Bioabsorbable particles and methods of use

Abstract

The present disclosure provides bioabsorbable polymeric microparticles containing a bioactive agent. The polymeric microparticles comprise a bioabsorbable polymer matrix and a lipid salt, both of which can be modified to affect particle properties. The polymeric microparticles can be administered to a patient for the treatment of a disease. Methods of making and using the polymeric microparticles are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Provisional Application No. 63 / 318,950, filed March 11, 2022, the entirety of which is incorporated herein by reference for all purposes. Incorporate into the subsections.

[0002] Field The present disclosure relates generally to bioabsorbable particles. More specifically, the present disclosure relates to particles comprising a polymer, a lipid salt and a therapeutic mRNA for therapeutic use. [Background technology]

[0003] background Numerous developments in the medical field are resulting in new technologies to fight disease. One such development is the use of mRNA as an active therapeutic agent. Messenger RNA (mRNA) has proven to be an effective vaccine, but is also being investigated for the treatment of various infections and diseases, such as influenza, tuberculosis, malaria, liver disease, HIV, and cancers, including skin, pancreatic, ovarian, and head and neck cancers.

[0004] A limitation of current mRNA therapy is the delivery mechanism of the mRNA to cells. Current delivery methods include encapsulating the mRNA in cationic lipids and / or encapsulating the mRNA in particles composed of polymers such as polysaccharides, cholesterol or polylactides. All current delivery mechanisms have limited stability and shelf life, especially above freezing temperatures. Current delivery mechanisms also exhibit low loading capacity - low numbers of mRNA contained within each delivery particle - necessitating large volumes to be administered to patients. Current delivery mechanisms also have limited ability to modify the timing of release of the therapeutic mRNA from the delivery mechanism after administration. Additionally, the high charge and / or increased acidity exhibited by current mRNA delivery mechanisms during mRNA release is also associated with increased inflammation at the delivery site in vivo. This application addresses the need for improved delivery mechanisms of therapeutic mRNA. Summary of the Invention

[0005] Abstract The present disclosure provides bioabsorbable particles (also referred to herein as polymeric microparticles) containing therapeutic mRNA. The polymeric microparticles include a bioabsorbable polymer matrix, such as a trimethylene carbonate (TMC)-based polymer, and a cationic lipid, both of which can be modified to affect the properties of the microparticle. The polymeric microparticles can be administered to a patient for the treatment of a disease. Methods of making and using the polymeric microparticles are also provided.

[0006] A formulation comprising a plurality of bioabsorbable polymeric microparticles, each polymeric microparticle comprising a polymeric matrix, a therapeutic mRNA encapsulated within the polymeric matrix, and a cationic lipid encapsulated within the polymeric matrix.

[0007] A method of producing a formulation of polymeric microparticles, the method comprising the steps of dissolving a polymer and a cationic lipid in a solvent, adding a therapeutic mRNA to the dissolved structural polymer and cationic lipid to create an oil solution, stirring the oil solution in a first mix, adding an aqueous solution to the oil solution, stirring the oil solution in a second mix, adding water to the oil solution, recovering a plurality of particles from the oil solution, and purifying the plurality of particles.

[0008] An additional method of producing a formulation of polymeric microparticles, the method comprising the steps of dissolving a polymer and a cationic lipid in a solvent, adding a therapeutic mRNA to the dissolved structural polymer and cationic lipid to create an oil solution, mixing, stirring or sonicating the oil solution, adding an aqueous solution to the oil solution, mixing, stirring or sonicating the oil solution again, adding water to the oil solution, recovering a plurality of particles from the oil solution, and purifying the plurality of particles.

[0009] A method of treating a patient, the method comprising the steps of delivering to the patient a plurality of particles, each particle comprising a polymer matrix, a therapeutic mRNA encapsulated within the polymer matrix, and a cationic lipid encapsulated within the polymer matrix, the ratio of the cationic lipid to the polymer matrix in each particle being about 1:4 to about 1:1 by weight, and allowing the polymer matrix to biodegrade in the patient to release the therapeutic mRNA. Further, in some embodiments, no cationic lipid may be used.

[0010] A formulation comprising a plurality of polymeric microparticles, which may be identical or different, each microparticle comprising a polymer matrix, a therapeutic mRNA encapsulated within the polymer matrix, a cationic lipid encapsulated within the polymer matrix, and a surface polymer attached to a surface of each of the bioabsorbable particles, the surface polymer comprising polyethylene glycol (PEG), each of the bioabsorbable particles comprising at least 5 percent by weight of PEG.

[0011] The above examples are merely examples and should not be construed as limiting or narrowing the scope of any of the inventive concepts otherwise provided by this disclosure. While multiple examples are disclosed, still other embodiments will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative examples. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive. [Brief description of the drawings]

[0012] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrating embodiments and, together with the description, serve to explain the principles of the disclosure.

[0013] [Figure 1] FIG. 1 is a schematic diagram of a bioabsorbable microparticle encapsulating a therapeutic mRNA according to at least one embodiment.

[0014] [Diagram 2] FIG. 2 is a schematic diagram of a bioabsorbable microparticle encapsulating a therapeutic mRNA that includes a surface polymer in accordance with at least one embodiment.

[0015] [Diagram 3] FIG. 3 is a flow chart of a method of making the bioabsorbable microparticles of FIG. 1 according to at least one embodiment.

[0016] [Figure 4] FIG. 4 is a partial flow chart similar to FIG. 3 for making the bioabsorbable microparticles of FIG. 2 according to at least one embodiment.

[0017] [Diagram 5] FIG. 5 is a flow chart of a method of treating a patient with the bioabsorbable microparticles of FIGS. 1-2 according to at least one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description Definitions and Terminology This disclosure is not intended to be read in a restrictive manner, for example, the terms used in this application should be interpreted broadly in the context of the meaning that one of ordinary skill in the art would give such terms.

[0019] With respect to imprecise terms, the terms "about" and "approximately" may be used interchangeably to refer to measurements that include the stated measurement and also include measurements that are reasonably close to the stated measurement. Measurements that are reasonably close to the stated measurement deviate from the stated measurement by a reasonably small amount, as understood and readily ascertained by one of ordinary skill in the relevant art. Such deviations may result, for example, from measurement errors, differences in calibration of measuring and / or manufacturing equipment, human error in reading and / or setting measurements, small adjustments made to optimize performance and / or structural parameters to account for differences in measurements associated with other components, specific implementation scenarios, imprecise adjustment and / or manipulation of objects by humans or machines, and / or the like. In cases where it is determined that the value of such reasonably small differences cannot be readily ascertained by one of ordinary skill in the relevant art, the terms "about" and "approximately" may be understood to mean plus or minus 10% of the stated value.

[0020] A therapeutic mRNA is an mRNA molecule that has a therapeutic activity in vivo, including, but not limited to, the mRNA being transcribed in vivo to produce a protein of interest, which has a therapeutic activity in the patient. By way of example, and without limiting the application or embodiment of the present invention, the mRNA molecule can encode a unique protein of a virus, such as a viral spike protein. After administration to a patient, the therapeutic mRNA is transcribed in vivo to produce the viral spike protein in the patient. The patient then generates antibodies against the spike protein, providing immunity to the virus in the patient.

[0021] Description of Various Embodiments Those skilled in the art will readily appreciate that the various aspects of the present disclosure may be implemented by any number of methods and devices configured to perform the intended functions. It should also be noted that the accompanying drawings referred to herein are not necessarily drawn to scale and may be exaggerated to illustrate various aspects of the present disclosure, and in that regard, the drawings should not be construed as limiting.

[0022] Referring first to Figure 1, an exemplary microparticle 100 is shown. The microparticle 100 includes a surface 110, a polymer matrix 120, a lipid 140, and a therapeutic mRNA 150. In an exemplary embodiment, the microparticle 100 has a size of about 2500 nm or less, such as 50 nm to 250 nm, 100 nm to 300 nm, 300 nm to 800 nm, or 800 nm to 2500 nm. The microparticle 100 can also have a neutral or near neutral charge, such as a zeta potential of about -20 mv to about +20 mv, about -10 mv to about +10 mv, or about 0 mv.

[0023] FIG. 1 is merely an illustration of a microparticle 100, and the depiction of the lipids 140 and therapeutic mRNA 150 as heterogeneous features within the polymer matrix 120 is for illustrative purposes only. The polymer matrix 120, lipids 140, and polynucleotide 150 can be in the form of a homogenous, heterogeneous, emulsion, suspension, or any other mixture, in whole or in part. Furthermore, the polymer matrix 120, lipids 140, and therapeutic mRNA 150 can be in any state of matter. For example, the therapeutic mRNA 150 can be a solid or liquid encapsulated within the polymer matrix 120 (e.g., a solid polymer matrix). Each of the polymer matrix 120, lipids 140, and polynucleotide 150 is further described below.

[0024] In a non-limiting embodiment, the polymer matrix 120 of the microparticle 100 is a bioabsorbable or biodegradable polymer. The polymer matrix 120 can be hydrophobic in nature to slow degradation compared to hydrophilic matrices. The polymer matrix 120 can also exhibit low acid degradation to minimize inflammation. Minimizing inflammation in the target cells and / or surrounding tissues can be important for both therapeutic function and patient comfort. Low acid degradation can also play a role in mRNA stability. In one embodiment, the polymer matrix 120 can be a trimethylene carbonate (TMC) based polymer, such as a copolymer of TMC and polylactic acid (PLA) (hereinafter "PLA:TMC"). PLA:TMC copolymers can be synthesized by methods well known in the art, for example, by combining TMC monomer with an appropriate lactic acid comonomer, such as L-lactic acid comonomer to produce poly(L-lactic acid-TMC) (hereinafter "L-PLA:TMC"), D-lactic acid comonomer to produce poly(D-lactic acid-TMC) (hereinafter "D-PLA:TMC"), L-lactic acid and D-lactic acid and TMC comonomers to produce poly(D / L-lactic acid-TMC) (hereinafter "D / L-PLA:TMC"), etc.

[0025] The number average molecular weight of the PLA:TMC copolymer can be greater than 20,000 g / mol, e.g., 20,000 g / mol or more, 30,000 g / mol or more, 40,000 g / mol or more, or 50,000 g / mol or more. The PLA:TMC copolymer can have a solubility of greater than about 2 wt% in the delivery fluid. In another embodiment, the polymer matrix 120 can include a terpolymer of PLA, TMC, and another polymer, such as polyethylene glycol (PEG), hereinafter "TMC:PLA:PEG." In yet another embodiment, the polymer matrix 120 can include a terpolymer of PLA, TMC, and polyglycolic acid (PGA), hereinafter "TMC:PLA:PGA."

[0026] The types of polymers used, the ratios of polymers used, and the method of manufacture of the polymer matrix 120 can all be varied to tailor the properties of the polymer matrix 120. Such properties can include the ability of the polymer matrix 120 to degrade or release the polynucleotides 150, the effect of pH on the polymer matrix 120, the size of the microparticle 100, the amount of lipids 140 and polynucleotides 150 loaded onto the microparticle 100, the physical properties of the microparticle 100 (e.g., density, melting point, glass transition temperature, elastic modulus, etc.), and the chemical properties of the microparticle 100 (e.g., molecular weight, polarity, charge, etc.).

[0027] In one embodiment, the lipid 140 of the microparticle 100 is a cationic lipid salt. In particular, the lipid 140 can be 1,2-dioleoyl-3-trimethylammonium propane chloride salt (DOTAP) or 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride salt (DOTMA). In other embodiments, any lipid 140 can be used. The amount of lipid 140 loaded into the microparticle 100 can be varied to adjust the properties of the microparticle 100, such as size, loading and / or release of modified mRNA 150, charge, zeta potential, etc. The loading amount of lipid 140 can be expressed as a ratio to the amount of polymer matrix 120. The microparticle 100 can be loaded with lipid 140 such that the ratio of lipid 140 to polymer matrix 120 is about 1:4 to about 1:1, or about 1:2 to about 3:4. The loading amount of lipid 140 can also be expressed as a ratio to the amount of modified mRNA 150. The microparticle 100 can be loaded with lipid 140 such that the ratio of lipid 140 to modified mRNA 150 is about 1:1 to about 10:1, such as about 2:1 to about 5:1, about 5:4 to about 7:4, or about 3:2. The loading of lipid 140 allows for finer tuning of the properties of the microparticle 100. In general, increasing the amount of lipid 140 loaded into the microparticle 100 corresponds to an increased loading of modified mRNA 150 and an increased zeta potential (FIGS. 7-8). It is typically desired that the microparticle 100 has a neutral or slightly negative charge, although this can be altered by the loading amount and type of lipid 140 in the microparticle 100.

[0028] The microparticles 100 can be loaded with at least 0.5 wt% modified mRNA 150, as described below, although this amount can be varied to tailor the properties of the microparticles 100. The microparticles 100 are configured to degrade and release the mRNA 150 for medical treatment. Additionally, the microparticles 100 can be configured to degrade, be absorbed, or release the mRNA in any organ of the patient. The microparticles 100 can be loaded with modified mRNA 150 that is transcribed in vivo into a protein with therapeutic activity. Through this in vivo transcription, the modified mRNA can be used to create in vivo active ingredients or drugs suitable for treating a wide variety of medical problems, including, but not limited to, influenza, tuberculosis, malaria, liver disease, HIV, and cancer, such as skin cancer, pancreatic cancer, ovarian cancer, and head and neck cancer.

[0029] Referring now to FIG. 2, another exemplary microparticle 100' is shown. The microparticle 100' of FIG. 2 is similar to the microparticle 100 of FIG. 1, and like reference numbers refer to like elements, except as otherwise described herein. As shown in FIG. 2, the microparticle 100' can also include a surface polymer 170'. In one embodiment, the surface polymer 170' is covalently attached to at least the surface 110' of the microparticle 100'. A non-limiting example of the surface polymer 170' is a copolymer of PLA and polyethylene glycol (PEG). When PEG is used as the surface polymer 170' (alone or as a copolymer), the surface 110' can be described as being "PEGylated." The surface polymer 170' can be added to the surface 110' of the microparticle 100' by forming the microparticle 100' in the presence of a physical blend of the surface polymer 170' or by forming a terpolymer with the surface polymer 170' and the polymer matrix 120'. Additionally, the surface polymer 170' can be attached to the surface 110' via coupling groups located on the microparticle 100'. Non-limiting examples of such coupling groups include 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), succinimidyl active ester, and NH2-PEG. In other embodiments, the surface polymer 170' can be added onto the surface 110' after the microparticle 100' is fabricated. The surface polymer 170' can aid in the migration of the microparticle 100', the bioabsorption of the microparticle 100', the degradation of the microparticle 100', or the modification of the physical or chemical properties of the microparticle 100'. Each microparticle 100' can include about 1 wt % to about 10 wt % of the surface polymer 170', as further described below.

[0030] Referring now to Figure 3, a method 300 of making the microparticles 100 of Figure 1 is shown. The method 300 includes dissolving 310 the polymer matrix 120 and lipid salts 140 in a suitable solvent, adding modified mRNA 150, sonicating, mixing or stirring the mixture to form an emulsion to produce an oil solution 320, adding an aqueous solution such as polyvinyl alcohol to produce a water-in-oil emulsion 330, sonicating, mixing or stirring the resulting oil emulsion 340, adding water 350, recovering 360 the microparticles 100 by drying, centrifugation or another suitable method, and purifying 370 the microparticles 100. While this method illustrates one method of making the microparticles 100, additional steps can be added to the method 300, including stirring the solution overnight after adding water 350, dispersing the microparticles 100 in water after purifying 370, and / or freeze-drying the microparticles 100 after purifying 370. The dissolving step 310 can be completed using any solvent that can effectively dissolve the polymer matrix 120 and lipids 140, such as, but not limited to, methylene chloride. The sonication, mixing or agitation steps (320 and 340) can be performed on an ice bath. In other embodiments, sonication can be performed at different temperatures, different times and different amplitudes. Once the microparticles 100 are produced, they can be analyzed as known in the art and as discussed in the Examples below.

[0031] Referring now to Figure 4, there is shown in part a method 300' for manufacturing the microparticle 100' of Figure 2. The method 300' shown in Figure 4 is similar to the method 300 of Figure 3, and like reference numbers identify like elements, except as otherwise described herein. As shown in Figure 4, a surface polymer 170' is added to the polymer matrix 120' and lipid salt 140' during dissolution 310'.

[0032] Referring now to Figure 5, a method of treatment 400 is shown. The method of treatment 400 is described with respect to the microparticle 100 of Figure 1, however the method of treatment 400 is equally applicable to the microparticle 100' of Figure 2. The method of treatment 400 includes administering 410 the microparticle 100 to a patient, degrading 420 the polymer matrix 120 within the patient, and subsequently releasing 430 the polynucleotides 150 from the microparticle 100 to the patient. The microparticle 100 may be administered orally 410 via a pill, tablet, or solution, or by injection 410.

[0033] The microparticle 100 is configured to be bioabsorbable, so that the polymer matrix 120, lipids 140 and / or modified mRNA 150 can be absorbed into the body. One possible method of absorption is by pinocytosis within the cell. In pinocytosis, the cell membrane expands and folds around the extracellular material, forming a sac that creates an internalized vesicle. The vesicle eventually fuses with a lysosome, where the contents (in this case the microparticle 100) are digested.

[0034] The microparticles 100, 100' shown in Figures 1-2 and the method shown in Figures 3-5 are provided as examples of various features of the device / method, and while combinations of these illustrated features are clearly within the scope of the invention, the examples and illustrations thereof are not intended to suggest that the inventive concepts provided herein are limited from fewer features, additional features, or alternative features to one or more of the features shown in Figures 1-5. For example, in various embodiments, the method steps shown in Figure 4 can include steps described with reference to Figure 3. It should also be understood that the reverse is true. Furthermore, one or more of the components shown in Figure 1 can be used in addition to or as a substitute for the components shown in Figure 2.

[0035] Polymer Matrix The present disclosure provides a method for producing a polypeptide of interest in a mammalian cell by contacting the mammalian cell with a polymeric microparticle comprising a polymeric matrix. The polymeric matrix can comprise a trimethylene carbonate (TMC)-based polymer, where the TMC-based polymer comprises a copolymer comprising repeating units of TMC and D / L-polylactide (D / L-PLA) (referred to herein as D / L-PLA:TMC or PLA:TMC).

[0036] The PLA:TMC copolymer can have a weight ratio of D / L-PLA to TMC of 3.25:1 to 0.75:1, for example, 3.25:1, 3.00:1, 2.75:1, 2.50:1, 2.25:1, 2.00:1, 1.75:1, 1.50:1, 1.25:1, 1.00:1, 0.75:1, or any range or value encompassed by these endpoints.

[0037] In other words, the PLA:TMC copolymer can be 45-60 wt% D / L-PLA and 40-55 wt% TMC, for example, the weight ratio of D / L-PLA to TMC can be 45% to 55% (45:55), 47% to 53% (47:53), 50% to 50% (50:50), 52% to 48% (52:48), 55% to 45% (55:45), 57% to 43% (57:43), 60% to 40% (60:40), or any value or range included within these endpoints.

[0038] Alternatively, the weight ratio of D / L-PLA:TMC can be from 3:1 to 12:1, for example, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:, 12:1, or any value or range including these endpoints.

[0039] In another embodiment, the TMC-based polymer can be a terpolymer that includes repeat units of TMC, D / L-PLA, and another polymer. In one embodiment, the terpolymer can include repeat units of TMC, D / L-PLA, and polyglycolic acid (PGA), also referred to herein as TMC:PLA:PGA. In another embodiment, the terpolymer can include TMC, D / L-PLA, and polyethylene glycol (PEG), also referred to herein as TMC:PLA:PGA.

[0040] The TMC:PLA:PGA terpolymer can contain an amount of PGA, as a percentage of the D / L-PLA:TMC copolymer, from 3 wt% to 19 wt%, for example, 3 wt% or more, 4 wt% or more, 5 wt% or more, 6 wt% or more, 7 wt% or more, 8 wt% or more, 9 wt% or more, 10 wt% or less, 11 wt% or less, 12 wt% or less, 13 wt% or less, 14 wt% or less, 15 wt% or less, 16 wt% or less, 17 wt% or less, 18 wt% or less, 19 wt% or less, or any range or value subsumed between these endpoints, when the D / L-PLA:TMC weight ratio is from 3.25:1 to 0.75:1.

[0041] The PLA:TMC:PEG terpolymer can include a PEG moiety attached to one end of each copolymer to form a PEG endcap. The PEG endcap can be located on the exterior surface of the microparticle. The PEG endcap can comprise about 1 wt% to 10 wt% of the total weight of the copolymer, such as 1 wt% or more, 2 wt% or more, 3 wt% or more, 4 wt% or more, 5 wt% or more, 6 wt% or less, 7 wt% or less, 8 wt% or less, 9 wt% or less, 10 wt% or less, or any range or value subsumed within these endpoints, where the D / L-PLA:TMC weight ratio is 3:1 to 12:1.

[0042] Encapsulated therapeutic mRNA The polymer matrix of the present disclosure can encapsulate therapeutic mRNA. It is well known in the art that for therapeutic use, mRNA must be modified to improve the stability of the molecule and increase the efficiency of in vivo delivery of the mRNA to target cells. Many methods and techniques for modifying mRNA for therapeutic use are known to those of skill in the art. See Pardi et al., mRNA vaccines- a new era in vaccinology, Nature Reviews Drug Discovery, 17, 261-279 (2018) and U.S. Pat. No. 9,271,996, both of which are incorporated herein by reference. The modified mRNA of the present invention can be a polynucleotide comprising more than 30 nucleotides.

[0043] The population of modified mRNAs can be combined with a lipid component to form an mRNA-lipid complex. The lipid component can include a cationic lipid. Suitable cationic lipids can include, for example, DOTAP or DOTMA.

[0044] The weight ratio of lipid component to polynucleotide component can be, for example, from about 1:1 to about 2:1, from about 5:4 to about 7:4, or about 3:2.

[0045] Polymer Microparticles The therapeutic mRNA of the present disclosure may be encapsulated within a polymeric matrix that includes a TMC-based polymer, such as the polymers described above, within the polymeric microparticles. The loading of the therapeutic mRNA (such as a modified mRNA) within the polymeric microparticles, as a percentage of the weight of the polymeric microparticle, can be about 0.5 wt% or more, for example, 0.5 wt%-5 wt%, 5 wt%-10 wt%, 10 wt%-25 wt%, or any range or value encompassed by these endpoints.

[0046] The weight ratio of therapeutic mRNA, e.g., mRNA-lipid complex, to TMC-based polymer can be about 1:4 to about 1:1, e.g., 1:400, 1:350, 1:300, 1:250, 1:20, 1:150, 1:800, 1:1000, 1:5000, etc.

[0047] The encapsulation efficiency of the polymeric microparticle polymer, as measured by gel electrophoresis, can be at least 25, for example, 25% to 70%, 75 to 80%, or any value or range included between these endpoints.

[0048] The polymeric microparticles of the present disclosure can have an average particle size of 25 μm or less, such as, for example, from 50 nm to 250 nm, from 100 nm to 300 nm, from 300 nm to 800 nm, or from 800 nm to 2500 nm.

[0049] The average particle size of the polymeric microparticles can vary slightly over time. The polymeric microparticles of the present disclosure can vary by no more than 10% over at least one day at temperatures between about 4° C. and about 20° C.

[0050] In addition to a stable average particle size, it is desirable for the encapsulated therapeutic mRNA, such as modified mRNA, to remain stable over a period of time. The encapsulated therapeutic mRNA, such as modified mRNA, of the present disclosure cannot degrade by more than 10% over at least one day at a temperature of about 3° C. to about 7° C. Furthermore, the encapsulated therapeutic material of the present disclosure, such as modified mRNA encapsulated within polymeric microparticles, can be 90% more stable in serum compared to unencapsulated modified mRNA.

[0051] How to use The polymer microparticles can be formulated to provide an injectable therapeutic mRNA. The TMC-based polymer can be dissolved in a solvent along with a cationic lipid. Suitable solvents can include, for example, methylene chloride (CH2Cl2), chloroform (CHCl3), or acetone (C3H6O). The therapeutic mRNA, such as a modified mRNA, can then be added to the solution of the TMC-based polymer and cationic lipid to create an oil solution. The oil solution can then be mixed, stirred, or sonicated, followed by adding an aqueous solution to the oil solution. Suitable aqueous solutions can include, for example, a solution of polyvinyl alcohol or cholic acid. After adding the aqueous solution, the mixture can be mixed, stirred, or sonicated a second time, followed by adding water. A plurality of particles can then be collected from the oil solution and cleaned to provide an injectable therapeutic mRNA.

[0052] The present disclosure further provides a method of treating a patient using the injectable therapeutic mRNA described herein. The method includes delivering a plurality of particles to a patient, the particles may be the same or different. Each particle may include a polymer matrix, a therapeutic mRNA encapsulated within the polymer matrix, and a cationic lipid encapsulated within the polymer matrix. The polymer matrix may biodegrade within the patient to release the therapeutic mRNA.

[0053] The therapeutic mRNA can be used to produce a polypeptide of interest in a mammalian cell. Specifically, the mammalian cell can be contacted with one or more polymeric microparticles encapsulating one or more therapeutic mRNAs. The polymeric microparticles can be formulated to provide an injectable therapeutic mRNA, as described above. In one embodiment, the injectable therapeutic can include a modified mRNA capable of encoding a polypeptide of interest. A population of modified mRNAs can be combined with a lipid component, such as a cationic lipid, to form an mRNA-lipid complex. The mRNA-lipid complex can be combined with a TMC-based polymer to produce a polymeric microparticle in which the mRNA-lipid complex is encapsulated within the TMC-based polymer.

[0054] The TMC-based polymer can include a copolymer including repeating units of trimethylene carbonate (TMC) and D / L-polylactide (D / L-PLA). Alternatively, the TMC-based polymer can include a copolymer including repeating units of TMC and D / L-PLA, where each copolymer is capped at one end with an attached PEG moiety. As yet another option, the TMC-based polymer can include a terpolymer including TMC, D / L-PLA, and PGA.

[0055] The injectable therapeutic mRNA can comprise one or more different polymer microparticle populations. Without wishing to be bound by theory, the use of different polymer microparticles can allow different time release characteristics of the encapsulated therapeutic mRNA. For example, the injectable therapeutic mRNA can comprise a first microparticle comprising a TMC-based copolymer that encapsulates one therapeutic mRNA and a second microparticle comprising a TMC-based terpolymer that encapsulates the therapeutic mRNA. The therapeutic mRNA encapsulated in the first microparticle can be the same or different from the therapeutic mRNA encapsulated in the second microparticle.

[0056] The polymeric microparticles can be selected to provide a designed release. In one embodiment, the polymeric microparticles can release the encapsulated therapeutic mRNA, e.g., modified mRNA, in a rapid release manner. Rapid release can be achieved by having a relatively high weight percentage of mRNA in the polymeric nanoparticles or by adding excipients. Excipients that can be added include polyethylene glycol (PEG), polylactic acid, or copolymers of polylactic acid and glycolic acid. The ratio and / or molecular weight of TMC:PLA can also be modified. Alternatively, the polymeric microparticles can release the encapsulated therapeutic mRNA, e.g., modified mRNA, in a time release manner, also referred to herein as delayed release. Time release can be achieved by having a relatively low weight percentage of mRNA in the polymeric nanoparticles. Additionally, TMC:PLA copolymers with a high percentage of TMC can be utilized. For example, the polymeric microparticles can release less than 50% of the modified mRNA from the encapsulation within a 48 hour period.

[0057] The injectable therapeutic mRNA of the present disclosure can cause less inflammation when contacted with mammalian cells. Without wishing to be bound by theory, TMC-based polymers can degrade into less acidic components, causing less irritation. Furthermore, the amount of lipids in the polymer microparticles is low, which can cause less inflammation.

[0058] Test Method Although particular methods and apparatus are described below, it will be appreciated that other methods or apparatus may be substituted as deemed suitable by those skilled in the art.

[0059] Inherent Viscosity Inherent viscosity (IV) was measured using a Cannon MiniPV-HX automated viscometer with hexafluoroisopropanol (HFIP) as the extraction solvent.

[0060] Microparticle size Microparticle size was measured using a Zetasizer Ultra (Malvern Panalytical) in multi-angle light scattering mode. Microparticles were diluted to 0.2 mg / mL in deionized water, and 1 mL of solution was used to measure size distribution.

[0061] charge The zeta potential of the microparticle dispersion was measured using a Zetasizer Ultra (Malvern Panalytical). The same microparticle solution used for size measurements was pipetted into a disposable collapsible capillary cell (Malvern) and the zeta potential was measured.

[0062] Release of oligonucleotides The amount and rate of oligonucleotides released from the microparticles were measured by gel electrophoresis. The microparticles were weighed and dissolved in 1 mL of dimethyl sulfoxide. The microparticle / DMSO solution was incubated in a 37°C water bath for 2 hours to completely dissolve the microparticles. To separate the oligonucleotides from the lipid salts, 200 μL of the DMSO / microparticle solution was added to 500 μL of a 100 mg / mL heparin and 100 mM octyl BD-galactoside solution in nuclease-free deionized water. The solution was vortexed for 5 minutes at room temperature, placed on a shaker table for 30 minutes at room temperature, and then vortexed for an additional 5 minutes before running on the gel and quantitating. Two oligonucleotide standards at concentrations of 2 ng / μL and 1 ng / μL in nuclease-free water are prepared for the gel. The standard solution should contain the same concentrations of heparin and octyl BD-galactoside as the nanoparticle samples to account for fluorescence due to the reagents. Place 10 μL of each sample or standard into a microcentrifuge tube. Add 2 μL of 6X DNA Orange loading dye to each sample and mix well. Add all 12 μL of each mixture to wells of a 10-well 4-20% TBE gel in 1X TBE buffer. Run the gel at 250V for 25 minutes. Remove the gel and stain with 1X Sybr Gold (50,000X stock diluted in 1X TBE buffer) for 20 minutes with gentle rocking. Image the gel using Image Ready software (Biorad) on a BioRad Gel Doc system. Create a standard curve using the standards and determine the concentrations of unknown samples from the standard curve.

[0063] PEG density characterization The PEG content on the microparticle surface was measured by UV assay. EXAMPLES

[0064] example Polymer synthesis The bioabsorbable polymers identified in Examples 1-6 below were synthesized according to the method described in “Analysis and characterization of resorbable DL-lactide trimethylene carbonate copolyesters”, Journal of Material Science: Materials in Medicine 4 (1993) pp. 381-88.

[0065] Example 1: Characterization of copolymers Five different copolymers were prepared with the components and ratios as shown in Table 1. Samples of each copolymer were analyzed to determine the IV, glass transition temperature (Tg), number average molecular weight (Mn), weight average molecular weight (Mw), z-average molecular weight and polydispersity (PD).

[0066] A purified sample of 75:25 L-PLA:TMC (Sample 1) was obtained by dissolving the copolymer in chloroform (CHCl3) at 2-5 wt%, precipitating in 10x isopropyl alcohol (IPA), followed by drying under vacuum. The analytical results of Sample 1 are shown in Table 1 below.

[0067] A purified sample of 50:50 L-PLA:TMC (Sample 2) was obtained by dissolving the copolymer in chloroform (CHCl3) at 2-5 wt%, precipitating in 10x isopropyl alcohol (IPA), followed by drying under vacuum. The analytical results of Sample 2 are shown in Table 1 below.

[0068] The analytical results for the 86.4:8.7:4.9 D / L-PLA:TMC-PEG5K copolymer (Sample 3) are shown in Table 1. Also shown in Table 1 are the analytical results for the 73.4:21.8:4.8 D / L-PLA:TMC-PEG5K copolymer (Sample 4) and the 86.5:8.5:5 D / L-PLA:TMC-PEG10K copolymer (Sample 5). [Table 1]

[0069] Example 2: Characterization of PEG surface density and coverage on PLA-TMC-PEG particles In this example, the characterization of PEG content on the surface of microparticles made from the polymer described in Example 1 is described.

[0070] PEGylated microparticles were prepared by dissolving approximately 160 mg of L-PLA:TMC-PEG5K 86.4:8.7:4.9 polymer (sample 3) in 6 mL of methylene chloride at room temperature. Approximately 15 mL of 0.5 w / v% aqueous cholic acid (available from Sigma Aldrich) was then added to the polymer methylene chloride solution. The resulting mixture was mixed for 1 min on an ice-water bath using the same sonicator and probe. Continuous mode was used for sonication at 45% amplitude to create an oil-in-water emulsion. Additionally, 230 mL of deionized water was added to the emulsion with magnetic stirring at 500 rpm. The emulsion was stirred overnight at room temperature to remove the methylene chloride.

[0071] The resulting hardened microparticles were clarified using a tangential flow filtration system. The system used a Minimate EVO system, product ID: OAPMPUNV, from Pall Corporation (Port Washington, NY), equipped with a membrane module MIDIKROS 20CM 500K MPES 0.5MM FLL x FLL, Repligen Corporation (Waltham, MA), product ID: D02-E500-05-N, mPES membrane, 500kD, 20cm. The particle emulsion was first concentrated from 400mL to 30mL. Then, 70mL of deionized water was added to the concentrated emulsion while stirring. The emulsion was concentrated again to 30mL. This process was repeated twice. The third time, the emulsion was concentrated from 100mL to approximately 15mL. The clarified particle emulsion was then pumped into a clean centrifuge tube.

[0072] PEGylated microparticles were also prepared from L-PLA:TMC-PEG5K polymer 73.4:21.8:4.8 (as described in sample 4) and L-PLA:TMC-PEG10K polymer 86.5:8.5:5 (sample 5) in the same manner as above. A UV assay based on the complexation reaction of PEO with molybdophosphate was used to detect the PEG content on the microparticles. The PEG concentration was determined from the absorbance difference between the blank and the test solution using a calibration curve. According to Koopal et al. (The Effect of Polyethylene Oxide Molecular Weight on Determination of its Concentration in Aqueous Solutions, Talanta, 1982, Vol. 29, p.495-501), this method is independent of the PEO molecular weight. Therefore, a calibration curve was made using different concentrations (10-500 μg / mL) of PEG with a molecular weight of 5K. The blank was obtained from deionized water.

[0073] The tested PEG content of each batch of microparticles is shown in Table 2. The PEG content on the particles was close to the content in the polymer, which means that most of the PEG was on the surface of the particles. [Table 2]

[0074] Example 3: Characterization of TMC:D / L-PLA:PGA terpolymer The TMC:D / L PLA:PGA terpolymers were compounded in a weight ratio of 63.75:21.25:15. The ratio of TMC to PLA was 3:1.

[0075] The inherent viscosity (IV) was measured on a Cannon MiniPV-HX automated viscometer using chloroform (CHCl3) as the solvent. In this example, the glass transition temperature of the polymer was determined to be -1°C with IV(CHCl3) = 1.155 dL / g. In vitro degradation studies showed that the terpolymer essentially decomposed over a period of 6-8 months.

[0076] Example 4: Characterization of TMC:D / L-PLA:PGA terpolymer The TMC:D / L PLA:PGA terpolymers were compounded in a weight ratio of 42.5:42.5:15. The ratio of TMC to PLA was 1:1.

[0077] The inherent viscosity (IV) was measured on a Cannon MiniPV-HX automated viscometer using chloroform (CHCl3) as the solvent. In this example, the glass transition temperature of the polymer was determined to be -1°C with IV(CHCl3) = 1.155 dL / g. In vitro degradation studies showed that the terpolymer essentially decomposed over a period of 6-8 months.

[0078] The characterization of the terpolymer is shown in Table 2 below. [Table 3]

[0079] Example 5: Microparticles of mRNA in L-PLA:TMC copolymer (no lipid salt) Microparticles with encapsulated mRNA were prepared by dissolving 40 mg of L-PLA-TMC copolymer in 5 mL of acetone. 50 μg of CleanCap® mCherry mRNA (996 bp) (TriLink) was dissolved in 2 mL of nuclease-free water. The aqueous mRNA solution was added dropwise to the polymer / acetone solution at a stirring speed of 400 rpm. This emulsion was then added to 10 mL of 2% w / v cholic acid sodium salt (CHA) in water as a stabilizer. The acetone was then completely evaporated by stirring at 400 rpm overnight. The microparticles were then cleaned by tangential flow filtration as described previously in Example 2.

[0080] Example 6: Microparticles of mRNA in L-PLA:TMC copolymer containing the lipid salt DOTAP The mRNA is encapsulated in microparticles made of L-PLA:TMC copolymer in the presence of high levels of the lipid salt DOTAP, specifically at a weight ratio of lipid salt DOTAP to mRNA of 2:1.

[0081] Microparticles are prepared by dissolving approximately 40 mg of L-PLA:TMC 75:25 polymer (Sample 1) and 200 micrograms of lipid salt DOTAP (1,2-dioleoyl-3-trimethylammonium-propane chloride salt, Avanti Polar Lipids Inc) in 6 mL of methylene chloride at room temperature. Then, 0.05 mL of 1 mg / mL CleanCap® mCherry mRNA (TriLink Biotechnologies, San Diego, CA, Cat. No. L-7203) of 996 nucleotides is pipetted into the polymer / DOTAP oil solution. The mixture is sonicated in an ice-water bath using a Branson SXF150 sonicator with a ¼ inch tapered probe. The water-in-oil (W / O) emulsion is placed on a shaker overnight. The separated mixture is sonicated again on ice water for 1 minute to produce a good dispersion. The separated mixture is sonicated again on ice water for 1 minute to produce a good dispersion. Alternatively, for mRNA that is sensitive to shear during sonication, a particle dispersion can be made by adding the aqueous mRNA solution dropwise to the polymer / acetone solution at a stirring speed of 400 rpm.

[0082] Approximately 15 mL of 0.5 wt / v% aqueous cholic acid (Sigma) is then added to the dispersion. The resulting mixture is sonicated for 1 min on an ice-water bath using the same sonicator and probe. Deionized water (230 mL) is added to the emulsion with magnetic stirring. The emulsion is stirred overnight at room temperature to remove the methylene chloride.

[0083] The resulting hardened microparticles are clarified using a tangential flow filtration system. This system used a Minimate EVO system, product ID: OAPMPUNV, from Pall Corporation (Port Washington, NY), equipped with a membrane module MIDIKROS 20CM 500K MPES 0.5MM FLL x FLL, Repligen Corporation (Waltham, MA), product ID: D02-E500-05-N, mPES membrane, 500kD, 20cm. The particle emulsion is first concentrated from 400mL to 30mL. Then, deionized water (70mL) is added to the concentrated emulsion while stirring. The emulsion is concentrated again to 30mL, and this process is repeated two more times. The third time, the emulsion is concentrated from 100mL to approximately 15mL. The clarified particle emulsion is then pumped into a clean centrifuge tube and frozen for further characterization.

[0084] Example 7: Size and charge characterization of the microparticles of Example 6 The size and zeta potential of the microparticles formed according to Example 6 can be determined after cleaning. The microparticles are characterized for size and zeta potential using dynamic light scattering using a Zetasizer Ultra (Malvern Panalytical, Malvern, UK).

[0085] Example 8: mRNA loading characterization of the microparticles of Example 6 The mRNA loading properties of the microparticles described in Example 6 can be characterized using the assays described below.

[0086] The microparticles are weighed and dissolved in 1 mL of dimethyl sulfoxide (Sigma Aldrich). The microparticle / dimethyl sulfoxide (DMSO) solution is incubated in a 37°C water bath for 2 hours to completely dissolve the microparticles. To separate the mRNA from the lipid salts, 200 μL of the DMSO / microparticle solution is added to 500 μL of a solution of 100 mg / mL heparin (Sigma Aldrich) and 100 mM octyl BD-galactoside (Sigma Aldrich) in nuclease-free deionized water (Invitrogen). The solution is vortexed for 5 minutes at room temperature, placed on a shaker table for 30 minutes at room temperature, and then vortexed for an additional 5 minutes before being run on an electrophoresis gel for quantification.

[0087] For electrophoresis, two mRNA standards are prepared in nuclease-free water (Invitrogen) at concentrations of 2ng / μL and 1ng / μL, respectively. The mRNA standard solution contains the same concentrations of heparin and octyl BD-galactoside contained in the microparticle samples to account for fluorescence due to the reagents. Load 10μL of each sample or standard into a microcentrifuge tube. Add 2μL of 6XDNA Orange Loading Dye (Invitrogen) to each sample and mix well. Add the entire 12μL of each mixture to a well of a 10-well 4-20% TBE gel (Invitrogen) in 1XTBE buffer. Run the gel at 250V for 25 minutes. Remove the gel and stain with 1X Sybr Gold (50,000X stock diluted in 1X TBE buffer, Invitrogen) for 20 minutes with gentle rocking. Gels are imaged on a BioRad Gel Doc system using Image Ready software (Biorad). Standards in Image Ready software (Biorad) are used to generate a standard curve, and concentrations of unknown samples are determined from the standard curve.

[0088] Example 9: Microparticles of mRNA in L-PLA:TMC copolymer containing the lipid salt DOTAP The modified mRNA is encapsulated in microparticles made of L-PLA:TMC copolymer in the presence of high levels of the lipid salt DOTAP, specifically at a weight ratio of lipid salt DOTAP to mRNA of 2:1.

[0089] Microparticles are prepared by dissolving approximately 40 mg of L-PLA:TMC 75:25 ("LT-75") polymer (as described in Example 1) and 400 micrograms of lipid salt DOTAP (1,2-dioleoyl-3-trimethylammonium-propane chloride salt, Aventi Polar Lipids Inc) in 6 mL of methylene chloride at room temperature. Then, 0.4 mL of 1 mg / mL CleanCap® mCherry mRNA (TriLink Biotechnologies, San Diego, CA, Catalog No. L-7203) of 996 nucleotides is pipetted into the polymer / DOTAP oil solution. The mixture is sonicated on an ice-water bath using a Branson SXF150 sonicator equipped with a ¼ inch tapered probe. The water-in-oil (W / O) emulsion is placed on a shaker overnight. The separated mixture is agitated again for 1 minute in ice-water to produce a good dispersion. The separated mixture is stirred again for 1 min on ice water to produce a good dispersion. Then, about 15 mL of 0.5 wt / v% cholic acid (Sigma) aqueous solution is added to the dispersion. The resulting mixture is sonicated for 1 min on an ice water bath using the same ultrasonic generator and probe. Additionally, 230 mL of deionized water is added into the emulsion under magnetic stirring. The emulsion is stirred overnight at room temperature to remove the methylene chloride. The resulting hardened microparticles are purified using a tangential flow filtration system. The system utilized a Minimate EVO system, product ID: OAPMPUNV, from Pall Corporation (Port Washington, NY), equipped with a membrane module MIDIKROS 20CM 500K MPES 0.5MM FLL X FLL, Repligen Corporation (Waltham, MA), product ID: D02-E500-05-N, mPES membrane, 500kD, 20cm. The particle emulsion is first concentrated from 400mL to 30mL. Next, add 70 mL of deionized water to the concentrated emulsion while stirring. Concentrate the emulsion again to 30 mL and repeat this process twice. The third time, concentrate the emulsion from 100 mL to approximately 15 mL.The clarified particle emulsion is then pumped into a clean centrifuge tube and frozen for further characterization.

[0090] Example 10: Release of mRNA Characterize the release of encapsulated mRNA at pH 7 and pH 4. To characterize the release of encapsulated mRNA at pH 7, 1 mL of 1 mg / mL mRNA microparticle dispersion made according to Example 5 is incubated in phosphate buffered saline (PBS) solution supplemented with 10% fetal bovine serum at 37° C. for 30 minutes, 1 hour, 4 hours, or 24 hours. Samples for analysis are collected in two ways for comparison. In the first method, the incubated sample is taken directly from the test tube and run on an electrophoresis gel as described above. This method takes into account the fact that the microparticles are too large to enter the gel, and the amount detected in the gel is only the "free" mRNA released in the assay. In the second method, the incubated sample is centrifuged at 10,000 rpm (Beckman Coulter) for 1 hour at 4° C. to separate the microparticles from the "free" mRNA. The supernatant of the centrifuged sample is then run on an electrophoresis gel. These two methods are used to determine whether either method artificially disassociates the mRNA from the microparticles by either force or current.

[0091] To confirm the release of encapsulated mRNA at pH 4, 1 mL of 1 mg / mL mRNA microparticle dispersion made according to Example 5 is incubated in phosphate citrate buffer at pH 4. The remaining analysis, including incubation and gel electrophoresis, is carried out in the same manner as described for the pH 7 sample.

[0092] Example 11: Microparticle size stability after storage The ability of microparticles, such as those formed according to Example 5, to maintain a consistent size when stored in a refrigeration device can be determined as follows.

[0093] The microparticles are placed in a standard refrigeration device (~4 °C) for ~24 h, after which samples are taken and analyzed. Samples are size characterized by dynamic light scattering using a Zetasizer Ultra (Malvern Panalytical, Malvern, UK). It is noted that particle size remains within 10-12% of the microparticles initially generated after cleaning.

[0094] Example 12: Stability of encapsulated mRNA in frozen microparticles The stability of mRNA encapsulated in microparticles when stored frozen can be determined as follows.

[0095] The microparticles are placed in a standard freezer (approximately -18°C) for approximately 24 hours. Samples are then analyzed according to the methods described above for total mRNA content. This content is compared to the same batch of microparticles produced according to Example 5 after purification but without storage. Note that the difference in mRNA content (loading) is less than 10%.

[0096] Example 13: Stability of Encapsulated mRNA in Refrigerated Microparticles The stability of the mRNA encapsulated within the microparticles when stored in a refrigerated device can be determined as follows.

[0097] A known mass of microparticles is placed in a standard freezer (approximately 4°C) for approximately 24 hours. Water is added to the sample to ensure a total volume of 1.5 mL, after which the sample is vortexed and centrifuged to recover the microparticles. A sample of the aqueous phase is taken to determine the mRNA content, occupying a total volume of 1.5 mL.

[0098] The microparticle mRNA content is then analyzed as described above to determine the mRNA content in the microparticles. The amount of mRNA detected in the aqueous phase is added to the encapsulated mRNA as prepared according to Example 5 after purification but without storage to obtain the total mRNA content. Note that the difference in mRNA content (loading) is less than 10%.

[0099] Example 14: Stability of encapsulated mRNA in serum at 37°C The stability of microparticle-encapsulated mRNA in serum at 37° C. can be determined as follows.

[0100] A known mass of microparticles is placed in a temperature controlled chamber (approximately 37° C.) for approximately 24 hours. Serum is added to the sample to ensure a total volume of 1.5 mL, after which the sample is vortexed and centrifuged to recover the microparticles. A sample of the aqueous phase is taken to determine the mRNA content occupying the total volume of 1.5 mL.

[0101] The mRNA content of the microparticles is then analyzed as described above to determine the mRNA content in the microparticles. The amount of mRNA detected in the aqueous phase is added to the encapsulated mRNA as prepared according to Example 5 after cleaning but without storage to obtain the total mRNA content. Note that the difference in mRNA content (loading) is less than 10%.

Claims

1. 1. A method for producing a polypeptide of interest in a mammalian cell, comprising contacting the mammalian cell with a polymer microparticle, the polymer microparticle comprising a trimethylene carbonate (TMC)-based polymer and a modified mRNA encoding the polypeptide of interest encapsulated within the TMC-based polymer, the TMC-based polymer comprising a copolymer comprising repeating units of TMC and D / L-polylactide (D / L-PLA).

2. The method of claim 1, wherein the copolymer has a D / L-PLA:TMC weight ratio of 3.25:1 to 0.75:

1.

3. The method of claim 1 , wherein the copolymer comprises 45-80 wt % D / L-PLA and 20-55 wt % TMC.

4. The method of claim 1 , wherein the TMC-based polymer is a terpolymer comprising repeating units of TMC, D / L-PLA, and polyglycolic acid (PGA).

5. The method of claim 1 , wherein each copolymer is capped at one end with a polyethylene glycol (PEG) bond.

6. The method of claim 1 , wherein the modified mRNA is greater than 30 nucleotides.

7. The method of claim 1, wherein the modified mRNA loading of the polymer microparticles is at least 0.5 wt%.

8. The method of claim 1, wherein the modified mRNA loading of the polymer microparticles is 0.5 wt% to 5 wt%.

9. The method of claim 1, wherein the modified mRNA loading of the polymer microparticles is between 5 wt% and 10 wt%.

10. The method of claim 1, wherein the modified mRNA loading of the polymer microparticles is between 10 wt% and 25 wt%.

11. The method of claim 1, wherein said polymeric microparticles have a percent encapsulation efficiency of at least 60-70%.

12. The method of claim 1, wherein said polymeric microparticles have a percent encapsulation efficiency of at least 75-80%.

13. The method of claim 1 , wherein the polymeric microparticles have an average particle size of 25 μm or less.

14. The method of claim 1, wherein the polymeric microparticles have an average particle size of 50 to 250 nm.

15. The method of claim 1, wherein the polymeric microparticles have an average particle size of 100 to 300 nm.

16. The method of claim 1, wherein the polymeric microparticles have an average particle size of 300 to 800 nm.

17. The method of claim 1, wherein the polymeric microparticles have an average particle size of 800 to 2500 nm.

18. 2. The method of claim 1, wherein the average particle size changes by no more than 10% over a temperature range of 4°C to 20°C over at least one day.

19. The method of claim 1, wherein the modified mRNA encapsulated within the polymer microparticles does not degrade by more than 10% over a period of at least one day at a temperature range of 3°C to 7°C.

20. The method of claim 1, wherein the modified mRNA encapsulated within the polymer microparticles is 90% more stable in serum than non-encapsulated modified mRNA.

21. The method of claim 1 , wherein the polymeric microparticles provide a delayed release of the modified mRNA from the encapsulation.

22. The method of claim 1 , wherein the polymeric microparticles release less than 50% of the modified mRNA from the encapsulation within 48 hours.

23. 1. A method for producing a polypeptide of interest in a mammalian cell, comprising contacting the mammalian cell with a polymeric microparticle, the polymeric microparticle comprising a trimethylene carbonate (TMC)-based polymer and a modified mRNA encoding the polypeptide of interest encapsulated within the TMC-based polymer, the TMC-based polymer comprising a terpolymer comprising TMC, D / L-polylactide (D / L-PLA) and polyglycolic acid (PGA).

24. 24. The method of claim 23, wherein the terpolymer comprises 3-19 wt % PGA and a TMC:D / L-PLA weight ratio of 3.25:1 to 0.75:

1.

25. 24. The method of claim 23, wherein the polymeric microparticles release less than 50% of the modified mRNA from the encapsulation within 48 hours.

26. 1. A method for producing a polypeptide of interest in a mammalian cell, comprising contacting the mammalian cell with a polymeric microparticle, the polymeric microparticle comprising a trimethylene carbonate (TMC)-based polymer and a modified mRNA encoding the polypeptide of interest encapsulated within the TMC-based polymer, the TMC-based polymer comprising a copolymer comprising repeating units of TMC and D / L-polylactide (D / L-PLA), each copolymer being capped at one end by attachment of polyethylene glycol (PEG).

27. 27. The method of claim 26, wherein the D / L-PLA:TMC copolymer comprises 1-10 wt % PEG endcaps and the D / L-PLA:TMC weight ratio is 3:1-12:

1.

28. The method of claim 26 , wherein the PEG endcaps of the copolymer are located on the exterior surface of the microparticle.

29. 27. The method of claim 26, wherein the polymeric microparticles release less than 50% of the modified mRNA from the encapsulation within 48 hours.

30. An encapsulated mRNA injectable therapeutic comprising a modified mRNA encoding a polypeptide of interest encapsulated within a trimethylene carbonate (TMC)-based polymer microparticle, the TMC-based polymer being a copolymer comprising repeating units of trimethylene carbonate (TMC) and D / L-polylactide (D / L-PLA).

31. The injectable bioactive agent of claim 30, wherein the TMC-based polymer comprises a copolymer comprising repeating units of TMC and D / L-polylactide (D / L-PLA), each copolymer being capped at one end with polyethylene glycol (PEG) attached thereto.

32. 31. The injectable bioactive agent of claim 30, wherein the TMC-based polymer comprises a terpolymer comprising (TMC), D / L-polylactide (D / L-PLA) and polyglycolic acid (PGA).

33. A method for producing an encapsulated mRNA injectable therapeutic, comprising the steps of: combining a population of modified mRNAs encoding a polypeptide of interest in a lipid component to generate an mRNA lipid complex; and and combining the mRNA-lipid complexes with a (TMC)-based polymer to produce microparticles, wherein the mRNA-lipid complexes are encapsulated within the TMC-based polymer, the copolymer comprising repeating units of trimethylene carbonate (TMC) and D / L-polylactide (D / L-PLA).

34. 34. The method of claim 33, wherein the TMC-based polymer comprises a copolymer comprising repeating units of TMC and D / L-polylactide (D / L-PLA), each copolymer being capped at one end with a polyethylene glycol (PEG) bond.

35. 34. The method of claim 33, wherein the TMC-based polymer comprises a terpolymer of (TMC), D / L-polylactide (D / L-PLA) and polyglycolic acid (PGA).

36. 34. The method of claim 33, wherein the lipid is a cationic lipid.

37. 37. The method of claim 36, wherein the cationic lipid is DOTAP or DOTMA.

38. 34. The method of claim 33, wherein the ratio of said mRNA-lipid complex to said TMC-based polymer is from 1:4 to about 1:1.

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