EP4489727A1

EP4489727A1 - Bioabsorbable particles and method of use

Info

Publication number: EP4489727A1
Application number: EP23715315.0A
Authority: EP
Inventors: Robert L. Cleek; Kayla E. STEWART
Original assignee: WL Gore and Associates Inc
Current assignee: WL Gore and Associates Inc
Priority date: 2022-03-11
Filing date: 2023-03-10
Publication date: 2025-01-15
Also published as: JP2025508139A; KR20240158984A; WO2023172747A1; CN118843454A

Abstract

The present disclosure provides bioabsorbable polymer microparticles with a bioactive agent. The polymer microparticles include a bioabsorbable polymer matrix and a lipid salt, both of which can be altered to affect the particle properties. The polymer microparticles may be administered to a patient for the treatment of diseases. Methods for making and using the polymer microparticles are also provided.

Description

BIOABSORBABLE PARTICLES AND METHOD OF USE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of Provisional Application No. 63/318,950, filed March 11 , 2022, which is incorporated herein by reference in its entirety for all purposes.

FIELD

[0002] The present disclosure relates generally to bioabsorbable particles. More specifically, the disclosure relates to particles that include a polymer, a lipid salt, and a therapeutic mRNA that are used for medical treatment.

BACKGROUND

[0003] Numerous developments in the medical field have led to new technologies for combatting diseases. One such development is the use of mRNA as an active therapeutic agent. Messenger RNA (mRNA) has proven to be an effective vaccine, but it is also being investigated to treat a range of infections and diseases such as influenza, tuberculosis, malaria, liver diseases, HIV, and cancers such as skin, pancreatic, ovarian, and head and neck cancer, for example.

[0004] A limitation of current mRNA therapies is the delivery mechanism for the mRNA to the cell. Current delivery methods include containing the mRNA in a cationic lipid and/or containing the mRNA in a particle composed of polysaccharide, cholesterol, or a polymer such as polylactide. All of the current delivery mechanisms have limitations in stability and shelf-life, particularly above freezing temperatures. Current delivery mechanisms also exhibit low loading capacity - low numbers of mRNA contained within each delivery particle - which necessitate a larger volume being administered to the patient. The current delivery mechanisms also provide limited ability to modify the timing of release of the therapeutic mRNA from the delivery mechanism after administration. Additionally, the high amount of charge and/or increase in acidity exhibited by current mRNA delivery mechanisms during release of the mRNA is also associated with increased inflammation at the delivery site in vivo. This application addresses the need for an improved delivery mechanism for therapeutic mRNA. SUMMARY

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

[0006] A formulation including a plurality of bioabsorbable polymer microparticles, each polymer microparticle including: a polymer matrix; a therapeutic mRNA encapsulated within the polymer matrix; and a cationic lipid encapsulated within the polymer matrix.

[0007] A method of making a formulation of polymer microparticles, the method including 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 mixing; adding an aqueous solution to the oil solution; stirring the oil solution in a second mixing; adding water to the oil solution; collecting a plurality of particles from the oil solution; and cleaning the plurality of particles.

[0008] An additional method of making a formulation of polymer microparticles, the method including 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; collecting a plurality of particles from the oil solution; and cleaning the plurality of particles.

[0009] A method of treating a patient, the method including the steps of: delivering a plurality of particles to the patient, each particle including: a polymer matrix; a therapeutic mRNA encapsulated within the polymer matrix; and a cationic lipid encapsulated within the polymer matrix, wherein a ratio of the cationic lipid to the polymer matrix in each particle is from about 1 :4 to about 1 :1 weight to weight and allowing the polymer matrix to biodegrade in the patient and release the therapeutic mRNA. Furthermore no cationic lipid may be utilized for some embodiments.

[00010] A formulation including a plurality of polymer microparticles which may be the same or different, each microparticle including: a polymer matrix; a therapeutic mRNA encapsulated within the polymer matrix; a cationic lipid encapsulated within the polymer matrix; and a surface polymer coupled to a surface of each of the bioabsorbable particles, wherein the surface polymer comprises polyethylene glycol (PEG) and each of the bioabsorbable particles comprises at least 5 weight percent of the PEG.

[00011] The foregoing Examples are just that and should not be read to limit or otherwise narrow the scope of any of the inventive concepts otherwise provided by the instant 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 rather than restrictive in nature.

BRIEF DESCRIPTION OF THE DRAWINGS

[00012] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description serve to explain the principles of the disclosure.

[00013] FIG. 1 is a schematic representation of a bioabsorbable microparticle encapsulating a therapeutic mRNA in accordance with at least one embodiment;

[00014] FIG. 2 is a schematic representation of a bioabsorbable microparticle encapsulating a therapeutic mRNA that includes a surface polymer in accordance with at least one embodiment;

[00015] FIG. 3 is a flowchart for a method of making the bioabsorbable microparticle of FIG. 1 in accordance with at least one embodiment;

[00016] FIG. 4 is a partial flowchart similar to FIG. 3 for making the bioabsorbable microparticle of FIG. 2 in accordance with at least one embodiment;

[00017] FIG. 5 is a flowchart for a method of treating a patient with the bioabsorbable microparticle of FIGS. 1-2 in accordance with at least one embodiment.

DETAILED DESCRIPTION

Definitions and Terminology

[00018] This disclosure is not meant to be read in a restrictive manner. For example, the terminology used in the application should be read broadly in the context of the meaning those in the field would attribute such terminology. [00019] With respect to terminology of inexactitude, the terms “about” and “approximately” may be used, interchangeably, to refer to a measurement that includes the stated measurement and that also includes any 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 individuals having ordinary skill in the relevant arts. Such deviations may be attributable to measurement error, differences in measurement and/or manufacturing equipment calibration, human error in reading and/or setting measurements, minor adjustments made to optimize performance and/or structural parameters in view of differences in measurements associated with other components, particular implementation scenarios, imprecise adjustment and/or manipulation of objects by a person or machine, and/or the like, for example. In the event it is determined that individuals having ordinary skill in the relevant arts would not readily ascertain values for such reasonably small differences, the terms “about” and “approximately” can be understood to mean plus or minus 10% of the stated value.

[00020] Therapeutic mRNA is an mRNA molecule that has a therapeutic activity in vivo. This therapeutic activity may include, but is not limited to, the mRNA being transcribed in vivo to create a protein of interest, wherein the protein of interest has a therapeutic activity in the patient. By way of example, and in no way a limitation to the application or embodiments of the present invention, the mRNA molecule may encode a distinctive protein of a virus, such as a viral spike protein. After administration to the patient, the therapeutic mRNA will be transcribed in vivo to produce the viral spike protein in the patient. The patient will then generate antibodies against the spike protein and confer immunity against the virus in the patient.

Description of Various Embodiments

[00021 ] Persons skilled in the art will readily appreciate that various aspects of the present disclosure can be realized by any number of methods and apparatuses configured to perform the intended functions. It should also be noted that the accompanying drawing figures referred to herein are not necessarily drawn to scale but may be exaggerated to illustrate various aspects of the present disclosure, and in that regard, the drawing figures should not be construed as limiting.

[00022] Referring first to FIG. 1 , an exemplary microparticle 100 is shown. The microparticle 100 comprises a surface 110, a polymer matrix 120, a lipid 140, and a therapeutic mRNA 150. In an exemplary embodiment, 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. Also, the microparticle 100 may have a neutral or nearneutral electrical charge, such as a zeta potential from about -20 mv to about +20 mv, from about -10 mv to about +10 mv, or about 0 mv.

[00023] It should be noted that FIG. 1 is only a representation of a microparticle 100, and the depiction of a lipid 140 and therapeutic mRNA 150 as heterogeneous features within the polymer matrix 120 is for illustrative purposes only. The polymer matrix 120, the lipid 140, and the polynucleotide 150 may be entirely or partially homogeneous, heterogenous, an emulsion, a suspension, or any other form of mixture. Additionally, the polymer matrix 120, the lipid 140, and the therapeutic mRNA 150 may be any state of matter. For example, the therapeutic mRNA 150 may be a solid or a liquid encapsulated within the polymer matrix 120 (e.g. solid polymer matrix). Each of the polymer matrix 120, the lipid 140, and the polynucleotide 150 is described further below.

[00024] In a non-limiting embodiment, the polymer matrix 120 of the microparticle 100 is a bioabsorbable or biodegradable polymer. The polymer matrix 120 may be hydrophobic in nature to promote slower degradation than a hydrophilic matrix. The polymer matrix 120 may also exhibit low acid degradation to minimize inflammation. Minimizing inflammation in the target cell and/or surrounding tissue may be important both to therapeutic function and to the comfort of the patient. Low acid degradation can also play a role in stability of mRNA. In one embodiment, the polymer matrix 120 may be a trimethylene carbonate (TMC)-based polymer, such as a copolymer of TMC and polylactic acid (PLA), hereinafter “PLA:TMC”. The PLA:TMC copolymer may be synthesized using methods well-known to the art, such as, for example, by combining TMC monomers with suitable comonomers of lactic acid, such as L-lactic acid comonomers creating poly(L-lactic acid-TMC) hereinafter “L-PLA:TMC”; D-lactic acid comonomers creating poly(D-Lactic acid — TMC) hereinafter “D-PLA:TMC”; and comonomers of L-lactic acid and D-lactic acid and TMC creating poly(D/L-lactic acid — TMC) hereinafter “D/L-PLA:TMC”.

[00025] The PLA:TMC copolymer may have a number average molecular weight greater than 20,000 g/mol, such as 20,000 g/mol or greater, 30,000 g/mol or greater, 40,000 g/mol or greater, or 50,000 g/mol or greater. The PLA:TMC copolymer may have a solubility in the delivery fluid greater than about 2 wt.%. In another embodiment, the polymer matrix 120 may comprise 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 may comprise a termpolymer of PLA, TMC, and polyglycolic acid (PGA), hereinafter “TMC: PLA: PGA”.

[00026] The type of polymer(s) used, the ratios of polymers used, and the method of making the polymer matrix 120 may all be altered to adjust the properties of the polymer matrix 120. Such properties may include: the ability of the polymer matrix 120 to degrade or otherwise release the polynucleotide 150, the effect of pH on the polymer matrix 120, the size of the microparticle 100, the amount of the lipid 140 and the polynucleotide 150 loaded into microparticle 100, the physical properties of the microparticle 100 (e.g., density, melting point, glass transition temperature, modulus, etc.), and the chemical properties of the microparticle 100 (e.g., molecular weight, polarity, charge, etc.).

[00027] In one embodiment, the lipid 140 of the microparticle 100 is a cationic lipid salt. In particular, lipid 140 may 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 may be used. The amount of lipid 140 loaded in the microparticle 100 may be altered to adjust characteristics of microparticle 100, such as size, loading and/or release of the modified mRNA 150, charge, zeta potential, etc. The loading of the lipid 140 may be expressed as a ratio with respect to the amount of the polymer matrix 120. The microparticle 100 may be loaded with the lipid 140 such that the ratio of the lipid 140 to the polymer matrix 120 is from approximately 1 :4 to approximately 1 :1 or from approximately 1 :2 to approximately 3:4. The loading of the lipid 140 may also be expressed as a ratio with respect to the amount of the modified mRNA 150. The microparticle 100 may be loaded with the lipid 140 such that the ratio of the lipid 140 to the modified mRNA 150 is from approximately 1 :1 to approximately 10:1 , such as from approximately 2:1 to approximately 5:1 , from approximately 5:4 to approximately 7:4, or approximately 3:2. The lipid 140 loading allows for more fine-tuning of microparticle 100 characteristics. Generally, an increase in the amount of the lipid 140 loaded in the microparticle 100 corresponds to an increase in loading of the modified mRNA 150, and an increase in zeta potential (FIGS. 7-8). It is typically desirable for the microparticle 100 to have a neutral or slightly negative charge, which can be altered by the loading and type of the lipid 140 in the microparticle 100.

[00028] The microparticle 100 may be loaded with at least 0.5 wt.% of the modified mRNA 150 as discussed further below, although this amount may be altered to adjust characteristics of the microparticle 100. The microparticle 100 is configured to degrade and to release the mRNA 150 for medical treatment. Further, the microparticles 100 may be configured to degrade, be absorbed, or release the mRNA within any organ of the patient. The microparticles 100 may be loaded with modified mRNA 150 that will be transcribed in vivo into a protein with therapeutic activity. Through this, transcription in vivo, the modified mRNA can used to create an active ingredient or drug in vivo suitable for treatment of a wide variety of medical issues including, but not limited to, influenza, tuberculosis, malaria, liver diseases, HIV, and cancers such as skin, pancreatic, ovarian, and head and neck cancer.

[00029] Referring next to FIG. 2, another exemplary microparticle 100’ is shown. Microparticle 100’ of FIG. 2 is similar to microparticle 100 of FIG. 1 , with like reference numerals identifying like elements, except as described herein. As shown in FIG. 2, the microparticle 100’ may also comprise a surface polymer 170’. In one embodiment, the surface polymer 170’ is covalently bonded to at least the surface 110’ of the microparticle 100’. A non-limiting example of a surface polymer 170’ is a copolymer of PLA and polyethylene glycol (PEG). When PEG is used as the surface polymer 170’ (either on its own or in a copolymer), the surface 110’ may be described as being “PEGylated”. The surface polymer 170’ may be added onto 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 terpolymers with the surface polymer 170’ and the polymer matrix 120’. Additionally, the surface polymer 170’ may be added to the surface 110’ through a coupling group located on the microparticle 100’. Nonlimiting examples of such coupling groups include 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), succinimidyl active esters, and NH2-PEG. In other embodiments, the surface polymer 170’ may be added onto the surface 110’ after the microparticle 100’ has been made. The surface polymer 170’ may assist in the migration of the microparticles 100’, in the bioabsorption of the microparticles 100’, in the degradation of the microparticles 100’, or in altering the physical or chemical characteristics of the microparticles 100’. Each microparticle 100’ may include from about 1 wt.% to about 10 wt.% of the surface polymer 170’, as discussed further below.

[00030] Referring now to FIG. 3, a method of making 300 the microparticle 100 of FIG. 1 is shown. The method of making 300 includes dissolving 310 in the polymer matrix 120 and the lipid salt 140 in a suitable solvent, adding the modified mRNA 150 and forming an emulsion 320 through sonication, mixing, or stirring the mixture to produce an oil solution, adding an aqueous solution 330 such as polyvinyl alcohol to produce a water-in-oil emulsion, sonicating, mixing, or stirring 340 the resulting oil emulsion, adding water 350, collecting 360 the microparticles 100 by drying, centrifuging, or another suitable method, and cleaning 370 the microparticles 100. While this method demonstrates one way to make the microparticles 100, additional steps may be added to the method 300 including, such as, stirring the solution overnight after adding water 350, dispersing the microparticles 100 in water after cleaning 370, and/or lyophilizing the microparticles 100 after cleaning 370. The step of dissolving 310 may be completed with any solvent capable of effectively dissolving the polymer matrix 120 and the lipid 140, such as, but not limited to, methylene chloride. The steps of sonicating, mixing or stirring (320 and 340) may be carried out over an ice bath. In other embodiments, the sonication may take place at various temperatures, over various lengths of time, and with various amplitudes. Once the microparticles 100 have been made, they can be analyzed as is known in the art and as discussed in the later examples.

[00031] Referring now to FIG. 4, a method of making 300’ the microparticle 100’ of FIG. 2 is shown in part. The method of making 300’ shown in FIG. 4 is similar to the method of making 300 of FIG. 3, with like reference numerals identifying like elements, except as described herein. As shown in FIG. 4, the surface polymer 170’ is added to the polymer matrix 120’ and the lipid salt 140’ during the dissolving 310’.

[00032] Referring now to FIG. 5, a method for treatment 400 is shown. This method of treatment 400 is described with respect to the microparticles 100 of FIG. 1 , but this method of treatment 400 is equally applicable to the microparticles 100’ of FIG. 2. The method of treatment 400 includes administering 410 the microparticles 100 to the patient, degrading 420 the polymer matrix 120 in the patient, and subsequently releasing 430 the polynucleotide 150 from the microparticle 100 and into the patient. The microparticles 100 may be administered 410 orally through a pill, tablet, or solution, or it may be administered 410 through injection.

[00033] The microparticles 100 are configured to be bioabsorbable such that the polymer matrix 120, the lipid 140, and/or the modified mRNA 150 may be absorbed by the body. One potential method of absorption is through pinocytosis within cells. In pinocytosis, the cell membrane extends and folds around extracellular material, forming a pouch that creates an internalized vesicle. The vesicles eventually fuse with the lysosome where the contents (in this case, the microparticles 100) are digested.

[00034] The microparticles 100, 100’ shown in FIGS. 1-2 and the methods shown in FIGS. 3-5 are provided as examples of the various features of the device/methods and, although the combination of those illustrated features is clearly within the scope of invention, that example and its illustration is not meant to suggest the inventive concepts provided herein are limited from fewer features, additional features, or alternative features to one or more of those features shown in FIGS. 1-5. For example, in various embodiments, the steps of the method shown in FIG. 4 may include the steps described with reference to FIG. 3. It should also be understood that the reverse is also true. In addition, one or more of the components depicted in FIG. 1 can be employed in addition to, or as an alternative to components depicted in FIG. 2.

Polymer matrix

[00035] The present disclosure provides a method of producing a polypeptide of interest in a mammalian cell by contacting said mammalian cell with a polymer microparticle comprising a polymer matrix. The polymer matrix may comprise a trimethylene carbonate (TMC)-based polymer, wherein said 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.

[00036] The PLA:TMC copolymers may have a weight ratio of D/L-PLA to TMC of from 3.25:1 to 0.75:1 , such as 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.

[00037] Stated alternatively, the PLA:TMC copolymers may have a weight ratio of D/L-PLA to TMC of from 45-60 wt.% D/L-PLA and from 40-55 wt.% TMC, such as a weight ratio of D/L-PLA to TMC of 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 encompassed by these endpoints.

[00038] Alternatively, the weight ratio of D/L-PLA:TMC may be from 3:1 to 12:1 , such as 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 11 :, 12:1 , or any value or range encompassing these endpoints.

[00039] In another embodiment, the TMC-based polymer may be a terpolymer comprising repeating units of TMC, D/L-PLA and another polymer. In one embodiment, the terpolymer may comprise repeating units of TMC, D/L-PLA, and polyglycolic acid (PGA), also referred to herein as TMC:PLA:PGA. In another embodiment, the terpolymer may comprise TMC, D/L-PLA, and polyethylene glycol (PEG), also referred to herein as TMC:PLA:PGA.

[00040] The TMC:PLA:PGA terpolymer may comprise PGA in an amount of 3 wt.% to 19% wt.%, such as 3 wt.% or greater, 4 wt.% or greater, 5 wt.% or greater, 6 wt.% or greater, 7 wt.% or greater, 8 wt.% or greater, 9 wt.% or greater, 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 encompassed by these endpoints, as a percentage of the D/L-PLA:TMC copolymer, when the D/L-PLA:TMC weight ratio is from 3.25:1 to 0.75:1.

[00041] The PLA:TMC:PEG terpolymer may comprise a PEG moiety attached at one end of each copolymer to form a PEG end cap. The PEG end caps may be located on the outer surface of the microparticles. The PEG end caps may comprise about 1 wt.% to 10 wt.% of the total weight of the copolymer, such as 1 wt.% or greater, 2 wt.% or greater, 3 wt.% or greater, 4 wt.% or greater, 5 wt.% or greater, 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 encompassed by these endpoints, wherein the D/L-PLA:TMC weight ratio is from 3:1 to 12:1.

Encapsulated therapeutic mRNA

[00042] The polymer matrices of the present disclosure may encapsulate a therapeutic mRNA. It is well known in the art that, for use as a therapeutic, the mRNA must be modified to improve stability of the molecule and increase efficiency of in vivo delivery of the mRNA to the target cells. Many methods and techniques for modifying the mRNA for use as a therapeutic are known to the skilled artisan. See Pardi, et al., mRNA vaccines- a new era in vaccinology, Nature Reviews Drug Discovery, 17, 261- 279 (2018); and US 9,271 ,996, both incorporated by reference herein. The modified mRNA of the present invention may be a polynucleotide comprising greater than 30 nucleotides.

[00043] A population of the modified mRNA may be combined with a lipid component to for an mRNA-lipid complex. The lipid component may comprise a cationic lipid. Suitable cationic lipids may include DOTAP or DOTMA, for example.

[00044] The weight ratio of the lipid component to the polynucleotide component may be from about 1 : 1 to about 2:1 , from about 5:4 to about 7:4, or about 3:2, for example.

Polymer microparticle

[00045] The therapeutic mRNA of the present disclosure may be encapsulated within a polymer matrix comprising a TMC-based polymer, such as those described above, in a polymer microparticle. The loading of the therapeutic mRNA, such as a modified mRNA, in the polymer microparticle may be about 0.5 wt.% or greater, such as 0.5 wt.% to 5 wt.%., 5 wt.% to 10 wt.%, 10 wt.% to 25 wt.%, or any range or value encompassed by these endpoints, as a percentage of the weight of the polymer microparticle.

[00046] The weight ratio of the therapeutic mRNA, such as the mRNA-lipid complex, to the TMC-based polymer may be from about 1 :4 to about 1 :1 , such as 1 :400, 1 :350, 1 :300, 1 :250, 1 :20, 1 :150, 1 :800, 1 :1000, 1 : 5000 for example.

[00047] The encapsulation efficiency of the polymer microparticle polymer may be at least 25, such as 25%-70%, 75-80%, or any value or range encompassed by these endpoints, as measured by gel electrophoresis.

[00048] The polymer microparticles of the present disclosure may have a mean particle size no more than 25 urn, such as 50 nm to 250 nm, 100 nm to 300 nm, 300 nm to 800 nm, or 800 nm to 2500 nm, for example.

[00049] The mean particle size of polymer microparticles may change slightly over time. The polymer microparticles of the present disclosure may change no more than 10% over a period of at least one day, at a temperature of about 4°C to about 20°C.

[00050] In addition to stable mean particle size, it is desirable that the encapsulated therapeutic mRNA, such as a modified mRNA, remains stable over a period of time. The encapsulated therapeutic mRNA of the present disclosure, such as modified mRNA, may not degrade more than 10% over a period of at least 1 day at a temperature of about 3°C to about 7°C. Further, the encapsulated therapeutic of the present disclosure, such as modified mRNA encapsulated within the polymer microparticle particles, may be stable in 90% serum relative to unencapsulated modified mRNA.

Methods of use

[00051 ] The polymer microparticles may be formulated to provide an injectable therapeutic mRNA. The TMC-based polymer may be dissolved, along with a cationic lipid, in a solvent. Suitable solvents may include methylene chloride (CH2CI2) chloroform (CHCI3), or acetone (CsHeO) for example. A therapeutic mRNA, such as a modified mRNA, may then be added to the solution of TMC-based polymer and cationic lipid to create an oil solution. The oil solution may then be mixed, stirred or sonicated, followed by the addition of an aqueous solution to the oil solution. Suitable aqueous solutions may include solutions of polyvinyl alcohol or cholic acid, for example. Following addition of the aqueous solution, the mixture may be subjected to a second mixing, stirring or sonication, followed by the addition of water. A plurality of particles may then be collected from the oil solution and cleaned to provide the injectable therapeutic mRNA.

[00052] The present disclosure further provides a method of treating a patient using the injectable therapeutic mRNA described herein. The method comprises: delivering a plurality of particles to the patient, wherein 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 in the patient and release the therapeutic mRNA.

[00053] The therapeutic mRNA may be used to produce a polypeptide of interest in a mammalian cell. Specifically, a mammalian cell may be contacted with one or more polymer microparticles encapsulating one or more therapeutic mRNA. The polymer microparticles may be formulated to provide an injectable therapeutic mRNA, as described above. In an embodiment, the injectable therapeutic may comprise a modified mRNA capable of encoding a polypeptide of interest. A population of the modified mRNA may be combined with a lipid component, such as a cationic lipid, to form an mRNA-lipid complex. The mRNA-lipid complex may be combined with a TMC- based polymer to product a polymer microparticle in which the mRNA lipid complex is encapsulated within the TMC-based polymer.

[00054] The TMC-based polymer may comprise a copolymer comprising repeating units of trimethylene carbonate (TMC) and D/L-polylactide (D/L-PLA). Alternatively, the TMC-based polymer may comprise a copolymer comprising repeating units of TMC and D/L-PLA, wherein each copolymer is capped with a PEG moiety attached at one end. As a further alternative, the TMC-based polymer may comprise a terpolymer comprising TMC, D/L-PLA, and PGA.

[00055] The injectable therapeutic mRNA may comprise one or more different polymer microparticle populations. Without wishing to be bound by theory, using different polymer microparticles may allow for different time release properties for the encapsulated therapeutic mRNA. For example, the injectable therapeutic mRNA may comprise a first microparticle comprising a TMC-based copolymer encapsulating one therapeutic mRNA and a second microparticle comprising a TMC-based terpolymer encapsulating a therapeutic mRNA. The therapeutic mRNA encapsulated in the first microparticle may be the same or different from the therapeutic mRNA encapsulated by the second microparticle. [00056] The polymer microparticle may be selected so as to provide a designed release. In an embodiment, the polymer microparticle may release the encapsulated therapeutic mRNA, such as modified mRNA, in a quick release manner. Quick release may be achieved by a relatively high mRNA weight percent in the polymer nanoparticle, or addition of an excipient. Excipients that may 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 could also be modified. Alternatively, the polymer microparticle may release the encapsulated therapeutic mRNA, such as modified mRNA, in a time-release manner, also referred to herein as a delayed release. Timerelease may be achieved by a relatively low mRNA weight percent in the polymer nanoparticle. Additionally, TMC:PLA copolymer with a high percentage of TMC could be utilized. For example, the polymer microparticle may release less than 50% of the modified mRNA from encapsulation in a 48-hour time period.

[00057] The injectable therapeutic mRNAs of the present disclosure may cause less inflammation when contacting mammalian cells. Without wishing to be bound by theory, the TMC-based polymers may break down into low-acid components, leading to less irritation. Furthermore, the low amount of lipids in the polymer microparticles may lead to less inflammation.

TEST METHODS

[00058] It should be understood that although certain methods and equipment are described below, other methods or equipment determined suitable by one of ordinary skill in the art may be alternatively utilized.

Inherent Viscosity

[00059] Inherent viscosity (IV) was measured using a Cannon MiniPV-HX Automatic Viscometer with hexafluoroisopropanol (HFIP) as the solvent for extraction.

Microparticle Size

[00060] Microparticle size was measured using a Zetasizer Ultra (Malvern Panalytical) at multiangle 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.

Electrical Charge

[00061] Zeta potential of the microparticle dispersion was measured using an Zetasizer Ultra (Malvern Panalytical) The same microparticle solution that was used for size was pipetted into a disposable folded capillary cell (Malvern) to measure zeta potential.

Oligonucleotide Release

[00062] The amount and rate of oligonucleotide released from the microparticles was measured through gel electrophoresis. Microparticles were weighed out and dissolved in 1 mL dimethyl sulfoxide. The microparticle/DMSO solution was incubated in a water bath at 37C for 2 hours to allow microparticles to fully dissolve. To decomplex the oligonucleotide from the lipid salt, 200 pL of the DMSO/microparticle solution was added to 500 pL of a 100 mg/mL heparin and 100 mM Octyl B-D-glactactoside solution in nuclease free, deionized water. The solution was vortexed for 5 minutes at room temperature, placed on the shaker table for 30 minutes at room temperature, and then vortexed for another 5 minutes before running on the gel for quantification. For the gel, prepare two oligonucleotide standards at concentrations of 2 ng/pL and 1 ng/pL in nuclease free water. Standard solutions should include heparin and Octyl B-D- glactactoside in the same concentration that will be in the nanoparticle samples to account for any fluorescence attributed to the reagents. Load 10 pL of each sample or standard into a microcentrifuge tube. Add 2 pL 6X DNA orange loading dye to each sample and mix well. Add all 12 pl of each mixture to the well of a 10-well 4-20% TBE gel in 1X TBE buffer. Run the gel at 250 V 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 the Image Ready software (Biorad) on the BioRad Gel Doc system. Using the standards, create a standard curve and determine the concentration of the unknown samples from the standard curve.

PEG Density Characterization

[00063] The amount of PEG content on microparticle surface was measured through a UV assay.

EXAMPLES Polymer Synthesis

[00064] The bioabsorbable polymers identified in the following Examples 1-6 were synthesized according to the methodologies described in “Analysis and characterization of resorbable DL-lactide trimethylene carbonate copolyesters”, Journal of Material Science: Materials in Medicine 4(1993) pp. 381-88. Example 1 : Characterization of co-polymers

[00065] Five different copolymers were prepared with the components and ratios as shown in Table 1 . A sample of each copolymer was analyzed to determine IV, glass transition temperature (Tg), number average molecular weight (Mn), weight average molecular weight (Mw), z-average molecular weight, and polydispersity (PD).

[00066] A purified sample of 75:25 L-PLA:TMC (Sample 1 ) was obtained by dissolving the copolymer in chloroform (CHCh) at 2-5 wt.% and precipitating in 10x isopropyl alcohol (IPA), followed by drying under vacuum. The results of the analysis of Sample 1 are shown below in Table 1.

[00067] A purified sample of 50:50 L-PLA:TMC (Sample 2) was obtained by dissolving the copolymer in chloroform (CHCh) at 2-5 wt.% and precipitating in 10x isopropyl alcohol (IPA), followed by drying under vacuum. The results of analysis of Sample 2 are shown below in Table 1 .

[00068] The results of analysis for an 86.4:8.7:4.9 D/L-PLA:TMC-PEG5K copolymer (Sample 3) are shown in Table 1 , as are the results of analyses for a 73.4:21.8:4.8 D/L-PLA:TMC-PEG5K copolymer (Sample 4) and an 86.5:8.5:5 D/L- PLA:TMC-PEG10K copolymer (Sample 5).

Table 1

Example 2: Characterization of PEG surface density and coverage on PLA-TMC- PEG particles

[00069] This example describes characterization of PEG content on the surface of microparticles made from the polymers described above in Example 1 . [00070] 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 methylene chloride at room temperature. Approximately 15 mL of 0.5 w/v% of cholic acid (available from Sigma Aldrich) aqueous solution was then added to polymer methylene chloride solution. The resultant mixture was mixed for 1 min using the same sonifier and probe over ice water bath. Continuous mode was used with 45% of amplitude for sonication to produce an oil in water emulsion. Additionally, 230 mL of deionized water was added into the emulsion under magnetic stirring at 500 rpm. The emulsion was stirred overnight at room temperature to remove the methylene chloride.

[00071] The resulting hardened microparticles were cleaned using a tangential flow filtration system. The system utilized a Pall Corporation (Port Washington, New York) Minimate EVO System, Product ID: OAPMPUNV, with a membrane module MIDIKROS 20CM 500K MPES 0.5MM FLL X FLL, Repligen Corporation (Waltham, Massachusetts), Product ID: D02-E500-05-N, mPES membrane, 500 kD, 20 cm. The particle emulsions were first concentrated from 400 mL to 30 mL. 70 mL of di-water was then added into the concentrated emulsion under stirring. The emulsion was concentrated again to 30 mL. This process was repeated twice. The third time the emulsion was concentrated from 100 mL to approximately 15 mL. The cleaned particle emulsion was then pumped into a clean centrifuge tube.

[00072] 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-PEG1 OK polymer 86.5:8.5:5 (Sample 5) in the same way as described above. The UV assay that utilized the complexation reactions of PEG with molybdophosphoric acid was used to detect PEG content on the microparticles. PEG concentration was found from the difference in absorbance between a 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, Taianta, 1982, Vol. 29, p.495-501), this method has no PEO molecular weight dependency. Therefore, the calibration curve was established using PEG of molecular weights of 5K at different concentration (10-500 ug/mL). The blank was obtained from di-water.

[00073] The tested PEG content of each batch microparticles is listed in Table 2. PEG content on the particles is close to its content in the polymer, which means most of the PEGs are located on the surface of particles.

Table 2

Example 3: TMC:D/L-PLA:PGA terpolymer characterization

[00074] A TMC:D/L PLA:PGA terpolymer was formulated with a weight ratio of 63.75:21 .25: 15. The TMC to PLA ratio was 3 to 1 .

[00075] Inherent viscosity (IV) was measured using a Cannon MiniPV-HX Automatic Viscometer with chloroform (CHCh) as the solvent. For this example, IV (CHCI3) = 1.155 dL/g and the glass transition for the polymer was determined to be - 1°C. In vitro degradation studies indicated that the terpolymer essentially degrades over a 6-8 month timeframe.

Example 4: TMC:D/L-PLA:PGA terpolymer characterization

[00076] A TMC:D/L PLA: PGA terpolymer was formulated with a weight ratio of 42.5:42.5:15. The TMC to PLA ratio was 1 to 1 .

[00077] Inherent viscosity (IV) was measured using a Cannon MiniPV-HX Automatic Viscometer with chloroform (CHCh) as the solvent. For this example, IV (CHCh) = 1.155 dL/g and the glass transition for the polymer was determined to be - 1°C. In vitro degradation studies indicated that the terpolymer essentially degrades over a 6-8 month timeframe.

[00078] The characteristics of the terpolymers are shown below in Table 2.

Table 2

Example 5: Microparticles of mRNA in L-PLA:TMC copolymer (no lipid salt) [00079] mRNA encapsulated microparticles were prepared by dissolving 40 mg of L-PLA-TMC copolymer in 5 mL of acetone. 50 ug of CleanCap® mCherry mRNA (996 bp) (TriLink) was dissolved in 2 mL of nuclease free water. The aqueous mRNA solution was added dropwise into the polymer/acetone solution with 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. It was then stirred overnight, at 400 rpm to fully evaporate the acetone. Microparticles were then cleaned via tangential flow filtration as previously described in Example 2.

Example 6: Microparticles of mRNA in L-PLA:TMC copolymer with lipid salt DOTAP

[00080] mRNA is encapsulated in microparticles from a L-PLA:TMC copolymer in the presence of high level of lipid salt DOTAP, specifically a 2:1 weight ratio of lipid salt DOTAP to mRNA.

[00081] 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 methylene chloride at room temperature. Then, 0.05mL of the 1 mg/mL of CleanCap® mCherry mRNA (TriLink Biotechnologies, San Diego, CA, catalog number L-7203), of 996 nucleotides, is pipetted into the polymer/DOTAP oil solution. The mixture is sonicated over ice water bath using a Branson SXF150 sonifier equipped with a 1/4” tapered probe. The water-in-oil (W/O) emulsion is placed on a shaker overnight. The separated mixture is sonicated again for 1 min over ice water to generate a good dispersion. The separated mixture is sonicated again for 1 min over ice water to generate a good dispersion. Alternatively, for mRNAs more susceptible to shearing during sonication, particle dispersions can also be made by adding the aqueous mRNA solution dropwise into the polymer/acetone solution with a stirring speed of 400 rpm.

[00082] Approximately 15 mL of 0.5 wt/v% of cholic acid (Sigma) aqueous solution is then added to the dispersion. The resultant mixture is sonicated for 1 min using the same sonifier and probe over ice water bath. Deionized water (230 mL) is added into the emulsion under magnetic stirring. The emulsion is stirred overnight at room temperature to remove the methylene chloride.

[00083] The resulting hardened microparticles are cleaned using a tangential flow filtration system. The system utilized a Pall Corporation (Port Washington, New York) Minimate EVO System, Product ID: OAPMPUNV, with a membrane module MIDIKROS 20CM 500K MPES 0.5MM FLL X FLL, Repligen Corporation (Waltham, Massachusetts), Product ID: D02-E500-05-N, mPES membrane, 500 kD, 20 cm. The particle emulsions are first concentrated from 400 mL to 30 mL. Deionized water (70 mL) is then added into the concentrated emulsion with stirring. The emulsion is concentrated again to 30 mL, and the process is repeated twice more. The third time, the emulsion is concentrated from 100 mL to approximately 15 mL. The cleaned particle emulsion is then pumped into a clean centrifuge tube and frozen for further characterization.

Example 7: Size and charge characterization of microparticles from Example 6

[00084] Size and zeta potential of the microparticles formed according to Example 6 may be determined after cleaning. The microparticles are characterized for size and zeta potential utilizing by dynamic light scattering with a Zetasizer Ultra (Malvern Panalytical, Malvern United Kingdom).

Example 8: mRNA loading characterization of microparticles from Example 6

[00085] The mRNA loading of the microparticles described in Example 6 may be characterized using the assay described below.

[00086] Microparticles are weighed out and dissolved in 1 mL dimethyl sulfoxide (Sigma Aldrich). The microparticle/dimethyl sulfoxide (DMSO) solution is incubated in a water bath at 37°C for 2 hours to allow the microparticles to fully dissolve. To decomplex the mRNA from the lipid salt, 200 pL of the DMSO/microparticle solution is added to 500 pL of a 100 mg/mL heparin (Sigma Aldrich) and 100 mM Octyl B-D- galactoside (Sigma Aldrich) solution in nuclease free, deionized water (Invitrogen). The solution is vortexed for 5 minutes at room temperature, placed on the shaker table for 30 minutes at room temperature, and then vortexed for another 5 minutes before running on an electrophoresis gel for quantification.

[00087] For the electrophoresis, two mRNA standards are prepared at concentrations of 2 ng/pL and 1 ng/pL, respectively, in nuclease free water (Invitrogen). The mRNA standard solutions include heparin and Octyl B-D-galactoside in the same concentration that will be in the microparticle samples to account for any fluorescence attributed to the reagents. 10 pL of each sample or standard is loaded into a microcentrifuge tube. 2 pL 6X DNA orange loading dye (Invitrogen) is added to each sample and mix well. The entire 12 pl of each mixture is added to the well of a 10-well 4-20% TBE gel (Invitrogen) in 1X TBE buffer. The gel is run at 250 V for 25 minutes. The gel is removed and stained with 1X Sybr Gold (50,000X stock diluted in 1X TBE buffer, Invitrogen) for 20 minutes with gentle rocking. The gel is imaged using the Image Ready software (Biorad) on the BioRad Gel Doc system. Using the standards of the Image Ready software (Biorad), a standard curve is created, and the concentration of the unknown samples are determined from the standard curve.

Example 9: Microparticles of mRNA in L-PLA:TMC copolymer with lipid salt DOTAP

[00088] Modified mRNA is encapsulated in microparticles from a L-PLA:TMC copolymer in the presence of high level of lipid salt DOTAP, specifically a 2:1 weight ratio of lipid salt DOTAP to mRNA.

[00089] 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 methylene chloride at room temperature. Then, 0.4 mL of the 1 mg/mL of CleanCap® mCherry mRNA (TriLink Biotechnologies, San Diego, CA, catalog number L-7203), of 996 nucleotides, is pipetted into the polymer/DOTAP oil solution. The mixture is sonicated over ice water bath using a Branson SXF150 sonifier equipped with a 1/4” tapered probe. The water-in-oil (W/O) emulsion is placed on a shaker overnight. The separated mixture is stirred again for 1 min over ice water to generate a good dispersion. The separated mixture is stirred again for 1 min over ice water to generate a good dispersion. Approximately 15 mL of 0.5 wt/v% of cholic acid (Sigma) aqueous solution is then added to the dispersion. The resultant mixture is sonicated for 1 min using the same sonifier and probe over ice water bath. 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. This resulting hardened microparticles are cleaned using a tangential flow filtration system. The system utilized a Pall Corporation (Port Washington, New York) Minimate EVO System, Product ID: OAPMPUNV, with a membrane module MIDIKROS 20CM 500K MPES 0.5MM FLL X FLL, Repligen Corporation (Waltham, Massachusetts), Product ID: D02-E500-05-N, mPES membrane, 500 kD, 20 cm. The particle emulsions are first concentrated from 400 mL to 30 mL. 70 mL of di-water is then added into the concentrated emulsion under stirring. The emulsion is concentrated again to 30 mL and the process is repeated twice. The third time the emulsion is concentrated from 100 mL to approximately 15 mL. The cleaned particle emulsion is then pumped into a clean centrifuge tube and frozen for further characterization.

Example 10: mRNA release

[00090] Release of encapsulated mRNA at pH 7 and pH 4 is characterized. In order to characterize release of encapsulated mRNA at pH 7, 1 mL of a 1 mg/mL of mRNA microparticle dispersion made according to Example 5 is incubated in a solution of phosphate buffered saline (PBS), supplemented with 10% fetal bovine serum, at 37°C for time points of 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 samples are taken directly from the test tubes and run on electrophoresis gel as described above. This method accounts for the fact that microparticles are too large to enter the gel, and the quantity found in the gel is only “free” mRNA released in the assay. In the second method, incubated samples are centrifuged at 4 °C for 1 hour at 10,000 rpm (Beckman Coulter) to separate microparticles from “free” mRNA. The supernatant of the centrifugation samples are then run on an electrophoresis gel. These two methods are used to determine if either method is artificially separating mRNA from microparticles either by force or through the electric current.

[00091] To check for encapsulated mRNA release at pH 4, 1 mL of a l mg/mL mRNA microparticle dispersion made according to from Example 5 are incubated in a phosphate citrate buffer at pH 4. The remaining analysis including incubation and gel electrophoresis is conducted in the same manner as that described for the pH 7 samples.

Example 11 : Stability of microparticle size following storage

[00092] The ability of microparticles, such as those formed according to Example 5, to retain consistent size when stored in a refrigerator may be determined as follows.

[00093] Microparticles are placed in a standard refrigerator (approximately 4 °C) for the period of about 24 hours, after which a sample is taken for analysis. The sample is characterized for size by dynamic light scattering with a Zetasizer Ultra (Malvern Panalytical, Malvern United Kingdom). It is noted that the particle size remains within 10-12% of the originally produced microparticles following cleaning.

Example 12: Stability of encapsulated mRNA within frozen microparticles [00094] The stability of mRNA encapsulated within microparticles when stored frozen may be determined as described below.

[00095] Microparticles are placed in a standard freezer (approximately -18°C) for a period of about 24 hours. The sample is then analyzed according to the method described above for total mRNA content. This is content is compared to the same batch of microparticles as made according to Example 5 following cleaning, but without storage. It is noted that the difference in mRNA content (loading) is not greater than 10%.

Example 13: Stability of encapsulated mRNA within refrigerated microparticles

[00096] The stability of mRNA encapsulated within microparticles when stored in a refrigerator may be determined as described below.

[00097] A known mass of microparticles is placed in a standard refrigerator (approximately 4°C) for a period of about 24 hours. Water is added to the sample to ensure that the total volume is 1 .5 mL, after which the sample is vortexed and centrifuged to collect the microparticles. A sample of the aqueous phase is taken, and mRNA content is determined, accounting for the total 1 .5 mL volume.

[00098] Microparticle mRNA content is then analyzed as described above to determine mRNA content in the microparticles. The amount of mRNA found in the aqueous phase is added to the encapsulated mRNA to obtain the total mRNA content, as made according to Example 5 following cleaning, but without storage. It is noted that the difference in mRNA content (loading) is not greater than 10%.

Example 14: Stability of encapsulated mRNA in serum at 37°C

[00099] The stability of mRNA encapsulated in microparticles at 37°C in serum may be determined as described below.

[000100] A known mass of microparticles is placed in a controlled temperature chamber (approximately 37°C) for a period of about 24 hours. Serum is added to the sample to ensure that the total volume is 1 .5 mL, after which the sample is vortexed and centrifuged to collect the microparticles. A sample of the aqueous phase is taken, and mRNA content is determined, accounting for the total 1 .5 mL volume.

[000101] Microparticle mRNA content is then analyzed as described above to determine mRNA content in the microparticles. The amount of mRNA found in the aqueous phase is added to the encapsulated mRNA to obtain the total mRNA content, as made according to Example 5 following cleaning, but without storage. It is noted that the difference in mRNA content (loading) is not greater than 10%.

Claims

WHAT IS CLAIMED IS:

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

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

3. The method of claim 1 wherein the copolymer comprises from 45-80 wt.% D/L-PLA and from 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 with a polyethylene glycol (PEG) attached at one end.

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 microparticle particle is at least 0.5 wt.%.

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

9. The method of claim 1 wherein the modified mRNA loading of the polymer microparticle particle is 5 wt.% to 10 wt.%.

10. The method of claim 1 wherein the modified mRNA loading of the polymer microparticle particle is 10 wt.% to 25 wt.%.

11 . The method of claim 1 wherein the polymer microparticle particle has a percent encapsulation efficiency of at least 60-70%.

12. The method of claim 1 wherein the polymer microparticle particle has a percent encapsulation efficiency of at least 75-80%.

13. The method of claim 1 wherein the polymer microparticle particles have a mean particle size no more than 25 urn.

14. The method of claim 1 wherein the polymer microparticle particles have a mean particle size of 50 to 250 nm.

15. The method of claim 1 wherein the polymer microparticle particles have a mean particle size of 100 to 300 nm.

16. The method of claim 1 wherein the polymer microparticle particles have a mean particle size of 300 to 800 nm.

17. The method of claim 1 wherein the polymer microparticle particles have a mean particle size of 800 to 2500 nm.

18. The method of claim 1 wherein the mean particle size changes no more than 10% for at least 1 day at a temperature range of 4 °C to 20 °C.

19. The method of claim 1 wherein the modified mRNA encapsulated within the polymer microparticle particle does not degrade over 10% for at least 1 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 microparticle particles are stable in 90% serum relative to unencapsulated modified mRNA.

21 . The method of claim 1 wherein the polymer microparticle particles provide delayed release of the modified mRNA from encapsulation.

22. The method of claim 1 wherein the polymer microparticle releases less than 50% of the modified mRNA from encapsulation in a 48-hour time period.

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

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

25. The method of claim 23 wherein the polymer microparticle releases less than 50% of the modified mRNA from encapsulation in a 48-hour time period.

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

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

28. The method of claim 26 wherein the copolymer PEG end caps are located on the outer surface of the microparticle particles.

29. The method of claim 26 wherein the polymer microparticle releases less than 50% of the modified mRNA from encapsulation in a 48-hour time period.

30. An encapsulated mRNA injectable therapeutic, comprising

A modified mRNA encoding a polypeptide of interest encapsulated within a trimethylene carbonate (TMC)-based polymer microparticle particle wherein the TMC- based polymer is copolymer comprising repeating units of trimethylene carbonate (TMC) and D/L-polylactide (D/L-PLA).

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

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

33. A method of producing an encapsulated mRNA injectable therapeutic comprising combining a population of a modified mRNA encoding a polypeptide of interest within a lipid component to form an mRNA lipid complex, and combining the mRNA lipid complex with a (TMC)-based polymer to produce a microparticle particle wherein the mRNA lipid complex is encapsulated within the TMC- based polymer wherein the copolymer comprises repeating units of trimethylene carbonate (TMC) and D/L-polylactide (D/L-PLA).

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

35. 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. The method of claim 33 wherein the lipid is a cationic lipid.

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

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