or from 1:2 to 1:50 or from 1:2 to 1:50 or from 1:2 to 1:40 or from 1:2 to 1:30 or from 1:2 to 1:20 or from 1:2 to 1:10 or from 1:2 to 1:5 or from 1:3 to 1:100 or from 1:3 to 1:1:90 or from 1:3 to 1:80 or from 1:3 to 1:70 or from 1:3 to 1:60 or from 1:3 to 1:50 or from 1:3 to 1:50 or from 1:3 to 1:40 or from 1:3 to 1:30 or from 1:3 to 1:20 or from 1:3 to 1:10 or from 1:3 to 1:5 or from 1:4 to 1:100 or from 1:4 to 1:1:90 or from 1:4 to 1:80 or from 1:4 to 1:70 or from 1:4 to 1:60 or from 1:4 to 1:50 or from 1:4 to 1:50 or from 1:4 to 1:40 or from 1:4 to 1:30 or from 1:4 to 1:20 or from 1:4 to 1:10 or from 1:5 to 1:100 or from 1:5 to 1:1:90 or from 1:5 to 1:80 or from 1:5 to 1:70 or from 1:5 to 1:60 or from 1:5 to 1:50 or from 1:5 to 1:50 or from 1:5 to 1:40 or from 1:5 to 1:30 or from 1:5 to 1:20 or from 1:5 to 1:10 or from 1:10 to 1:100 or from 1:10 to 1:1:90 or from 1:10 to 1:80 or from 1:10 to 1:70 or from 1:10 to 1:60 or from 1:10 to 1:50 or from 1:10 to 1:50 or from 1:10 to 1:40 or from 1:10 to 1:30 or from 1:10 to 1:20 or from 1:20 to 1:100 or from 1:20 to 1:1:90 or from 1:20 to 1:80 or from 1:20 to 1:70 or from 1:20 to 1:60 or from 1:20 to 1:50 or from 1:20 to 1:50 or from 1:20 to 1:40 or from 1:20 to 1:30 or from 1:30 to 1:100 or from 1:30 to 1:1:90 or from 1:30 to 1:80 or from 1:30 to 1:70 or from 1:30 to 1:60 or from 1:30 to 1:50 or from 1:30 to 1:50 or from 1:30 to 1:40.

[0076] In the formulations of the present invention nucleic acids are encapsulated in LNPs. The higher the encapsulation efficiency the greater the amount of nucleic acid that is encapsulated in the LNP. A high percentage encapsulation efficiency is desirable. In some embodiments of the present invention the nucleic acid is encapsulated in the LNP at an efficiency of from about 40-100%. The encapsulation efficiency may be measured at various times prior to administration of the formulation on the MAP. When the nucleic acid-LNP composition is first formulated and coated onto the MAP the encapsulation efficiency can be about 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater or 95% or greater. The formulated composition encapsulation efficiency may be from about 50% to 100% or from 60% to 100% or from 70% to 100% or from 80% to 100% or from 90% to 100% or from about 40% to 95% or from 50% to 95% or from 60% to 95% or from 70% to 95% or from 80% to 95% or from 90% to 95% or from about or from 50% to 90% or from 60% to 90% or from 70% to 90% or from 80% to 90% or from about 50% to 85% or from 60% to 85% or from 70% to 85% or from 80% to 85% or from 50% to 80% or from 60% to 80% or from 70% to 80% or from 50% to 75% or from 60% to 75% or from 70% to 75% orC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 18 - from 50% to 70% or from 60% to 70% or from 50% to 60%. Over time as the formulation is stored the encapsulation efficiency may decrease. As with the original formulation it is desirable to maintain the encapsulation efficiency during storage where the formulation has been coated onto the MAP.

[0077] The methods and compositions of the present invention provide microprojection arrays that can be coated with nucleic acid / LNP vaccines that are stable over time. The vaccine compositions of the present invention are stable at at least 2-8°C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months at various temperatures and conditions. The stability of the vaccine formulations may be measured by a variety of techniques.

[0078] The methods and compositions of the present invention provide microprojection arrays that can be coated with multiple vaccines that are stable over time. The vaccine compositions of the present invention are stable at at least 25°C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months at various temperatures and conditions. The stability of the vaccine formulations may be measured by a variety of techniques.

[0079] Reduction of encapsulation efficiency for the nucleic acid / LNP formulations of the present invention upon rapid drying can be about 0% or less than about 5% or less than about 10% or less than about 15% or less than about 20% or less than about 25% or less than about 30% or less than about 35% or less than about 40% or less than about 45% or less than about 50% or less than about 55% or less than about 60% or less than about 65% or less than about 70% or less than about 75% or less than about 80% or less than about 85% or less than about 90%.

[0080] Reduction of encapsulation efficiency for the nucleic acid / LNP formulations of the present invention upon rapid drying and storage at at least 2-8°C for at least 1 or at least 2 orC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 19 - at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 11 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months can be about 0% or less than about 5% or less than about 10% or less than about 15% or less than about 20% or less than about 25% or less than about 30% or less than about 35% or less than about 40% or less than about 45% or less than about 50% or less than about 55% or less than about 60% or less than about 65% or less than about 70% or less than about 75% or less than about 80% or less than about 85% or less than about 90%.

[0081] Reduction of encapsulation efficiency for the formulations / antigens of the present invention upon rapid drying and storage at at least 25°C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 11 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months can be about 0% or less than about 5% or less than about 10% or less than about 15% or less than about 20% or less than about 25% or less than about 30% or less than about 35% or less than about 40% or less than about 45% or less than about 50% or less than about 55% or less than about 60% or less than about 65% or less than about 70% or less than about 75% or less than about 80% or less than about 85% or less than about 90%.

[0082] Reduction of encapsulation efficiency for the formulations / antigens of the present invention upon rapid drying and storage at at least 40°C for at least 1 or at least 2 or at least 3 or at least 4 weeks or at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 11 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months can be about 0% or less than about 5% or less than about 10% or less than about 15% or less than about 20% or less than about 25% or less than about 30% or less than about 35% or less than about 40% or less than about 45% or less than about 50% or less than about 55% or less than about 60% or less than about 65% or less than about 70% or less than about 75% or less than about 80% or less than about 85% or less than about 90%.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 20 -

[0083] In the formulations of the present invention nucleic acids / LNPs are coated onto a high density microprojection array. The higher the delivery efficiency the greater the amount of nucleic acid / LNP that is delivered below the skin of the patient thus providing an immunological response in the patient. A high percentage delivery efficiency is desirable. In some embodiments of the present invention the nucleic acid / LNP coated onto the MAP is delivered into the skin at an efficiency of from about 40-100%. When the nucleic acid / LNP composition is delivered into the skin of a patient via MAP delivery, the efficiency can be about 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater or 95% or greater. The formulated composition may be delivered into the skin with an efficiency of from about 50% to 100% or from 60% to 100% or from 70% to 100% or from 80% to 100% or from 90% to 100% or from about 40% to 95% or from 50% to 95% or from 60% to 95% or from 70% to 95% or from 80% to 95% or from 90% to 95% or from about or from 50% to 90% or from 60% to 90% or from 70% to 900% or from 80% to 90% or from about 50% to 85% or from 60% to 85% or from 70% to 85% or from 80% to 85% or from 50% to 80% or from 60% to 80% or from 70% to 80% or from 50% to 75% or from 60% to 75% or from 70% to 75% or from 50% to 70% or from 60% to 70% or from 50% to 60%. A small amount of the nucleic acid / LNP formulation may left on the skin (about 2-5%).

[0084] Particle sizes for nucleic acid / LNP formulation may vary. For example, particle sizes for mRNA / LNP vaccines are often less than 200nm. The sizes vary depending on drying method, storage conditions and excipients. The optimal size range for LNPs may be from about 60-150 nm. The size of the LNPs can be tailored with process variables in LNP manufacture, lipids, buffer and additives. The size of LNP may not change with nucleic acids of choice. The sizes of the particles will likely vary by LNP type. For example, some LNP particles have a size in the order of 70nm while other LNP particles are usually 90-100nm. The particle size of the mRNA / LNP that are eluted from MAPs is larger than prior to drying them. That size also depends on the buffer used to elute the LNPs from the MAPs. Particle size may potentially be used as a measure of stability; large particle sizes may indicate agglomeration or individual particles.

[0085] In preferred embodiments the microprojections of the microprojection array are coated by an aseptic print-head type device which rapidly provides small droplets which dry quicklyC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 21 - on the microprojections. In preferred embodiments the coating such as a vaccine formulation rapidly dries on the top portion of the microprojection to increase the amount of vaccine that can be delivered. The aseptic print head device may deliver multiple drops to the microprojections either sequentially or in an alternating fashion. A suitable coating device for coating the formulations of the present invention onto high density microprojection arrays is described in US Patent No.11,254,126.

[0086] The microprojections of the array of the present invention may be of any shape including cylindrical or conical. Other geometries are also possible. The microprojection arrays may have substrate with a plurality of microprojections protruding from the substrate wherein the microprojections have a tapering hexagonal shape and comprise a tip and a base wherein the base has two substantially parallel sides with a slight draught angle of approximately 1 to 20 degrees up to a transition point at which point the angle increases to from about 20 degrees to about 70 degrees. A sharp blade-like tip will allow for enhanced penetration of the microprojections into the skin while also generating an enhanced localized cell death / bystander interaction in the skin with a different profile than conical microprojection arrays. In a preferred embodiment the microprojections are made of a polymer and are slightly blunted at the tip with a step or discontinuity near the end of the projection on which the coating material may attach such that the coating material does not drip down the microprojection and onto the base of the microprojection array.

[0087] In specific embodiments, the material coated onto the MAP is selected from antigens including exogenous antigens that are foreign to that host. The antigens may be in the form of soluble peptides or polypeptides or polynucleotides from which an expression product (e.g., protein or RNA) is producible. Exemplary pathogenic organisms include, but are not limited to, viruses, bacteria, fungi parasites, algae and protozoa and amoebae. Illustrative viruses include viruses responsible for diseases including, but not limited to, flavivirus, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Epstein-Barr virus and other herpesviruses such as papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBankC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 22 - Accession No. M24444), hantavirus, Sendai virus, respiratory syncytial virus, orthomyxoviruses, vesicular stomatitis virus, visna virus, cytomegalovirus and human immunodeficiency virus (HIV) (e.g., GenBank Accession No. U18552). Any suitable antigen derived from such viruses are useful in the practice of the present invention. For example, illustrative retroviral antigens derived from HIV include, but are not limited to, antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components. Illustrative examples of hepatitis viral antigens include, but are not limited to, antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA. Illustrative examples of influenza viral antigens include; but are not limited to, antigens such as hemagglutinin and neuraminidase and other influenza viral components. Illustrative examples of measles viral antigens include, but are not limited to, antigens such as the measles virus fusion protein and other measles virus components. Illustrative examples of rubella viral antigens include, but are not limited to, antigens such as proteins E1 and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components. Illustrative examples of cytomegaloviral antigens include, but are not limited to, antigens such as envelope glycoprotein B and other cytomegaloviral antigen components. Non- limiting examples of respiratory syncytial viral antigens include antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components. Illustrative examples of herpes simplex viral antigens include, but are not limited to, antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components. Non-limiting examples of varicella zoster viral antigens include antigens such as 9PI, gpII, and other varicella zoster viral antigen components. Non-limiting examples of Japanese encephalitis viral antigens include antigens such as proteins E, M-E, M-E- NS 1, NS 1, NS 1-NS2A, 80% E, prM, prM-E, and other Japanese encephalitis viral antigen components. Representative examples of rabies viral antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. Illustrative examples of papillomavirus antigens include, but are not limited to, the L1 and L2 capsid proteins as well as the E6 / E7 antigens associated with cervical cancers, See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M., 1991, Raven Press, New York, for additional examples of viral antigens.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 23 -

[0088] Illustrative examples of fungi include Acremonium spp., Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces dermatidis, Candida spp., Cladophialophora carrionii, Coccoidiodes immitis, Conidiobolus spp., Cryptococcus spp., Curvularia spp., Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp., Fonsecaea compacta, Fonsecaea pedrosoi, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Hortaea werneckii, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia furfur, Microsporum spp., Neotestudina rosatii, Onychocola canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa, Piedraia hortae, Piedra iahortae, Pityriasis versicolor, Pseudallesheria boydii, Pyrenochaeta romeroi, Rhizopus arrhizus, Scopulariopsis brevicaulis, Scytalidium dimidiatum, Sporothrix schenckii, Trichophyton spp., Trichosporon spp., Zygomcete fungi, Absidia corymbifera, Rhizomucor pusillus and Rhizopus arrhizus. Thus, representative fungal antigens that can be used in the compositions and methods of the present invention include, but are not limited to, candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.

[0089] Illustrative examples of bacteria include bacteria that are responsible for diseases including, but not restricted to, diphtheria (e.g., Corynebacterium diphtheria), pertussis (e.g., Bordetella pertussis, GenBank Accession No. M35274), tetanus (e.g., Clostridium tetani, GenBank Accession No. M64353), tuberculosis (e.g., Mycobacterium tuberculosis), bacterial pneumonias (e.g., Haemophilus influenzae.), cholera (e.g., Vibrio cholerae), anthrax (e.g., Bacillus anthracis), typhoid, plague, shigellosis (e.g., Shigella dysenteriae), botulism (e.g., Clostridium botulinum), salmonellosis (e.g., GenBank Accession No. L03833), peptic ulcers (e.g., Helicobacter pylori), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), Other pathogenic bacteria include Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. Thus, bacterial antigens which can be used in the compositions and methods of the inventionC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 24 - include, but are not limited to: pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components, streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components, pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumiococcal bacterial antigen components; Haemophilus influenza bacterial antigens such as capsular polysaccharides and other Haemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.

[0090] Illustrative examples of protozoa include protozoa that are responsible for diseases including, but not limited to, malaria (e.g., GenBank Accession. No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis, leishmaniasis, giardiasis (GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis. Thus, protozoal antigens which can be used in the compositions and methods of the invention include, but are not limited to: Plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, blood-stage antigen pf 155 / RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; Leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components;C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 25 - and Trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components. Examples Example 1 Cell Based Production of eGFP from Disc Eluate

[0091] A study was conducted on Moderna-like LNPs containing different eGFP mRNA sources. These are able to transfect & express in HEK293T cells with 72 hours post transfection giving clearest eGFP expression. eGFP-LNPs eluted from LCP discs post-drying are able to successfully transfect and express in-vitro demonstrating that the encapsulated mRNA remains viable after drying and subsequent elution (rehydration from dried state). Example 2 Additive Stability Studies on Disc

[0092] An additive screen was conducted with the additives listed below for LNP-RNA. Additives were tested with LNP-mRNA through multiple rounds of screening on LCP discs.

[0093] Nine additives including 2-OH propyl beta-cyclodextrin (HPBCD), sulfobutyl ether beta cyclodextrin (SBECD), Dextran 40, PEG-3350, glycerol, mannitol, sorbitol, sucrose and trehalose were evaluated alone and in combinations.

[0094] A stock was prepared of each additive and combined with LNP-mRNA with various mRNA to additive ratios (1:5, 1:10, 1:20, 1:40 and 1:60 w / w. Discs were coated with 5µl of each formulation and dried with N2 gas. The discs were packed with desiccant and stored at 2- 8°C for 0-28 days and 40°C for accelerated stability. After the allotted time elapsed the discs were rehydrated in a buffer solution (DPBS).

[0095] GFP mRNA LNP was formulated with sugars and cyclodextrins at various ratios on discs. The choice of additives affects particle size measurement. Sucrose and trehalose (1:20) do not prevent LNPs from changes in particle size measurement (possibly from agglomeration,C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 26 - or possibly from other causes) upon dry down and reconstitution. HPBCD is relatively robust in maintaining particle size and LNP integrity (%EE).

[0096] Luciferase mRNA LNP was formulated with sugars and cyclodextrins at various ratios on discs as described above at 2-8°C and 40°C. HPBCD ratio is important, 1:20 ratio (mRNA:HPBCD) provided the most stable composition as determined by particle size and encapsulation efficiency. Not all ratios of HPBCD stabilize the mRNA-LNP formulation.

[0097] Stealth LNPs appear stable during dry down with sucrose but no expression was observed in HEK293Ts with LNPs before or after drying. Example 3 Additive Combination Stability Studies on Discs

[0098] An additive study (including a DOE approach) was conducted with various combinations of trehalose, sucrose, SBECD, HPBCD up to 1:20 ratio for 7days. The results of the study are shown in Figure 1 which is a plot particle size (DLS) versus excipients stored at 2-8°C. Cyclodextrin excipients are preferred over sugars and in this study HPBCD is the preferred additive which could be combined with SBECD. Temperature had no significant effect on particle size or encapsulation efficiency. The length of time of storage affected particle size as the particle size increased with increasing time. Liquid control (LC) with no drying exhibited Z-averages that remained stable (96-128nm) for 7 days. Glycerol and hydroxypropyl β cyclodextrin (HPBCD) exhibited Z-averages which remained closest to those of LC samples (D1-D7). Trehalose, sucrose, glycerol and SBECD showed particle sizes that were closer to those of LC samples compared to other additives and the absence of additives.

[0099] Figure 2 is a plot of encapsulation efficiency versus additives stored at 2-8°C. The plot demonstrates that liquid control encapsulation efficiency remained stable for 7 days (88-90%). HPBCD showed encapsulation efficiency values close to LC samples at D0 (87% vs 90%) and maintained encapsulation efficiency above 70% for 7 days. Trehalose, sucrose, glycerol and SBECD formulations also maintained encapsulation efficiency of greater than 50% for 7 days. No improvement in encapsulation efficiency is seen with sorbitol, ectoin, PEG-3350 and no additive.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 27 -

[0100] Figure 3 is a plot particle size (DLS) versus additives stored at 40°C. The plot demonstrates that HPBCD had Z-averages closest to liquid control samples (D1-D3), but with increased sizes compared to D0. Sucrose, glycerol, and SBECD exhibited stable particle sizes from D0 to D3, and closer to LC samples compared to other additives and no additives.

[0101] Figure 4 is a plot of encapsulation efficiency versus additives stored at 40°C. Figure 4 demonstrates that mRNA-LNP (LC) which contained no additive and stored at 2-8°C not at 40°C. SBECD showed similar encapsulation efficiency to the LC samples at D1-D3 (77% vs. 90%). Trehalose, sucrose, sorbitol, glycerol, and HPBCD formulations exhibited comparable encapsulation efficiencies of greater than 60% for 3 days. There was no improvement of encapsulation efficiency with respect to ectoin, PEG-3350, and no additive.

[0102] With respect to particle size a unique sharp peak was observed for LC, trehalose, sucrose, glycerol, SBECD, and HPBCD. Multiple peaks were present for sorbitol, ectoin, PEG- 3350 and samples without additive. Example 4 Coating of mRNA-LNP Formulations on microprojection arrays (MAPs)

[0103] Studies were conducted to develop a coating process that provides consistent dried formulation coating morphology on the microprojections as measured by scanning electron microscopy (SEM) as well as providing reliable delivery of the mRNA-LNP formulation into skin.

[0104] Successful coating of microprojections (good morphology, good encapsulation efficiency and reasonable delivery efficiency) has been accomplished with research scale DirectJet technology. The coating of the microprojections may be affected by plasma treatment of HD-MAP surface, different additive concentrations and viscosity modifiers. DirectJet has low throughput since the device coats one projection at a time and there is a long setup / calibration for each run.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 28 - Example 5 Stability of mRNA-LNP Formulations Coated on Microprojection Arrays (MAPs)

[0105] A study was conducted (using 150 and 70 MAPs) with eGFP which looked at encapsulation efficiency on MAPs at Day 0, Day 7 and Day 28 timepoints as well as Day 60. Encapsulation efficiency of coated MAPs dropped to about 65% after drying at Day 0. A second study using luciferase was conducted in which the encapsulation efficiency of coated MAPs at Day 0 decreased to about 45%. When dried on discs, encapsulation efficiency dropped to 83-85% for the same formulations. On MAPs there appears to be an immediate encapsulation efficiency loss on drying, but after the initial drop the encapsulation efficiency appears to stabilize. It is possible that aggregation or separation of LNP material during processing may occur. The use of the DirectJet method is slow and has limited throughput and works with only small volumes, however the method permits the adjustment of different parameters. Studies demonstrated that the addition of carboxymethylcellulose improved coating morphology at 0.4%. In studies with 0.4% CMC the encapsulation efficiency of luciferase mRNA and hemagglutinin mRNA was greater than 80% and greater than 55% respectively. Delivery efficiencies were from approximately 65-85%. The use of the MJet method is fast and has high throughput and works with large volumes, however the method permits few adjustments of different parameters. In some iterations the MAP is coated in quadrants of about 100 microprojections in which the microprojections are coated simultaneously. Studies demonstrated that the addition of carboxymethylcellulose improved coating morphology at 0.4%. In studies with 0.4% CMC the encapsulation efficiency of eGFP mRNA, luciferase mRNA and hemagglutinin mRNA was about 50 to 60%, about 50 to 60% and about 55% respectively. Delivery efficiencies were from approximately 37 to 70%. Example 6 Stability of mRNA-LNP Formulations Coated by MJet on Microprojection Arrays (MAPs)

[0106] Figure 5A is a plot of encapsulation efficiency for liquid formulations (before printing) including unformulated control, trehalose control, HPBCD control, trehalose and HPBCD. Figure 5B is a plot of encapsulation efficiency for unformulated / no excipient, trehalose, HPBCD for LNP dried HD-MAP post MJet printing. Shear stresses caused by microdropletC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 29 - printing do not substantively impact eGFP-LNP particle size. Variable (passive) drying post- printing of eGFP-LNP appears to affect particle size. Active or faster drying may be necessary to protect LNPs from aggregation. There were comparable DLS (particle size) results for LCP discs and MAPs. Encapsulation efficiency was performed on liquid dispensing controls and on MAPs. Example 7 Viscosity Studies

[0107] The addition of CMC at 0.4-0.6% increases viscosity of the mRNA-LNP formulation to about 2 cP, which is close to the viscosity of solutions known to coat the microprojections well. Tested successfully with eGFP and luciferase mRNA LNPs. Table 1 – Formulations and Viscosity (data here are from a single experiment)

[0108] Three (3) formulations were selected that were in the 2-3 cP range for further coating development (highlighted in bold). Carboxymethylcellulose (CMC) increases viscosity and improves coating (MJet and DirectJet) Example 8 Stability Studies with CMC

[0109] Selected formulations were assessed for stability for up to 3 months at 5°C, 25°C / 60%RH and 40°C / 75%RH. Primary evaluation of stability was particle size and mRNA encapsulation upon rehydration. Formulations capable of coating the microprojections wereC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 30 - tested for the ability to transfect and express protein in a cell-based assay (eGFP). The formulations were evaluated for up to 24 months. A preferred embodiment of the formulation is stability of ≥ 12 months at 5°C plus ≥ 3 days at 40°C / 75%RH.

[0110] Stability data on discs showed good stability for material with HPBCD. After adding a viscosity modifier (in this case, CMC) to improve coating when printing the formulation onto microprojections, another disc study was conducted to compare HPBCD plus CMC with HPBCD alone. The HPBCD + CMC formulation showed good stability on discs at 2-8°C and 40°C. Higher concentrations of HPBCD in combination with CMC were tested but these gave poorer stability outcomes. Combinations of stabilizing additive and viscosity modifier were found to have minimal impact on thermostability including 1:20 HPBCD with 0.4% CMC. The formulation had ex vivo and in vivo delivery efficiency of ~65-85% in studies where the formulation was applied to the MAP by DirectJet coating. Studies conducted with MJet printing on MAP had immediate loss upon drying, but has a relatively stable encapsulation efficiency for 28 days

[0111] Coating solution had an approximately 70-80% encapsulation efficiency which dropped to encapsulation efficiency of about 50-60% after printing and rehydrating.

[0112] The addition of CMC does not appear to negatively impact mRNA-LNP stability while increasing HPBCD reduces particle integrity. Before further coating development for preclinical studies occurred, the inital impact on LNP stability of CMC added to the formulation was assessed. Increasing cyclodextrin ratio was also assessed for stability improvements. Formulations were applied to LCP discs (2 µg) and dried before storing at 2- 8°C & 40°C. The eluted discs were compared to GF-S1G-05 liquid control stored at 2-8°C. Particle size increased on dry down, however encapsulation retained greater than 85% in presence of CMC even after 14 days at 40°C for 1:20 HPBCD formulation. Increasing HPBCD may be detrimental for encapsulation efficiency.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 31 - Table 2 – Particle Size and Encapsulation Efficiency for FormulationsTable 3 – Particle Size and Encapsulation Efficiency for Formulations with CMCExample 9 In vivo Expression Studies in Mice

[0113] Studies demonstrated delivery of mRNA by HD-MAP via luminescence imaging in mice with the coated product using the reporter gene luciferase to confirm the transfer of mRNA into skin in vivo. Immunogenicity studies in mice demonstrate immunogenicity of HD- MAP vaccine delivery in mice with the mRNA-carrier formulations dried onto HD-MAPs. Serum IgG response (ELISA) is the primary measure. Cell-mediated responses are assessed by ELISPOT or intracellular cytokine staining as a secondary measure. In vivo expression studies (mice) demonstrated expression of luciferase for up to 9 days (limit of study duration) at two dose levels (target 1ug and 0.5ug) in HPBCD / CMC formulation coated via DirectJet. An eGFP mRNA-LNP delivery study in ex vivo pig was performed to estimate delivery efficiency at approximately 65-80% delivery efficiency. The luciferase mRNA expression studyC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 32 - demonstrated approximately 75% delivery. An in vivo immunogenicity hemagglutinin (HA) delivery study using MAP coated by DirectJet provided an approximately 65% delivery rate and an approximately 57% encapsulation efficiency. Additional MAPs were coated via MJet printing for two doses of HA at two dose levels, by HD-MAP with ID and IM cases. Lower encapsulation efficiency was seen. Example 10 Ex vivo Expression Studies in Pigs

[0114] A study was conducted to test delivery efficiency of two formulations using 2.5K HD- MAPs: 2ug of GF-S1G-051:20 HPBCD, 0.4% CMC and 2ug of GF-S1G-051:40 HPBCD, 0.4% CMC. Coated HD-MAPs were applied to the inside hind leg of ex vivo pig. The coated HD-MAPs were applied for 120s. Pre- and post-application SEM images were used to assess coating removal. Post-application the RiboGreen method was used to determine removal of mRNA and delivery efficiency. eGFP mRNA-carrier material is effectively delivered into ex vivo pig skin at about 65-80% delivery efficiency. Delivery efficiency was determined by calculating % difference of total RNA (by RiboGreen®) on unapplied vs applied MAPs. Figure 6 is a plot of delivery efficiency for 2ug of GF-S1G-05 (green fluorescent protein mRNA in LNP) 1:20 HPBCD, 0.4% CMC and 2ug of GF-S1G-05 (green fluorescent protein mRNA in LNP) 1:40 HPBCD, 0.4% CMC. Example 11 In vivo Luciferase Expression Studies in Mouse

[0115] A study was conducted to deliver ≥2 dose levels of luciferase mRNA by HD-MAP and assess for successful expression in vivo. Groups were nominally (1) Naïve, (2) luciferase mRNA Moderna-like LNPs 1 ug mRNA, (3) luciferase mRNA Moderna-like LNPs 0.5 ug mRNA, and (4) luciferase mRNA Moderna-like LNPs 1 ug mRNA delivered IM. HD-MAPs were coated with LU-B1G-01 formulated in 1:20 HPBCD, 0.4% CMC, 10 mM Tris-HCl pH7.4. The MAPs were coated using DirectJet in two groups: 1 and 0.5 µg mRNA. The positive control (Group 4) was applied in two subsets (to provide information if HD-MAPs were not effective): (a) 1 µg MAP eluted with injectable saline into 50 µL, and the eluateC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 33 - administered IM, and (b) the coating solution used to coat Group 2, administered IM (not dose matched for delivery efficiency or other losses). HD-MAPs and controls were applied to 5 mice per group. Luciferase expression was to be observed, from 4h post vaccination, by luciferin administered IP. Delivery efficiency was determined by SEM and residual RNA (RiboGreen). All treated mice expressed luciferase and there was an increase in expression over time up to around 72h post dosing, which then decreased over time, but all treatment groups had some luciferase expression until the study end after Day 9. Example 12 In vivo Hemagglutinin (HA) Expression Studies in Mouse (DirectJet coating)

[0116] A delivery study was conducted to obtain estimates for delivery efficiency by delivering 0.5-1 ug HA mRNA by eluting MAPs post application. HA mRNA Moderna-like LNPs were delivered at 1.0ug and 0.5ug and delivered ID on MAP n=5. The HA was H1N1 A / Auckland / 1 / 09 strain. Doses were selected based on previous mRNA dose experience and on ‘coatability’ using direct jet. Coating solution (CS) the same: Group 1 vs Group 2 differ in ‘number of coats’ per projection only. A comparison with a cutaneous route (ID) was explored. Formulation behaved very differently during coating / dispensing process despite being ‘Moderna-like LNPs. No aggregates were observed during the coating process. Coating solution was prepared daily before coating. The actual amount of mRNA per MAP was determined after application due to time constraint / limited MAP availability. Table 4 – Particle size and Encapsulation Efficiency for Formulations

[0117] Post application SEM indicated low-moderate delivery efficiency of about 65% for both groups. Delivered dose was targeted at 1µg and 0.5µg but actual delivered dose was ~0.3µg and 0.2µg, respectively. The actual coated dose was for Group 1= 0.48µg and Group 2C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 34 - = 0.34µg, respectively. Increase coats for immune study. Figure 7 is a plot of delivery efficiency for 1ug and 0.5ug of hemagglutinin mRNA in LNP. Example 16 In vivo Hemagglutinin (HA) Expression Studies in Mouse (MJet Coating)

[0118] A delivery study was conducted to obtain estimates for delivery efficiency by delivering 0.5-2 ug HA mRNA by eluting MAPs post application. HA mRNA Moderna-like LNPs were delivered at 2.0ug, 1.0ug and 0.5ug and delivered ID on MAP n=6. The HA was H1N1 A / Auckland / 1 / 09 strain and the formulation was coating using MJet method. 2, 1 and 0.5ug delivered by MAP and ID; negative IM control (2 groups) and no treatment control. The encapsulation efficiency of the formulated coated MAPs was around 55%. Example 17 Stability Studies on MAP

[0119] mRNA LNP formulations on HD-MAP were tested for stability at 2-8°C and 28+ days at 40°C. The stability study assessed particle size, encapsulation efficiency, mRNA integrity of the mRNA LNP materials of coated MAPs stored at 2-8°C, 25°C, and 40°C. The formulations included eGFP mRNA LNP (Moderna-like) + HPBCD (1:20) + (3µg mRNA / MAP) and eGFP mRNA LNP (Moderna-like) + HPBCD (1:20) + 0.4% CMC (3µg mRNA / MAP). The formulations remained stable for 6 months at 2-8°C and for 28 days at 40°C.

[0120] Figure 9 is a plot of Z average (particle size) at 2-8°C, 25°C and at 40°C over 180 days. MAPs coated with eGFP mRNA mLNP when stored at 2-8°C maintained a Z-average of < 200 nm in the presence or absence of 0.4% CMC. MAPs coated with eGFP mRNA mLNP when stored at 25°C maintained a particle size for the mRNA-LNP 0f approximately 200 nm after 6 months of storage. MAPs coated with eGFP mRNA mLNP without 0.4% CMC stored at 40 °C showed the particle size ~ 228 nm after storage for 6 months. MAPs coated with eGFP mRNA mLNP with (+) 0.4% CMC stored at 40 °C showed an increase in particle size to 336 nm after storage for 6 monthsC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 35 -

[0121] Figure 10 is a plot of encapsulation efficiency at 2-8°C, 25°C and at 40°C over180 days. Figure 11 is a plot of Z average (particle size) at 2-8°C, 25°C and 40°C over 180 days and is derived from the content of Figures 9 and 10. The figures indicate that MAPs coated with eGFP mRNA mLNPs stored at 2-8°C maintained a consistent encapsulation efficiency throughout the 6-month storage period in the presence or absence of 0.4% CMC. MAPs coated with eGFP mRNA mLNPs formulated with (+) 0.4% CMC when stored at 25°C also maintained a consistent encapsulation efficiency of about 50%. MAPs coated with eGFP mRNA mLNPs formulated with (+) 0.4% CMC when stored at 40°C showed decreasing encapsulation efficiency at about 41%. MAPs coated with eGFP mRNA mLNPs formulated without (-) 0.4% CMC when stored at 25°C and 40°C was shown to have a similar trend in encapsulation efficiency with a decreasing trend observed past the T7 time point then an increase at T180. Overall, the formulations are stable with respect to particle size and encapsulation efficiency for 180 days at 2-8°C and 25°C and 28+ days at 40°C.

[0122] The majority of MAP eluates from day 180 showed distinct bands consistent with the liquid control and naked mRNA. MAPs stored at 40°C showed smearing or faint bands at day180 while distinct bands were observed at day 90 indicating some degradation. No bands observed for CS stored at 25°C and 40°C consistent with day 90 results. Expression at day 180 is consistent with expression observed at day 90. MAPs coated with eGFP mRNA mLNPs stored at 2-8°C and 25°C showed expression after 48 hours post transfection. MAPs coated with eGFP mRNA mLNPs stored at 40°C showed no expression throughout the 72-hour observation period. LNPs without any excipients also showed expression after 48 hours post transfection. GF-S1G-05 used here has been stored at 2-8°C for ~8 months. Example 18 Luciferase Stability Study

[0123] Particle size of luciferase mRNA in Moderna-like LNPs coated on MAPs stored at 2- 8°C remained consistent (<200 nm) in the presence or absence of CMC. mLNPs coated on MAPs without CMC stored at 40°C showed an increasing particle size to ~287 nm. mLNPs coated on MAPs with 0.4% CMC stored at 40°C showed a relatively consistent particle size ~ 163 nm. Figure 12 is a plot of the Z-average (particle size) at 2-8°C and 40°C over 185 days.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 36 - Luciferase mRNA in Moderna-like LNPs dried on MAPs were observed to be relatively consistent when stored at 2-8°C with or without the addition of CMC.

[0124] Figure 13 is a plot of encapsulation efficiency of luciferase mRNA in Moderna-like LNPs coated on MAPs stored at 2-8°C and 40°C for 185 days. mLNPs coated on MAPs stored at 40°C also similarly showed a consistent encapsulation efficiency regardless of addition of CMC.

[0125] Luciferase mRNA in Moderna-like LNPs coated on MAPs formulated without CMC when stored at 2-8°C showed slight smearing indicative of slight mRNA degradation. mRNA / LNPs applied to MAPs formulated without CMC stored at 40°C showed faint bands with more intense smearing. mRNA / LNPs dried on MAPs formulated with 0.4% CMC stored at 2-8°C showed single bands indicative of good mRNA integrity. mRNA / LNPs dried on MAPs formulated with 0.4% CMC stored at 40°C showed no banding with some smearing. mRNA / LNP formulations on HD-MAP remain stable for 6 months at 2-8°C and 28+ days at 40°C, based on particle size, encapsulation efficiency, TapeStation mRNA integrity analysis, and qualitative GFP expression (for GFP; no expression data for luciferase case). Formulation with CMC appears to be slightly better for stability maintenance. Example 19 mRNA Concentration Effect on Encapsulation Efficiency on MAPs

[0126] A study was conducted to assess the effects of increasing working stock mRNA concentration (0.4, 0.6 and 0.8 mg / mL) with constant HPBCD ratio of 1:20 on encapsulation efficiency of eGFP mRNA in a Pfizer-like LNP formulation.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 37 - Table 5 – Formulations

[0127] Increasing the concentration of mRNA did not improve encapsulation efficiency. Encapsulation efficiency of formulations without CMC had higher EE than those with 0.4% CMC. Note drying time was ~10-13 mins as before. Example 20 Excipient Concentration Effect on Encapsulation Efficiency

[0128] A study was conducted to elucidate the effects of different excipient ratios of HPBCD including 1:10; 1:20; 1:40: 1:60 on encapsulation efficiency of eGFP mRNA in a Pfizer-like LNP formulation (mRNA concentration was kept at 0.4 mg / ml). Table 6 – FormulationsC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 38 -

[0129] Increasing the ratios to 1:40 or 1:60 (mRNA : HPBCD) does not improve encapsulation efficiency of dried formulations on LCP discs. Encapsulation efficiency of coating solutions decrease after being on ice for 3 hours. See Figure 14A and B. Example 21 Excipient to mRNA Ratio Effect on Encapsulation Efficiency

[0130] A study was conducted to assess the impact of decreasing mRNA to excipient ratio on Pfizer-like LNP stability (1:5, 1:10, 1: 20) with or without CMC (%). Table 7 – Formulations

[0131] Formulations with HPBCD (1:5) with or without CMC was not stable. Encapsulation efficiency of formulations with HPCD (1:10 and 1:20) with or without CMC were similar (65- 70%). See Figure 15. Example 22 Additive to mRNA Ratio Effect on Encapsulation Efficiency

[0132] A study was conducted to assess the impact of different temperatures (on ice and RT) over a 4-hour duration with various ratios of HPBCD and 0.4% CMC on Encapsulation efficiency of liquid formulations.

[0133] Formulation (with HPBCD 1:5 + 0.4% CMC) was stable after 4h on ice or at RT. Formulations seem more stable at RT than on ice.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 39 -

[0134] Studies successfully printed mRNA-LNPs on HD-MAPs and demonstrated temperature stability at 2-8°C and 25°C for 6 months and 40°C for one month on HD-MAPs. Skin delivery efficiency was demonstrated ex vivo in pig and in vivo in mice, in line with protein vaccines (65-85%). Luciferase mRNA was successfully delivered with functional expression in mice (up to 9+ days after coating the MAP. HA mRNA was successfully delivered and immunogenicity of HA mRNA in mice demonstrated, with B-cell and T-cell responses. Example 23 Encapsulation Efficiency Across Excipient Range and mRNA Range

[0135] The encapsulation efficiency of mRNA-LNP formulations with different concentrations of HPGCD (1:5 to 1:80) were tested by drying the formulations on LCP discs. The formulations included 0.4% CMC. Figure 18 is a plot of the encapsulation efficiency of an mRNA-LNP formulation at various concentrations of HPGCD at ratios up to 1:80 (with a fixed concentration of 0.4% CMC). The encapsulation efficiency of the initial liquid containing the mRNA-LNP with HPGCD and 0.4% CMC was about 97%. When the formulation contains no HPGCD the encapsulation of the mRNA is reduced to zero. In the presence of various concentrations of HPGCD and 0.4% CMC the encapsulation efficiency is greater than 90%.

[0136] The encapsulation efficiency of mRNA-LNP formulations with different amounts of mRNA (3.0, 5.0, 6.5 and 10.0 µg) were tested by drying the formulations on LCP discs. The formulations included HPGCD and 0.4% CMC. Figure 19 is a plot of the encapsulation efficiency of an mRNA-LNP formulation at various amounts of mRNA in which HPGCD and 0.4% CMC were in the formulation. In each case the encapsulation efficiency was greater than 80%. The lesser amount of mRNA seemed to improve the encapsulation efficiency.

[0137] Within this disclosure, any indication that a feature is optional is intended provide adequate support (e.g., under 35 U.S.C.112 or Art.83 and 84 of EPC) for claims that include closed or exclusive or negative language with reference to the optional feature. Exclusive language specifically excludes the particular recited feature from including any additional subject matter. For example, if it is indicated that A can be drug X, such language is intended to provide support for a claim that explicitly specifies that A consists of X alone, or that A doesC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 40 - not include any other drugs besides X. "Negative" language explicitly excludes the optional feature itself from the scope of the claims. For example, if it is indicated that element A can include X, such language is intended to provide support for a claim that explicitly specifies that A does not include X. Non-limiting examples of exclusive or negative terms include "only," "solely," "consisting of," "consisting essentially of," "alone," "without", "in the absence of (e.g., other items of the same type, structure and / or function)" "excluding," "not including", "not", "cannot," or any combination and / or variation of such language.

[0138] Similarly, referents such as "a," "an," "said," or "the," are intended to support both single and / or plural occurrences unless the context indicates otherwise. For example "a dog" is intended to include support for one dog, no more than one dog, at least one dog, a plurality of dogs, etc. Non-limiting examples of qualifying terms that indicate singularity include "a single", "one," "alone", "only one," "not more than one", etc. Non-limiting examples of qualifying terms that indicate (potential or actual) plurality include "at least one," "one or more," "more than one," "two or more," "a multiplicity," "a plurality," "any combination of," "any permutation of," "any one or more of," etc. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.

[0139] Where ranges are given herein, the endpoints are included. Furthermore, it is to be understood that 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.

[0140] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 41 -

[0141] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that the various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

[0142] Further advantages of the present immunological compositions and adjuvants of the present invention can be achieved by those skilled in the art based upon the embodiments described herein and are thus specifically within the scope of the present invention.

[0143] Throughout this specification and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers. As used herein and unless otherwise stated, the term "approximately" means ±20%.

[0144] It will of course be realised that whilst the above has been given by way of an illustrative example of this invention, all such and other modifications and variations hereto, as would be apparent to persons skilled in the art, are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims

C:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 42 - THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1) A composition comprising an RNA molecule and a lipid nanoparticle (LNP) and an additive, wherein the RNA molecule is encapsulated in the LNP. 2) The composition of claim 1, wherein the RNA molecule is a self-amplifying RNA (samRNA). 3) The composition of claim 1, wherein the RNA molecule is a messenger RNA (mRNA), a small interfering RNA (siRNA), a short hairpin RNA or small hairpin RNA (shRNA), a microRNA (miRNA), a miRNA inhibitor (antagomirs / antimirs), or a messenger-RNA- interfering complementary RNA (micRNA). 4) The composition of any one of claims 1 to 3, wherein the RNA molecule encodes a polypeptide or a fragment thereof. 5) The composition of any one of claims 1 to 4, wherein the RNA molecule encodes a polypeptide antigen. 6) The composition of any one of claims 1 to 5, wherein the RNA molecule encodes a viral polypeptide. 7) The composition of claim 6, wherein the viral polypeptide is or is derived from a spike protein of a virus. 8) The composition of claim 7, wherein the virus is an influenza virus. 9) The composition of claim 5, wherein the virus is a coronavirus. 10) The composition of claim 9, wherein the coronavirus is SARS-CoV-2. 11) The composition of any one of claims 4 to 9, wherein the RNA molecule is a vaccine against a virus comprising the viral polypeptide or fragment thereof to be administered to a subject in need thereof. 12) The composition of any one of claims 1 to 11, further comprising a second RNA molecule encapsulated in the LNP. 13) The composition of any one of claims 1 to 12, wherein the additive comprises one or more of sucrose, glucose, galactose, fructose, trehalose, maltose, histidine, sodium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium sulfate, sodium succinate, Tris, human albumin, hydrolyzed gelatin, protamine sulfate, benzalkonium chloride, Brij 35, Polaxamer 188, Polysorbate 20, Polysorbate 80, sodium docusate, Triton X-100, glycerol, mannitol, sorbitol, calcium chloride, DTT, EDTA, magnesium chloride, arginine, asparticC:\Users\txe\AppData\Roaming\iManage\Work\Recent\35636923PCT RNA-LNP Formulations Delivered by HD-MAP\VX-HDMAP-RNALNP PCT specification - 35636923(27543248.1).docx-9 / 09 / 2025 - 43 - acid, glutamic acid, glycine, histidine, isoleucine, lactic acid, lysine, maleic acid, malic acid, methionine, proline, urea, arginine, glutamic acid, isoleucine, gamma-cyclodextrin, 2-OH propyl beta cyclodextrin (HPBCD), sulfobutyl ether beta-cyclodextrin (SBECD), carboxymethylcellulose, dextran sulfate, dextran 40, PEG-3350, sodium hyaluronate, betaine, firoin, ectoine, gamma-cyclodextrin, alpha-cyclodextrin, methyl beta cyclodextrin (MBCD), random methylated beta-cyclodextrin (RAMEB), 2-OH propyl gamma cyclodextrin (HPGCD) and carboxymethyl beta cyclodextrin (CMBCD), hydroxypropyl methylcellulose (HPMC), maltodextrin, polyvinylpyrrolidone, guar gum, methylcellulose, beta-cyclodextrin and combinations thereof. 14) The composition of claim 13 wherein the additive is a cyclodextrin. 15) The composition of claim 14 wherein the cyclodextrin is selected from the group consisting of sulfobutyl ether beta-cyclodextrin (SBECD), 2-OH propyl beta cyclodextrin (HPBCD), methyl beta cyclodextrin (MBCD), random methylated beta-cyclodextrin (RAMEB), 2- OH propyl gamma cyclodextrin (HPGCD) and carboxymethyl beta cyclodextrin (CMBCD) and combinations thereof. 16) The composition of claim 15 wherein the RNA:cyclodextrin ratio is from 1:5 to 1:

80. 17) The composition of claim 16 wherein the RNA:cyclodextrin ratio is 1:

20. 18) The composition of claim 17 further comprising a non-cyclodextrin additive. 19) The composition of claim 18 wherein the non-cyclodextrin additive is carboxymethyl cellulose. 20) The composition of claim 19 wherein the carboxymethyl cellulose is present at a concentration of between 0.2 and 0.6%. 21) The composition of claim 20 wherein the carboxymethyl cellulose is present at a concentration of 0.4%. 22) The composition of claim 15 wherein the composition is a liquid composition that has been dried onto a surface. 23) The composition of claim 22 wherein the mRNA is encapsulated in the LNP at an encapsulation efficiency of greater than 70%. 24) The composition of claim 23 wherein the mRNA is encapsulated in the LNP at an encapsulation efficiency of from 80% to 100%.

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