Lipid nanoparticle compositions and methods for mrna delivery.

COMPOSITIONS OF LIPID NANOPARTICLES AND METHODS FOR ADMINISTRATION OF mRNA

DESCRIPTIVE MEMORY

New approaches and therapies are still needed for the treatment of protein and enzyme deficiencies. For example, lysosomal storage diseases are a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function, usually due to a deficiency of an enzyme necessary for metabolism. Fabry's disease is a lysosomal storage disease that results from a deficiency of the alpha galactosidase enzyme (GLA), which causes a glycolipid known as globotriaosylceramide to accumulate in blood vessels and other tissues, leading to various painful manifestations. For certain diseases, such as Fabry's disease, a protein or enzyme that is normally secreted by cells in the bloodstream needs to be replaced. Therapies, such as gene therapy, that increase the level or production of an affected protein or enzyme could provide a treatment or even cure for such disorders. However, there have been several limitations to the use of conventional gene therapy for these purposes.

Conventional gene therapy involves the use of DNA for insertion of desired genetic information into host cells. The DNA introduced into the cell is generally integrated to some extent in the genome of one or more transfected cells, allowing a lasting action of the genetic material introduced into the host. Although there may be substantial benefits from such sustained action, the integration of exogenous DNA into a host genome can also cause many harmful effects. For example, it is possible that the introduced DNA is inserted into an intact gene, resulting in a mutation that prevents or even completely eliminates the function of the endogenous gene. Therefore, gene therapy with DNA can result in an alteration of a vital genetic function in the treated host, such as, for example, elimination or harmful reduction of the production of an essential enzyme or disruption of a gene fundamental for regulation of cell growth, resulting in increased or cancerous cell proliferation. In addition, with conventional DNA-based gene therapy, effective expression of the desired gene product is necessary to include a strong promoter sequence, which can again lead to undesirable changes in the regulation of normal gene expression in the cell. It is also possible that the DNA-based genetic material results in the induction of unwanted anti-DNA antibodies which, in turn, can trigger a possibly fatal immune response. Gene therapy approaches using viral vectors can also result in an adverse immune response. In some circumstances, the viral vector can even be integrated into the host genome. In addition, the production of clinical grade viral vectors is also expensive and time consuming. Directed administration of the genetic material introduced using viral vectors can also be difficult to control. Therefore, although DNA-based gene therapy has been evaluated for administration of secreted proteins using viral vectors (US Patent No. 6,066,626; US2004 / 0110709), these approaches may be limited for these various reasons.

Another apparent obstacle in these previous approaches in the administration of nucleic acids that encode secreted proteins is in the levels of protein that are ultimately produced. It is difficult to achieve considerable levels of the desired protein in the blood, and the amounts do not sustain over time. For example, the amount of protein produced by nucleic acid administration does not reach normal physiological levels. See, for example, US2004 / 0110709.

Unlike DNA, the use of RNA as a gene therapy agent is substantially safer because (1) the RNA does not involve the risk of stably integrating into the genome of the transfected cell, thus eliminating the concern that the introduced genetic material will alter the normal functioning of an essential gene, or cause a mutation that results in harmful or oncogenic effects; (2) the use of external promoter sequences is not necessary for the effective translation of the encoded protein, again avoiding possible harmful side effects; (3) Unlike plasmid DNA (pDNA), messenger RNA (mRNA) lacks immunogenic CpG motifs so that no anti4 antibodies are generated

RNA; and (4) any harmful effects that result from mRNA-based gene therapy will be of limited duration due to the relatively short half-life of the RNA. In addition, it is not necessary for mRNA to enter the nucleus to carry out its function, while DNA must overcome this important barrier.

One reason why mRNA-based gene therapy has not been used more in the past is that mRNA is much less stable than DNA, especially when it reaches the cytoplasm of a cell and is exposed to degrading enzymes. The presence of a hydroxyl group in the second carbon of the remaining sugar in the mRNA causes a spherical impediment that prevents the mRNA from forming the most stable double helix structure of the DNA and, consequently, makes the mRNA more prone to hydrolytic degradation As a result, until recently, it was widely believed that mRNA was too labile to support transfection protocols. Advances in the modifications for RNA stabilization have caused a greater interest in the use of mRNA instead of plasmid DNA in gene therapy. Certain administration vehicles, such as polymeric administration vehicles or cationic lipids, can also help protect transfected mRNA from endogenous RNases. However, despite the increased stability of the modified mRNA, the administration of mRNA to cells in vivo in a way that enables therapeutic levels of protein production is still a challenge, especially for mRNA encoding full-length proteins. Although the administration of mRNA encoding secreted proteins (US2009 / 0286852) has been achieved, the levels of full-length secreted protein that would actually occur through administration of mRNA in vivo are unknown and there is no reason to expect levels exceed those observed with DNA-based gene therapy.

To date, considerable progress has been made using mRNA gene therapy only in applications where low levels of translation are not a limiting factor, such as immunization with antigens encoding mRNA. Clinical trials involving vaccination against tumor antigens by intradermal injection of naked mRNA or complexing with protamine have demonstrated viability, absence of toxicity and promising results. X. Su et al., Mol. Pharmaceutics 8: 774787 (2011). Unfortunately, low levels of translation have greatly restricted the exploitation of mRNA-based gene therapy in other applications that require higher levels of sustained expression of the protein encoded by the mRNA to exert a biological or therapeutic effect.

The invention provides methods of administration of mRNA gene therapeutic agents that lead to the production of therapeutically effective levels of secreted proteins through a "prolonged effect." In embodiments of the invention, mRNA encoding a protein secreted in lipid nanoparticles is loaded and administered to the target cells in vivo. The target cells then act as a long-acting source for the production of soluble secreted protein in the circulatory system at therapeutic levels. In some embodiments, the levels of secreted protein produced are above normal physiological levels.

The invention provides compositions and methods for intracellular administration of mRNA in a liposomal transfer vehicle to one or more target cells for the production of therapeutic levels of secreted functional protein.

The compositions and methods of the invention are useful in the management and treatment of a large number of diseases, in particular diseases resulting from protein and / or enzymatic deficiencies, where the protein or enzyme is normally secreted. Individuals suffering from such diseases may have underlying genetic defects that lead to the compromised expression of a protein or enzyme, including, for example, the non-synthesis of the secreted protein, the reduced synthesis of the secreted protein or the synthesis of a secreted protein. that lacks or has diminished biological activity. In particular, the methods and compositions of the invention are useful for the treatment of lysosomal storage disorders and / or urea cycle metabolic disorders that occur as a result of one or more defects in the biosynthesis of secreted enzymes involved in the urea cycle.

The compositions of the invention comprise an mRNA, a transfer vehicle and, optionally, an agent to facilitate contact with and subsequent transfection of a target cell. The mRNA can encode a clinically useful secreted protein. For example, the mRNA can encode a functional secreted enzyme from the urea cycle or a secreted enzyme that participates in lysosomal storage disorders.

The mRNA can encode, for example, erythropoietin (for example, human EPO) or α-galactosidase (for example, human α-galactosidase (human GLA).

In some embodiments, the mRNA may comprise one or more modifications that confer stability to the mRNA (for example, compared to a natural or native version of the mRNA) and may also comprise one or more modifications with respect to the naturally occurring one that correct an implied defect. in the associated aberrant expression of the protein. For example, the nucleic acids of the present invention may comprise modifications in one or both regions not translated into 5 'and 3'.

Such modifications may include, but are not limited to, the inclusion of a partial sequence of an immediate early cytomegalovirus (CMV) 1 (IE1) gene, a poly A end, a Capí structure or a sequence encoding a human growth last (hGH)). In some embodiments, the mRNA is modified to decrease the immunigenicity of the mRNA.

Methods of treating a subject comprising administering a composition of the invention are also contemplated. For example, methods are provided for treating or preventing conditions in which the production of a specific secreted protein and / or the use of a specific secreted protein is inadequate or dangerous. In one embodiment, the methods provided herein can be used to treat a subject that has a deficiency in one or more of the urea cycle enzymes or in one or more enzymes deficient in a lysosomal storage disorder.

In a preferred embodiment, the mRNA in the compositions of the invention is formulated in a liposomal transfer vehicle to facilitate administration to a target cell. The transfer vehicles contemplated may comprise one or more cationic lipids, non-cationic lipids and / or PEG modified lipids. For example, the transfer vehicle may comprise at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000 or HGT5001. In embodiments, the transfer vehicle comprises cholesterol (col) and / or a PEG modified lipid. In some embodiments, transfer vehicles comprise DMG-PEG2K. In certain embodiments, the transfer vehicle comprises one of the following lipid formulations: C12-200, DOPE, col, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, col, DMG-PEG2K, HGT5001, DOPE, col, DMG-PEG2K.

The invention also provides compositions and methods useful for facilitating the transfection and administration of one or more mRNA molecules to target cells capable of exhibiting the "prolonged effect." For example, the compositions and methods of the present invention contemplate the use of targeting ligands capable of enhancing the affinity of the composition with one or more target cells. In one embodiment, the targeting ligand and apolipoprotein-B or apolipoprotein-E and the corresponding target cells express low-density lipoprotein receptors, thereby facilitating recognition of the targeting ligand. The methods and compositions of the present invention can be used to preferentially target a vast number of target cells. For example, the target cells contemplated include, by way of non-taxation, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

In embodiments, the secreted protein is produced by the target cell for a sustained amount of time. For example, the secreted protein can be produced for more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours or more than 72 hours after administration. In some embodiments, the polypeptide is expressed at a peak level approximately six hours after administration. In some embodiments, polypeptide expression is sustained at least at a therapeutic level. In some embodiments, the polypeptide is expressed at least one therapeutic level for more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours or more than 72 hours after administration. In some embodiments, the polypeptide is detected at that level in the patient's serum or tissue (eg, liver or lung). In some embodiments, the detected polypeptide level is of continuous expression from the mRNA composition for periods of time of more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours or more than 72 hours after administration.

In certain embodiments, the secreted protein is produced at levels above normal physiological levels. The level of secreted protein can be increased compared to a control.

In some embodiments the control is the physiological reference level of the polypeptide in a normal individual or in a population of normal individuals. In other embodiments, the control is the physiological reference level of the polypeptide in an individual who has a deficiency in the relevant protein or in a population of individuals who have a deficiency in the relevant protein or polypeptide. In some embodiments, the control may be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. In other embodiments, the control is the level of expression of the polypeptide after another therapeutic intervention, for example, the direct injection of the corresponding polypeptide, in one or more comparable time points.

In certain embodiments, the polypeptide is expressed by the control cell at a level that is at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, 30 times, at least 100 times, at least 500 times, at least 5000 times, at least 50 000 times or at least 100,000 times greater than a witness. In some embodiments, the increase in expression greater than the control is sustained for more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours or more than 72 hours after administration . For example, in one embodiment, the levels of secreted protein are detected in the serum at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, 30 times, at least 100 times , at least 500 times, at least 5000 times, at least 50 000 times or at least 100,000 times greater than a witness for at least 48 hours or 2 days. In certain embodiments, the levels of secreted protein are detectable at 3 days, 4 days, 5 days or 1 week or more after administration. Increased levels of secreted protein can be observed in the serum and / or in a tissue (for example, liver, lung).

In some embodiments, the method provides a sustained half-life of the desired secreted protein. For example, the secreted protein can be detected for more hours or more days than the half-life observed through subcutaneous injection of the secreted protein. In embodiments, the half-life of the secreted protein is sustained for more than 1 day, 2 days, 3 days, 4 days, 5 days or 1 week or more.

In some embodiments, administration comprises a single dose or repeated doses. In certain embodiments, the dose is administered intravenously or by pulmonary administration.

The polypeptide can be, for example, one or more of erythropoietin, α-galactosidase, LDL receptor, Factor VIII, Factor IX, a-Liduronidase (for MPS I), iduronate sulfatase (for MPS II), heparan-Nsulfatase (for MPS IIIA), α-Ν-acetylglucosaminidase (for MPS IIIB), galactose 6-sultatase (for MPS IVA), lysosomal acid lipase, arylsulfatase-A.

Certain embodiments relate to compositions and methods that provide mRNA to a cell or subject, at least a portion thereof encoding a functional protein, in an amount that is substantially less than the amount of corresponding functional protein generated from said mRNA. In other words, in certain embodiments, the mRNA administered to the cell can produce an amount of protein that is substantially greater than the amount of mRNA administered to the cell. For example, in a given amount of time, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or 24 hours after the administration of mRNA to a cell or subject, the amount of corresponding protein generated by said mRNA may be at least 1.5, 2, 3, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500 or more times greater than the amount of mRNA actually administered to the cell or subject. This can be measured based on mass, based on moles and / or based on molecules. Protein can be measured in several ways. For example, for a cell, the measured protein can be measured as an intracellular protein, such as an extracellular protein or a combination of the two. For a subject, the measured protein may be whey measured protein; in a specific tissue or tissues such as the liver, kidneys, heart or brain; in a specific cell type such as one of several cell types of the liver or brain; or in any combination of serum, tissue and / or cell type. In addition, a reference amount of endogenous protein in the cell or subject can be measured before administration of the mRNA and then subtracted from the protein measured after administration of the mRNA to provide the corresponding amount of protein generated from the mRNA. In this way, the mRNA can provide a source of reservoir or reservoir of a large amount of therapeutic material for the cell or subject, for example, compared to the amount of mRNA administered to the cell or subject. The deposit source can act as a continuous source for polypeptide expression from mRNA for sustained periods of time.

The above and many other features and related advantages of the present invention will be better understood in reference to the following detailed description of the invention when taken in conjunction with the accompanying examples. The various embodiments described herein are complementary and may be used in combination or used together in a manner understood by the expert by virtue of the explanations contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a 5 'CMV sequence (SEQ ID NO: 1), where X, if present, is GGA.

FIG. 2 shows the nucleotide sequence of a 3 'hGH sequence (SEQ ID NO: 2).

FIG. 3 shows the nucleotide sequence of human erythropoietin mRNA (EPO) (SEQ ID NO: 3). This sequence may be flanked at the 5 'end by SEQ ID NO: 1 and at the 3' end by SEQ ID NO: 2.

FIG. 4 shows the nucleotide sequence of human alphagalactosidase mRNA (GLA) (SEQ ID NO: 4). This sequence may be flanked at the 5 'end by SEQ ID NO: 1 and at the 3' end by SEQ ID NO: 2.

FIG. 5 shows the nucleotide sequence of human antitrypsin alpha-1 mRNA (A1AT) (SEQ ID NO: 5). This sequence may be flanked at the 5 'end by SEQ ID NO: 1 and at the 3' end by SEQ ID NO: 2.

FIG. 6 shows the nucleotide sequence of mRNA factor

IX human (FIX) (SEQ ID NO: 6). This sequence may be flanked at the 5 'end by SEQ ID NO: 1 and at the 3' end by SEQ ID NO: 2.

FIG. 7 shows the quantification of secreted hEPO protein levels, such as measured through ELISA. The protein detected is the result of its production through hEPO mRNA administered intravenously through a single dose of several lipid nanoparticle formulations. The formulations C12-200 (30 ug), HGT4003 (150 ug), ICE (100 ug), DODAP (200 ug) are represented as the cationic / ionizable lipid component of each test article (Formulations 1-4). Values ​​are based on blood samples four hours after administration.

FIG. 8 shows hematocrit measurement of mice treated with a single IV dose of lipid nanoparticles loaded with human EPO mRNA (Formulations 1-4). Samples of whole blood were taken at 4 hr (Days 1), 24 hr (Day 2), 4 days, 7 days and 10 days after administration.

FIG. 9 shows hematocrit measurements of mice treated with lipid nanoparticles loaded with human EPO mRNA with a single IV dose or three injections (day 1, day 3, day 5). Samples of whole blood were taken before injection (day -4), day 7 and day 15. Formulation 1 was administered: (30 ug, single dose) or (3 x 10 ug, dose day 1, day 3 , day 5); Formulation 2 was administered: (3 x 50 ug, dose day 1, day 3, day 5).

FIG. 10 shows the quantification of levels of secreted human ogalactosidase protein (hGLA), as measured through ELISA. The protein detected is the result of production from hGLA mRNA administered through lipid nanoparticles (Formulation 7;

single intravenous dose of 30 ug, based on encapsulated mRNA). HGLA protein is detected for 48 hours.

FIG. 11 shows the activity of hGLA in serum. HGLA activity was measured using the 4-methylumbelliferyl-aD-galactopyranoside (4-MU-o-gal) substrate at 37 ° C. The data is an average of 6 to 9 individual measurements.

FIG. 12 shows the quantification of serum hGLA protein levels as measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200 (C12-200: DOPE: Col: DMGPEG2K, 40: 30: 25: 5 (Formulation 7); 30 ug of mRNA based on Encapsulated mRNA, single dose IV). The hGLA protein is monitored for 72 hours, by single intravenous dose, based on encapsulated mRNA). The hGLA protein is monitored for 72 hours.

FIG. 13 shows the quantification of hGLA protein levels in the liver, kidneys and spleen, as measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200 (Formulation 1; 30 ug mRNA based on encapsulated mRNA, single dose IV). The hGLA protein is monitored for 72 hours.

FIGS. 14A-14B show a dose response study that monitors hGLA protein production as human GLA protein derived from serum MRT (Figure 14A) and liver is secreted (Figure

14Β). Samples were measured 24 hours post-administration (Formulation 1; single dose, IV, N = 4 mice / group) and quantified by ELISA.

FIG. 15 shows the pharmacokinetic profiles of Alphagalactosidase based on ERT in nude hairless mice (40 ug / kg dose) and hGLA protein produced from MRT (Formulation 1; mRNA dose 1.0 mg / kg).

FIG. 16 shows the quantification of hGLA protein levels in mice with Fabry treated with MRT, such as measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200 (Formulation 1; 10 ug of mRNA per single intravenous dose based on encapsulated mRNA). The serum is monitored for 72 hours.

FIG. 17 shows the quantification of hGLA protein levels in the liver, kidneys, spleen and heart of mice with Fabry-inactivated genes treated with MRT, such as measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200 (Formulation 1; 30 ug mRNA based on encapsulated mRNA, single dose IV). The hGLA protein is monitored for 72 hours. The literature values ​​that represent normal physiological levels are plotted as dashed lines.

FIG. 18 shows the quantification of hGLA protein levels secreted in Fabry mice treated with MRT and Alpha-galactosidase, as measured using ELISA. Both therapies were dosed with a single intravenous dose of 1.0 mg / kg.

FIG. 19 shows the quantification of hGLA protein levels in the liver, kidneys, spleen and heart of mice with Fabry-inactivated genes treated with MRT and ERT (Alpha-galactosidase), such as measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles (Formulation 1; 1.0 mg / kg mRNA based on encapsulated mRNA, single dose IV).

FIG. 20 shows the relative quantification of globotrioasilceramide (Gb3) and Iiso-Gb3 in the kidneys of treated and untreated mice. Mice with male Fabry deactivated genes were treated with a single dose of lipid nanoparticles loaded with GLA mRNA or Alpha-galactosidase at 1.0 mg / kg. The amounts reflect the amount of Gb3 / smooth-Gb3 one week after administration.

FIG. 21 shows the relative quantification of globotrioasilceramide (Gb3) and Iiso-Gb3 in the heart of treated and untreated mice. Mice with male Fabry deactivated genes were treated with a single dose of lipid nanoparticles loaded with GLA mRNA or Alpha-galactosidase at 1.0 mg / kg. The amounts reflect the amount of Gb3 / smooth-Gb3 one week after administration.

FIG. 22 shows a dose response study that monitors the production of GLA protein as human GLA protein derived from serum MRT is secreted. Samples were measured 24 hours after administration (single dose, IV, N = 4 mice / group) of lipid nanoparticles based on HGT4003 (Formulation 3) or HGT5000 (Formulation 5) and quantified through ELISA.

FIGS. 23A-23B show the production of hGLA protein as measured in the serum (Figure 23A) or in the liver, kidneys and spleen (Figure 23B). Samples were measured 6 hours and 24 hours after administration (single dose, IV, N = 4 mice / group) of lipid nanoparticles based on HGT5001 (Formulation 6) and quantified through ELISA.

FIG. 24 shows the quantification of secreted human Factor IX protein levels measured using ELISA (mean ng / mL ± standard deviation). The FIX protein is produced from FIX mRNA administered through lipid nanoparticles based on C12-2O0 (C12200: DOPE: Col: DMGPEG2K, 40: 30: 25: 5 (Formulation 1; 30 ug of mRNA per single intravenous dose based on encapsulated mRNA) FIX protein is monitored for 72 hours (n = 24 mice)

FIG. 25 shows the quantification of levels of secreted human a1-antitrypsin protein (A1AT), as measured using ELISA. The A1AT protein is produced from A1AT mRNA administered through lipid nanoparticles based on C12-200 (C12200: DOPE: Col: DMGPEG2K, 40: 30: 25: 5 (Formulation 1; 30 ug of mRNA per single intravenous dose based on encapsulated mRNA.) The A1AT protein is monitored for 24 hours.

FIG. 26 shows the ELISA-based quantification of the hEPO protein detected in the lungs and serum of mice treated after intratracheal administration of nanoparticles loaded with hEPO mRNA (measured mlU) (lipid nanoparticles based on C12-200, HGT5000 or HGT5001; Formulations 1 , 5, 6 respectively). Animals were sacrificed 6 hours after administration (n = 4 mice per group).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for intracellular administration of mRNA in a liposomal transfer vehicle to one or more target cells for the production of therapeutic levels of secreted functional protein.

The term "functional", as used herein to qualify a protein or enzyme, means that the protein or enzyme has biological activity, or alternatively, is capable of performing the same or a function similar to that of a protein. protein or enzyme with normal functioning. The mRNA compositions of the invention are useful for the treatment of various metabolic or genetic disorders and, in particular, those genetic or metabolic disorders that involve non-expression, altered expression or deficiency of a protein or enzyme. The term "therapeutic levels refers to the levels of protein detected in the blood and tissues that are above the control levels, where the control may be normal physiological levels, or levels in the subject prior to administration of the composition. mRNA The term "secreted" refers to the protein that is detected outside the target cell, in the extracellular space. The protein can be detected in the blood or tissues. In the context of the present invention, the term produced is used in its broadest sense to refer to the translation of at least one mRNA into a protein or enzyme. As provided herein, the compositions include a transfer vehicle. As used herein, the term "transfer vehicle" includes any of the standard pharmaceutical carriers, diluents, excipients and the like that are generally used in connection with the administration of biologically active agents, including nucleic acids. The compositions and, in particular, the transfer vehicles described herein are capable of administering mRNA to the target cell. In embodiments, the transfer vehicle is a lipid nanoparticle.

MRNA

The mRNA in the compositions of the invention can encode, for example, a hormone, enzyme, receptor, polypeptide, peptide or other secreted protein of interest that is normally secreted. In one embodiment of the invention, the mRNA may optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of said mRNA or that improve or otherwise facilitate the production of protein.

The methods of the invention provide optional co-administration of one or more unique mRNAs to target cells, for example, by combining two unique mRNAs in a single transfer vehicle. In one embodiment of the present invention, a first therapeutic mRNA and a second therapeutic mRNA can be formulated in a single transfer vehicle and administered. The present invention also contemplates the co-administration of a first therapeutic mRNA and a second nucleic acid to facilitate and / or enhance the function or administration of the first therapeutic mRNA. For example, said second nucleic acid (for example, exogenous or synthetic mRNA) can encode a transporter protein that after expression (for example, translation of exogenous or synthetic mRNA) facilitates the administration or enhances the biological activity of the first mRNA. Alternatively, the The first therapeutic mRNA can be administered with a second nucleic acid that functions as a "chaperone", for example, to direct the fold of the first messenger mRNA.

The methods of the invention also provide the administration of one or more therapeutic nucleic acids to treat a single disorder or deficiency, wherein each of said nucleic acids functions by a different mechanism of action. For example, the compositions of the present invention may comprise a first therapeutic mRNA that, for example, is administered to correct an endogenous protein or enzymatic deficiency, and that is accompanied by a second nucleic acid, which is administered to deactivate or "inactivate" an endogenous nucleic acid with altered function and its protein or enzyme product. Said "second" nucleic acids may encode, for example, the mRNA or

SiRNA,

After transfection, a natural mRNA in the compositions of the invention can be decomposed with a half-life of between 30 minutes and several days. The mRNA of the compositions of the invention preferably retains at least some ability to be translated, thus producing a functional secreted protein or enzyme. Therefore, the invention provides compositions comprising and methods of administration of a stabilized mRNA. In some embodiments of the invention, mRNA activity is prolonged for an extended period of time. For example, the activity of the mRNA can be prolonged so that the compositions of the present invention are administered to a subject semi-weekly or bi-weekly or preferably, monthly, bi-monthly, quarterly or annually. The extended or prolonged activity of the mRNA of the present invention is directly related to the amount of protein or secreted functional enzyme produced from said mRNA. Similarly, the activity of the compositions of the present invention can be extended or further extended by modifications made to improve or enhance the translation of mRNA. In addition, the amount of protein or functional enzyme produced by the target cell is a function of the amount of mRNA administered to the target cells and the stability of said mRNA. To the extent that the mRNA stability of the present invention can be improved or enhanced, the half-life, activity of the secreted protein or enzyme produced and the dosage frequency of the composition can be further extended.

Therefore, in some embodiments, the mRNA in the compositions of the invention comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms "modification" and "modified" refer to the nucleic acids provided herein, include at least one alteration that preferably enhances the example and provides a more stable mRNA (for example, resistant to nuclease digestion) than the naturally occurring version of the mRNA. As used herein, the terms "stable" and "stability" refer to the nucleic acids of the present invention and particularly in relation to mRNA, refer to the increased or enhanced resistance to degradation by, for example , nucleases (ie, endonucleases or exonucleases) that are normally capable of degrading said mRNAs. Increased stability may include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (eg, endonucleases or exonucleases) and conditions within the target cell or tissue, thereby increasing or enhancing the residence of said mRNA in the cell. , tissue, subject and / or target cytoplasm. The stabilized mRNA molecules provided herein demonstrate more extensive half-lives with respect to their unmodified counterparts of natural origin (eg, the natural version of the mRNA). They also contemplate the terms "modification and" modified as such that refer to the mRNA of the present invention, alterations that improve or enhance the translation of the mRNA nucleic acids, including, for example, the inclusion of sequences that function in the initiation of protein translation (for example, the Kozac consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

In some embodiments, the mRNA of the invention has undergone a chemical or biological modification that has become more stable. Exemplary modifications in an mRNA include the reduction of a base (for example, by deletion or by substitution of one nucleotide with another) or modification of a base, for example, the chemical modification of a base. The phrase "chemical modifications", as used herein, includes modifications that introduce chemicals that differ from those naturally occurring in the mRNA, for example, covalent modifications such as the introduction of modified nucleotides, (eg, analogs nucleotides or the inclusion of pendant groups that are not naturally found in said mRNA molecules).

Additionally, suitable modifications include alterations in one or more nucleotides of a codon so that the codon encodes the same amino acid but is more stable than the codon found in the natural version of the mRNA. For example, an inverse relationship between RNA stability and a larger amount of cytidine (C) and / or uridine (U) residues has been demonstrated, and it was found that RNA lacking residues of C and U is more stable to most RNasas (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994)). In some embodiments, the amount of C and / or U residues in an mRNA sequence is reduced. In another embodiment, the amount of C and / or U residues is reduced by replacing a codon encoding a specific amino acid with another codon encoding the same or a related amino acid. The modifications contemplated in the mRNA nucleic acids of the present invention also include the incorporation of pseudouridines. The incorporation of pseudouridines into the nucleic acids of the mRNA of the present invention can enhance stability and translational capacity, as well as decrease immunogenicity in vivo. See, for example, Karikó, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008). Substitutions and modifications in the mRNA of the present invention can be carried out by methods known to one or more skilled in the art.

The restrictions on reducing the amount of C and U residues in a sequence will probably be greater within the coding region of an mRNA, compared to an untranslated region, (that is, it will probably not be possible to eliminate all residues C and U present in the messenger while still maintaining the ability of the messenger to encode the desired amino acid sequence). However, the degeneracy of the genetic code presents an opportunity to allow the reduction of the amount of C and / or U residues that are present in the sequence, while maintaining the same coding capacity (that is, depending on the amino acid encoded by a codon, there are several possibilities of modifying the RNA sequences that may be possible). For example, codons for Gly can be altered for GGA or GGG instead of GGU or GGC.

The term modification also includes, for example, the incorporation of non-nucleotide ligatures or modified nucleotides into the mRNA sequences of the present invention (eg, modifications at one or both 3 'and 5' ends of an mRNA molecule encoding a protein or functional secreted enzyme). Such modifications include the addition of bases to an mRNA sequence (for example, the inclusion of a poly A end or a longer poly A end), the alteration of UTR in 3 'or UTR in 5', the formation of a complex between mRNA and an agent (for example, a complementary nucleic acid protein or molecule), and the inclusion of elements that change the structure of an mRNA molecule (for example, that form secondary structures).

It is believed that the poly A end stabilizes natural messengers. Therefore, in one embodiment, a long poly A end can be added to a mRNA molecule, thus providing a more stable mRNA. Poly A ends can be added using a variety of techniques known in the art. For example, long poly A ends can be added to synthetic mRNA or transcribed in vitro using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A ends. In addition, poly A ends can be added by transcription directly from PCR products. In one embodiment, the length of the poly A end is at least about 90, 200, 300, 400, at least 500 nucleotides. In one embodiment, the length of the poly A end is adjusted to control the stability of a modified mRNA molecule of the invention and, consequently, protein transcription. For example, since the length of the poly A end can influence the half-life of an mRNA molecule, the length of the poly A end can be adjusted to modify the level of resistance of the mRNA to nucleases and thus control the expression time protein in a cell In one embodiment, stabilized mRNA molecules are sufficiently resistant to degradation in vivo (eg, by nucleases), so that they can be administered to the target cell without a transfer vehicle.

In one embodiment, an mRNA can be modified by incorporating untranslated sequence (UTR) into 3 '15 and / or 5' that are not naturally found in the natural mRNA. In one embodiment, a 3 'and / or 5' flanking sequence that naturally flanks an mRNA and encodes a second untranslated protein can be incorporated into the nucleotide sequence of an mRNA molecule that encodes a therapeutic or functional protein for the purpose of modify it For example, 3 'or 5' sequences of mRNA molecules that are stable (eg, globin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) can be incorporated into the 3 'region and / or 5 'of a mRNA nucleic acid molecule encoding to increase the stability of the molecule

MRNA coding. See, for example, US2003 / 0083272.

In some embodiments, the mRNA in the compositions of the invention include modification of the 5 'end of the mRNA to include a partial sequence of an immediate early CMV gene 1 (IE1) or a fragment thereof (eg, SEQ ID NO: 1) to improve nuclease resistance and / or improve mRNA half-life. In addition to increasing the stability of the mRNA nucleic acid sequence, it was surprisingly found that the inclusion of a partial sequence of a CMV gene early 1 (IE1) enhances the translation of the mRNA and the expression of the functional protein or enzyme. Also contemplated herein is the inclusion of a human growth hormone (hGH) gene sequence or a fragment thereof (e.g., SEQ ID NO: 2) to the 3 'ends of the nucleic acid (e.g., MRNA) to further stabilize the mRNA. Generally, preferred modifications improve the stability and / or pharmacokinetic properties (eg, half-life) of mRNA with respect to its unmodified counterparts, and include, for example, modifications made to improve the resistance of said mRNA to digestion by nucleases in vivo.

In addition, variants of the nucleic acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 are contemplated, wherein the variants maintain the functional properties of nucleic acids, including mRNA stabilization and / or pharmacokinetic properties (by example, half-life). Variants can have more than 90%, more than 95%, more than 98% or more than

99% sequence identity! with SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the composition may comprise a stabilizing reagent. The compositions may include one or more formulation reagents that bind directly or indirectly and stabilize the mRNA, thereby enhancing the residence time in the target cell. Such reagents preferably lead to an improved half-life of mRNA in the target cells. For example, the stability of an mRNA and the efficiency of translation can be increased by incorporating "stabilizing reagents" that form complexes with the mRNA that occur naturally within a cell (see, for example, US Patent No. 5,677 .124). The incorporation of a stabilizing reagent can be achieved, for example, by combining the poly A and a protein with the mRNA so that it is stabilized in vitro before loading or encapsulating the mRNA within a transfer vehicle. Exemplary stabilizing reagents include one or more proteins, peptides, aptamers, translational accessory proteins, mRNA binding proteins and / or translation initiation factors.

The stabilization of the compositions can also be improved by the use of opsonization-inhibition moieties, which are typically large hydrophilic polymers that chemically or physically bind to the transfer vehicle (for example, by the intercalation of a lipid soluble anchor itself). membrane, or by direct binding with active groups of membrane lipids). These hydrophilic opsonization-inhibition polymers form a protective surface layer that significantly decreases the absorption of liposomes by the macrophage-monocyte system and the reticuloendothelial system (for example, as described in US Patent No. 4,920,016, the full disclosure of which is incorporated herein by reference). Modified transfer vehicles with opsonization-inhibition residues, therefore, remain in the circulation much longer than their unmodified counterparts.

When RNA is hybridized with a complementary nucleic acid molecule (for example, DNA or RNA) it can be protected from nucleases. (Krieg, et al. Melton. Methods in Enzymology. 1987; 155, 397-415). The stability of the hybridized mRNA is likely due to the inherent single strand specificity of most RNases. In some embodiments, the stabilizing reagent selected to form a complex with mRNA is a eukaryotic protein, (eg, a mammalian protein). In another additional embodiment, the mRNA can be modified by hybridization with a second nucleic acid molecule. If an entire mRNA molecule was hybridized with a complementary nucleic acid molecule, translation initiation could be reduced. In some embodiments, the 5 'untranslated region and the AUG start region of the mRNA molecule may optionally be left unhybridized. After the start of the translation, the unwinding activity of the ribosomal complex can work even in high affinity duplexes so that the translation can proceed. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. J Biol Chem. 1993; 268: 14514-22.)

It will be understood that any of the methods described above to improve mRNA stability can be used alone or in combination with one or more of any of the other methods and / or compositions described above.

The mRNA of the present invention can optionally be combined with a reporter gene (for example, towards 3 'or 5' of the mRNA coding region) which, for example, facilitates the determination of mRNA administration to target cells or tissues. . Suitable reporter genes may include, for example, fluorescent green protein mRNA (GFP mRNA), Renilla Luciferase mRNA (Luciferase mRNA), Firefly Luciferase mRNA or any combination thereof. For example, GFP mRNA can be fused with an mRNA encoding a secretable protein to facilitate confirmation of the location of the mRNA in the target cells that will act as a reservoir for protein production.

As used herein, the terms "transfect" or "transfection" mean the intracellular introduction of an mRNA into a cell or preferably into a target cell. The introduced mRNA can be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of mRNA absorbed by the target cell that undergoes the transfection. In practice, transfection efficiency is estimated by the amount of a reporter nucleic acid product expressed by the target cells after transfection. Preferred embodiments include compositions with high transfection efficiencies and, in particular, those compositions that minimize adverse effects that are mediated by non-target cell transfection. Compositions of the present invention demonstrating high transfection efficiencies improve the likelihood of administering adequate dosages of mRNA to the target cell, while minimizing potential systemic adverse effects. In one embodiment of the present invention, the transfer vehicles of the present invention are capable of administering large mRNA sequences (e.g., mRNA of at least 1kDa, 1.5kDa, 2kDa, 2.5kDa, 5kDa, 10kDa, 12kDa, 15kDa , 20kDa, 25kDa, 30kDa or more). The mRNA can be formulated with one or more acceptable reagents, which provide a vehicle for administering said mRNA to the target cells. Appropriate reagents are generally selected in relation to several factors, including, among other things, the biological or chemical properties of the mRNA, the expected route of administration, the anticipated biological environment to which the mRNA will be exposed and the specific properties of the cells. intended target. In some embodiments, transfer vehicles, such as liposomes, encapsulate the mRNA without compromising biological activity. In some embodiments, the transfer vehicle demonstrates preferential and / or substantial binding to a target cell with respect to non-target cells. In a preferred embodiment, the transfer vehicle manages its content to the target cell so that the mRNA is administered in the appropriate subcellular compartment, such as the cytoplasm.

Transfer vehicle

In embodiments, the transfer vehicle in the compositions of the invention is a liposomal transfer vehicle, for example, a lipid nanoparticle. In one embodiment, the transfer vehicle can be selected and / or prepared to optimize the administration of mRNA to a target cell. For example, if the target cell is a hepatocyte, the properties of the transfer vehicle (eg, size, load and / or pH) can be optimized to effectively administer said transfer vehicle to the target cell, reduce immune clearance and / or promote retention of said target cell. Alternatively, if the target cell is the central nervous system (for example, mRNA is administered for the treatment of neurodegenerative diseases, it can be directed specifically towards the brain or spinal tissue), the selection and preparation of the transfer vehicle should consider penetration. and retention within the blood brain barrier and / or the use of alternative means of direct administration of said transfer vehicle to said target cell. In one embodiment, the compositions of the present invention may be combined with agents that facilitate the transfer of exogenous mRNA (for example, agents that alter or improve the permeability of the blood-brain barrier and thereby enhance the transfer of exogenous mRNA to the target cells ).

The use of liposomal transfer vehicles to facilitate the administration of nucleic acids to the target cells is contemplated in the present invention. Liposomes (for example, liposomal lipid nanoparticles) are generally useful in a variety of applications in research, industry and medicine, particularly for their use as transfer vehicles for diagnostic or therapeutic compounds in vivo (Lasic, Trends BiotechnoL, 16: 307 -321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and are generally characterized as microscopic vesicles that have an internal aqueous space isolated from an external medium by means of a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin comprising spatially separated hydrophilic and hydrophobic domains (Lasic, Trends BiotechnoL, 16: 307-321, 1 998). The bilayer membranes of the liposomes can also be formed by polymers and amphiphilic surfactants (eg, polymerosomes, niosomes, etc.).

In the context of the present invention, a liposomal transfer vehicle typically serves to transport mRNA to the target cell. For the purposes of the present invention, liposomal transfer vehicles are prepared to contain the desired nucleic acids. The process of incorporating the desired entity (for example, a nucleic acid) into a liposome is often referred to as "loading" (Lasic, et al., FEBS Lett., 312: 255-258, 1992). Nucleic acids incorporated into the liposome can be located completely or partially in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the outer surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as "encapsulation" where the nucleic acid is completely contained within the interior space of the liposome. The purpose of incorporating an mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment that may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause rapid excretion of nucleic acids. Accordingly, in a preferred embodiment of the present invention, the selected transfer vehicle is capable of enhancing the stability of the mRNA contained therein. The liposome may enable the encapsulated mRNA to reach the target cell and / or preferably may allow the mencapsulated mRNA to reach the target cell or alternatively limit the administration of said mRNA to other sites or cells where the presence of the administered mRNA may be useless or unwanted In addition, the incorporation of the mRNA into a transfer vehicle, such as, for example, a cationic liposome, also facilitates the administration of said mRNA to a target cell.

Ideally, liposomal transfer vehicles are prepared to encapsulate one or more desired mRNAs so that the compositions demonstrate high transfection efficiency and enhanced stability. Although liposomes can facilitate the introduction of nucleic acids into the target cells, the addition of polycations (for example, poly L-lysine and protamine), as a copolymer, can facilitate and, in some cases, markedly enhance the transfection efficiency of several types of cationic liposomes 2-28 times in several cell lines in vitro and in vivo. (See NJ Caplen, et a /., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.)

Lipid Nanoparticles

In a preferred embodiment of the present invention, the transfer vehicle is formulated as a lipid nanoparticle. As used herein, the phrase "lipid nanoparticle" refers to a transfer vehicle comprising one or more lipids (eg, cationic lipids, non-cationic lipids and PEG modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more mRNAs to one or more target cells. Examples of suitable lipids include, for example, phosphatidyl compounds (for example, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides). The use of polymers as transfer vehicles, either alone or in combination with other transfer vehicles, is also contemplated. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethyleneimine. In one embodiment, the transfer vehicle is selected based on its ability to facilitate the transfection of an mRNA into a target cell.

The invention contemplates the use of lipid nanoparticles as transfer vehicles comprising a cationic lipid to encapsulate and / or enhance the administration of mRNA in a target cell that will act as a reservoir for protein production. As used herein, the phrase "cationic lipid refers to any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH. The contemplated lipid nanoparticles can be prepared including multicomponent lipid mixtures of varying ratios using one or more cationic lipids, non-cationic lipids and PEG modified lipids. Several cationic lipids have been described in the literature, many of these are commercially available.

Cationic lipids particularly suitable for use in the compositions and methods of the invention include those described in international patent publication WO 2010/053572, incorporated herein by reference and more particularly, C12-200 described in paragraph [00225 ] of WO 2010/053572. In certain embodiments, the compositions and methods of the invention employ lipid nanoparticles comprising an ionizable cationic lipid described in US provisional patent application 61 / 617,468, filed March 29, 2012 (incorporated herein by reference), such as, for example, (15Ζ, 18Z) -N, N-dimethyl-6- (9Z, 12Z) -octadeca-9,12-dien-1-yl) tetracosa-15,18dien-1-amino (HGT5000) , (15Z, 18Z) -N, N-dimethyl-6 - ((9Z, 12Z) -octadeca-9,12dien-1-yl) tetracosa-4,15,18-trien-1-amine (HGT5001) and (15Z, 18Z) -N, N-dimethyl6 - ((9Z, 12Z) -octadeca -9,12-dien-1-yl) tetracosa-5,15,18-t laugh-1-amino (HGT5002).

In some embodiments, the cationic lipid N [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride or DOTMA chloride is used. (Felgner et al. (Proc. Nat'l Acad. Sci. 84: 7413 (1987); U.S. Patent No. 4,897,355). DOTMA can be formulated alone or can be combined with neutral lipid, dioleoylphosphatidyl ethanolamine or "DOPE" or other cationic or non-cationic lipids in a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the administration of nucleic acids. to target cells. Other suitable cationic lipids include, for example, 5-carboxypermylglycinadioctadecylamide or "DOGS, 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethl-1-propanaminium or" DOSPA "( Behr et al. Proc. Nat.'l Acad. Sci. 86. 6982 (1989); U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium- Propane or "DODAP", 1,2-Dioleoyl-3-Trimethylammonium-Propane or "DOTAP". The cationic lipids contemplated also include 1,2-distearyloxy-N, N-dimethyl-3-amnopropane or "DSDMA", 1,2-diioleyloxy-N, N-dimethyl-3-aminopropane or DODMA ", 1,2- dilinoleyloxy-N, Ndimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N, N-dimethyl-3aminopropane or "DLenDMA", N-diolel, N-dimethylammonium or "DODAC" , N, N-distearyl-N, N-dimethylammonium bromide or "DDAB" bromide, N- (1,2-dm -ristyloxypropyl-3-yl) -N, N-dimethyl-N-hydroxy ethyl ammonium bromide "DMRIE", 3dimethylamino-2- (colest-5-en-3-beta-oxibutan-4-oxi) -1- (c¡ s, cis-9,12octadecadienoxy) propane or “CLinDMA”, 2- [5 '- (colest- 5-en-3-beta-oxy) -3'oxapentoxy) -3-dimethyl-1- (cis, cis-9 ', 1-2'-octadecadienoxy) propane or "CpLinDMA, N, N-dimethyl-3, 4-dioleyloxybenzylamine or "DMOBA", 1,2-N, N'dioleylcarbamil-3-dimethylaminopropane or "DOcarbDAP", 2,3-Dylinoleoyloxy-N, Ndimethylpropylamine or "DLinDAP", 1,2-N, N ' -Dilinoleilcarbamil-3dimethylaminopropane or “DLincarbDAP”, 1,2-Dilinoleoylcarbamil-3dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] dioxolane or "DLin-K-DMA", 2,2-dil¡nole¡l-4-dimet Lamnoethyl- [1,3] -dioxolane or "DLin-K-XTC2-DMA" and 2- (2,2-di ((9Z, 12Z) -octadeca-9,12-dien-141) -1 , 3dioxolan-4-yl) -N, N-dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple etal., Nature Biotech. 28: 172-176 (2010)) or mixtures thereof . (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., Et al., Nat. Biotechnol. 23 (8): 1003-1007 (2005); PCT Publication WO2005 / 121348A1).

The use of cationic cholesterol-based lipids is also contemplated in the present invention. Such cationic cholesterol-based lipids can be used either alone or in combination with other cationic or non-cationic lipids. Cholesterol-based cationic lipids include, for example, DC-Col (N, N-methyl-N-et-carboxyamidocholesterol), 1,4bis (3-N-oxylamino-propyl) piperazine (Gao, et al. Biochem. Biophys Res.

Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335) or ICE.

In addition, there are several reagents available in the market to enhance transfection efficiency. Suitable examples include LIPOFECTIN (DOTMAOOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPAOOPE) (Invitrogen), LIPGFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS) and EFFECTENE.

Cationic lipids such as dialkylamino-based, imidazole-based and guanidinium-based lipids are also contemplated. For example, certain embodiments are directed to a composition comprising one or more cationic lipids based on imidazole, for example, imidazole cholesterol ester or ICE lipid "(3S, 10R, 13R, 17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro1 H-cyclopenta [a] phenanthren-3 -yl 3- (1H-imidazol-4-yl) propanoate, as represented by structure (I) below. In a preferred embodiment, a transfer vehicle for mRNA administration may comprise one or more cationic lipids based on imidazole, for example, imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R) -10, 13- dimethyl17 - ((R) -6-methylheptan-2-yl) -2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17tetradecahydro-1 H-cyclopenta [a ] phenanthren-3-yl 3- (1 H-imidazol-4-yl) propanoate, as represented by structure (I).

<img file="MX367605B_D0001.tif" />

Without adhering to any specific theory, it is believed that the fusogenicity of the cationic lipid based on imidazole ICE is related to the endosomal alteration that is facilitated by the imidazole group, which has a lower pKa relative to traditional cationic lipids. The endosomal alteration in turn promotes osmotic swelling and alteration of the liposomal membrane, followed by intracellular transfection or release of the nucleic acid (s) contents charged thereto to the target cell.

The cationic lipids based on imidazole are also characterized by their reduced toxicity with respect to other cationic lipids. The cationic lipids based on imidazole (for example, ICE) can be used as the only cationic lipid in the lipid nanoparticle or, alternatively, can be combined with traditional cationic lipid, non-cationic lipids and PEG-modified lipids. The cationic lipid may comprise a molar ratio of about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40% of the total lipid present in the transfer vehicle or preferably about 20% to about 70% of the total lipid present in the transfer vehicle.

Similarly, certain embodiments are directed to lipid nanoparticles comprising the cationic lipid HGT4003 2 - ((2,3Bis ^ OZ. ^ Zj-octadeca-O. ^ - dien-l-yloxyijpropiOdisulfaniQ-N. Ndimethylethanamine, as depicted by structure (II) below and as further described in US Provisional Application No. 61 / 494,745, filed on June 8, 2011, whose full disclosures are incorporated herein by reference in their entirety:

<img file="MX367605B_D0002.tif" />

(I)

In other embodiments, the compositions and methods described herein are directed to lipid nanoparticles comprising one or more cleavage lipids, such as, for example, one or more cationic lipids comprising a cleavage disulphide (SS) functional group (for example, HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as further described in US Provisional Application No: 61 / 494,745, whose full disclosure is incorporated herein by reference in its entirety.

The use of phospholipids modified with polyethylene glycol (PEG) and derived lipids such as derived ceramides (PEG-CER), including N

Octanoyl-sphingosine-1- [Siiccinil (Methox Polyethylene Glycol) -2000] (ceramide C8 PEG-2000) is also contemplated in the present invention, either alone or preferably in combination with other lipids together comprising the transfer vehicle (by example, a lipid nanoparticle). The PEG-modified lipids contemplated include, by way of non-taxation, a polyethylene glycol chain up to 5 kDa in length covalently coupled with a C6-C20 alkyl chain (s) in length. The addition of such components can prevent the accumulation of complexes and can also provide a means to increase the circulation life and increase the administration of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237) or can be selected to quickly swap out of the formulation in vivo (see US Patent No. 5,885,613). Particularly useful interchangeable lipids are PEG ceramides having shorter acyl chains (for example, C14 or C18). The phospholipid modified by PEG and derived lipids of the present invention may comprise a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 1%, about 4% to about 10% or about 2% of the total lipid present in the liposomal transfer vehicle.

The present invention also contemplates the use of non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of several lipid species that carry a net negative charge at a selected pH, such as physiological pH. The noncationic lipids include, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoiloleoilfosfatidilcolina (POPC), palmitoyloleoyl-phosphatidylethanolamine ( POPE), dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimiristoylphosphoethanolamine (DMPE), distearoyl phosphatidyl ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), cholesterol or a mixture thereof. Such non-cationic lipids can be used alone, but preferably they are used in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipid present in the transfer vehicle.

Preferably, the transfer vehicle (for example, a lipid nanoparticle) is prepared by combining multiple lipid and / or polymer components. For example, a transfer vehicle can be prepared using C12-200, DOPE, col, DMG-PEG2K at a molar ratio of 40: 30: 25: 5, or DODAP, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 18 : 56: 20: 6, or HGT5000, DOPE, col, DMG-PEG2K at a molar ratio of 40: 20: 35: 5, or HGT5001, DOPE, col, DMG-PEG2K at a molar ratio of 40:20:35 :5. The selection of cationic lipids, non-cationic lipids and / or PEG modified lipids comprising the lipid nanoparticle, as well as the relative molar ratio of said lipids to each other, is based on the characteristics of the selected lipid (s), the nature of the intended target cells, the characteristics of the mRNA that. It will be administered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid (s). Therefore, molar relationships can be adjusted accordingly. For example, in embodiments, the percentage of cationic lipid in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60% or greater than 70 %. The percentage of non-cationic lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30% or greater than 40%. The percentage of cholesterol in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30% or greater than 40%. The percentage of lipid modified by PEG in the lipid nanoparticle may be greater than 1%, greater than 2%, greater than 5%, greater than 10% or greater than 20%.

In certain preferred embodiments, the lipid nanoparticles of the invention comprise at least one of the following cationic lipids: 012-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000 or HGT5001. In embodiments, the transfer vehicle comprises cholesterol and / or a PEG modified lipid. In some embodiments, transfer vehicles comprise DMG-PEG2K. In certain embodiments, the transfer vehicle comprises one of the following lipid formulations: C12200, DOPE, col, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, col, DMG-PEG2K, HGT5001, DOPE, col, DMG-PEG2K.

Liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques that are currently known in the art. Multilamellar vesicles (MLV) can be prepared by conventional techniques, for example, by depositing a selected lipid into the inner wall of a suitable container or container by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film inside. of the container or by spray drying. An aqueous phase can then be added to the vortex vessel that results in the formation of MLV. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments of the present invention, the compositions of the present invention comprise a transfer vehicle wherein the mRNA is associated on the surface of the transfer vehicle and encapsulated within the same transfer vehicle. For example, during the preparation of the compositions of the present invention, cationic liposomal transfer vehicles can be associated with mRNA through electrostatic interactions

In certain embodiments, the compositions of the invention can be loaded with a diagnostic radionuclide, fluorescent materials or other materials that are detectable in in vitro and in vivo applications. For example, diagnostic materials suitable for use in the present invention may include Rhodamine-dioleoylphospha-tidylethanolamine (Rh-PE), green fluorescent protein mRNA (GFP mRNA), Renilla Luciferase mRNA and Firefly Luciferase mRNA.

For the selection of the appropriate size of a liposomal transfer vehicle, the site of the target cell or tissue and to some extent the application for which the liposome is being created must be taken into account. In some embodiments, it may be desirable to limit mRNA transfection to certain cells or tissues. For example, in order to target hepatocytes, a liposomal transfer vehicle may have a size such that its dimensions are smaller than the fenestrations of the endothelial layer lining the hepatic sinusoids in the liver; consequently, the liposomal transfer vehicle can easily penetrate said endothelial fenestrations to reach the target hepatocytes. Alternatively, a liposomal transfer vehicle may have a size such that the dimensions of the liposome are of a diameter sufficient to expressly limit or prevent distribution to certain cells or tissues. For example, a liposomal transfer vehicle may have a size such that its dimensions are larger than the fenestrations of the endothelial layer that lines the sinusoids to thereby limit the distribution of the liposomal transfer vehicle to hepatocytes. Generally, the size of the transfer vehicle is within the range of about 25 to 250 nm, preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10 nm.

A variety of alternative methods known in the art are available to adjust the size of a population of liposomal transfer vehicles. One such method for adapting the size is described in US Patent No. 4,737,323, incorporated herein by reference. The sonication of a liposomal suspension by bath or sonication probe produces a progressive reduction in size to small ULVs with less than about 0.05 microns in diameter. Homogenization is another method that depends on shear energy to fragment large liposomes into small ones. In a typical homogenization procedure, MLVs are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomal vesicles can be determined by quasi electric light diffraction (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10: 421-450 (1981), incorporated herein by reference. The average liposome diameter can be reduced by sonication of the formed liposomes. Intermittent sonication cycles can be altered with QELS assessments to perform efficient liposomal synthesis.

Target cells

As used herein, the term "target cell" refers to a cell or tissue to which a composition of the invention is directed. In some embodiments, the target cells are deficient in a protein or enzyme of interest. For example, when it is desired to administer a nucleic acid to a hepatocyte, the hepatocyte represents the target cell. In some embodiments, the compositions of the invention are transfected to the target cells on a discriminatory basis (ie, they are not transfected to non-target cells). The compositions of the invention can also be prepared to preferentially target a variety of target cells, including, but not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, cells mesenchymal, neural cells (for example, meninges, astrocytes, motor neurons, ganglion cells of the posterior root and motor neurons of the anterior horn), photoreceptor cells (e.g., cones and rods), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells , testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

The compositions of the invention can be prepared to be preferentially distributed to target cells such as in the heart, lungs, kidneys, liver and spleen. In some embodiments, the compositions of the invention are distributed to liver cells to facilitate administration and subsequent expression of mRNA comprised therein by liver cells (eg, hepatocytes). Target hepatocytes can function as a biological "reservoir" or "reservoir" capable of systemically producing and excreting a functional protein or enzyme. Accordingly, in one embodiment of the invention, the liposomal transfer vehicle can be directed to hepatocytes and / or preferentially distributed to liver cells after administration. After transfection of the target hepatocytes, the mRNA loaded in the liposomal vehicle is translated and a functional protein product is produced, which is excreted and distributed systemically. In other embodiments, cells other than hepatocytes (eg, lung, spleen, heart, eye, or central nervous system cells) can serve as a location of the reservoir for protein production.

In one embodiment, the compositions of the invention facilitate endogenous production in the subject of one or more functional proteins and / or enzymes and, in particular, the production of proteins and / or enzymes that demonstrate less immunogenicity with respect to their recombinantly prepared counterparts. In a preferred embodiment of the present invention, transfer vehicles comprise mRNA encoding a protein or deficient enzyme. Upon distribution of said compositions to the target tissues and the subsequent transfection of said target cells, the exogenous mRNA loaded in the liposomal transfer vehicle (eg, a lipid nanoparticle) can be translated in vivo to produce a functional protein or enzyme encoded by mRNA administered exogenously (for example, a protein or enzyme in which the subject is deficient). Accordingly, the compositions of the present invention exploit a subject's ability to translate mRNA exogenously or recombinantly prepared to produce an endogenously translated protein or enzyme and thus produce (and when appropriate, excrete) a functional protein or enzyme. The expressed or translated proteins or enzymes can also be characterized by the in vivo inclusion of natural post-translational modifications that can often be absent in recombinantly prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme.

The administration of mRNA encoding a protein or deficient enzyme avoids the need to deliver the nucleic acids to specific organelles within a target cell (eg, mitochondria). Instead, after transfection of a target cell and administration of nucleic acids to the cytoplasm of the target cell, the mRNA content of a transfer vehicle can be translated and a functional protein or enzyme can be expressed.

The present invention also contemplates discriminatory targeting of target cells and tissues by passive and active targeting means. The phenomenon of passive addressing exploits the patterns of natural distributions of a transfer vehicle in vivo without relying on the use of additional excipients or means to enhance recognition of the transfer vehicle by the target cells. For example, transfer vehicles that undergo phagocytosis by cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen and, therefore, can provide means to passively direct the administration of compositions to said target cells.

Alternatively, the present invention contemplates active addressing, which involves the use of additional excipients, referred to herein as "targeting ligands" that can be attached (covalently or non-covalently) to the transfer vehicle to stimulate the location of said transfer vehicles in certain target cells or target tissues. For example, targeting may be mediated by the inclusion of one or more endogenous targeting ligands (eg, apolipoprotein E) in or on the transfer vehicle to stimulate distribution to target cells or tissues. The recognition of the targeting ligand by the target tissues actively facilitates the tissue distribution and cellular absorption of the transfer vehicle and / or its contents in the target cells and tissues (eg, the inclusion of an apolipoprotein-E targeting ligand in or on the transfer vehicle stimulates recognition and binding of the transfer vehicle to endogenous low density lipoprotein receptors expressed by hepatocytes). As provided herein, the composition may comprise a ligand capable of enhancing the affinity of the composition with the target cell. The targeting ligands can be ligated to the outer bilayer of the lipid particle during formulation or post-formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusogenic polymers such as PEAA, hemagglutinin, other lipopeptides (see US Patent Applications Nos. Series 08 / 835,281 and 60 / 083,294, which are incorporated herein by reference) and other features useful for in vivo and / or intracellular administration. In other various embodiments, the compositions of the present invention demonstrate improved transfection efficiencies and / or demonstrate enhanced selectivity towards the target cells or tissues of interest. Therefore, compositions comprising one or more ligands (for example, peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents with cells and tissues are contemplated objective. Appropriate ligands may optionally be attached or ligated to the surface of the transfer vehicle. In some embodiments, the addressing ligand may encompass the surface of a transfer vehicle or may be encapsulated within the transfer vehicle. Appropriate ligands are selected based on their physical, chemical or biological properties (for example, selective affinity and / or recognition of markers or surface characteristics of the target cell). The specific cell targeting sites and their corresponding targeting ligand can vary widely. Appropriate targeting ligands are selected in order to exploit the distinctive characteristics of a target cell, thus allowing the composition to distinguish between target and non-target cells. For example, the compositions of the invention may include surface markers (for example, apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition or affinity with hepatocytes (eg, by recognition and receptor-mediated binding of said surface markers ). Additionally, the use of galactose as the targeting ligand is expected to direct the compositions of the present invention towards parenchymal hepatocytes or, alternatively, it is expected that the use of sugar containing sugar residues as the targeting ligand will direct the compositions of the present invention. to liver endothelial cells (for example, mannose containing sugar residues that may preferentially bind to the asialoglycoprotein receptor present in hepatocytes). (See Hillery AM, et al. "Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists" (2002) Taylor &amp; Francis, Inc). Therefore, the presentation of said targeting ligands that have been conjugated with moieties present in the transfer vehicle (eg, a lipid nanoparticle) facilitates the recognition and absorption of the compositions of the present invention in the target cells and tissues . Examples of appropriate targeting ligands include one or more peptides, proteins, aptamers, vitamins and oligonucleotides.

Application and Administration

As used herein, the term "subject" refers to any animal (eg, a mammal), including, but not limited to, humans, non-human primates, rodents and the like, to which the compositions are administered. and methods of the present invention. Typically, the terms "subject and" patient "are used interchangeably herein in reference to a human subject.

The compositions and methods of the invention provide mRNA administration to treat various disorders. In particular, the compositions and methods of the present invention are suitable for the treatment of diseases or disorders related to the deficiency of proteins and / or enzymes that are excreted or secreted by the target cell in the surrounding extracellular fluid (eg, mRNA that encodes hormones and neurotransmitters). In embodiments, the disease may involve a defect or deficiency in a secreted protein (eg, Fabry disease or ALS). In certain embodiments, the disease may not be caused by a defect or deficit in a secreted protein, but may benefit from the provision of a secreted protein. For example, the symptoms of a disease can be improved by providing the compositions of the invention (for example, cystic fibrosis). Disorders for which the present invention is useful include, but are not limited to disorders such as Huntington's disease; Parkinson's disease; muscular dystrophies (such as, for example, Duchenne and Becker); hemophilic diseases (such as, for example, hemophilia B (FIX), hemophilia A (FVIII); spinal muscular atrophy related to SMN1 (SMA); amyotrophic lateral sclerosis (ALS); galactosemia related to GALT; Cystic fibrosis (CF); disorders related to SLC3A1 including cystinuria; disorders related to COL4A5 including Alport syndrome; galactocerebrosidase deficiencies; adrenoleukodystrophy and adrenomyeloneuropathy linked to the X chromosome; Friedreich's ataxia; Pelizaeus-Merzbacher disease; tuberous sclerosis related to TSC1 and TSC2; Sanfilippo B syndrome (MPS IIIB); cystinosis related to CTNS; FMR1-related disorders that include fragile X chromosome syndrome, Tremor / ataxia syndrome associated with fragile X chromosome and premature ovarian insufficiency syndrome of fragile X chromosome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick Type C1 disease, diseases related to neuronal zeroide lipofuscinosis including childhood neuronal zeroide lipofuscinosis (JNCL), childhood Batten disease, SantavuoriHaltia disease, Jansky-Bielschowsky disease and PTT-1 and TPP1 deficiencies; childhood ataxia related to EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5 with hypomyelination of the central nervous system / evanescent white matter; Episodic ataxia related to CACNA1A and CACNB4 Type 2; MECP2-related disorders including classical Rett syndrome, severe neonatal encephalopathy related to MECP2 and PPM-X syndrome; Atypical Rett syndrome related to CDKL5; Kennedy disease (SBMA); Autosomal dominant cerebral arteriopathy with subcortical infarctions and Notch-3-related leukoencephalopathy (CADASIL); seizure disorders related to SCN1A and SCN1B; Polymerase G-related disorders that include Alpers-Huttenlocher syndrome, sensory ataxic neuropathy associated with POLG, dysarthria and ophthalmoparesis and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agamaglobulinemia; Wilson's disease; and Fabry's disease. In one embodiment, the nucleic acids and, in particular, the mRNA of the invention can encode functional proteins or enzymes that are secreted in the extracellular space. For example, secreted proteins include coagulation factors, complement pathway components, cytokines, chemokines, chemotaxins, protein hormones (eg, EGF, PDF), whey protein components, antibodies, secretable toll-like receptors and others. In some embodiments, the compositions of the present invention may include mRNA encoding erythropoietin, a1-antitrypsin, carboxypeptidase N or human growth hormone.

In embodiments, the invention encodes a secreted protein that is constituted by subunits that are encoded by more than one gene. For example, the secreted protein may be a heterodimer, wherein each chain or subunit is encoded by a separate gene. It is possible that more than one mRNA molecule is administered in the transfer vehicle and that the mRNA encodes separate units of the secreted protein. Alternatively, a single mRNA can be genomanipulated to encode more than one sub-unit (for example, in the case of a single chain Fv antibody). In certain embodiments, separate mRNA molecules encoding individual subunits can be administered in separate transfer vehicles. In one embodiment, the mRNA can encode full-length antibodies (both light and heavy chains of the variable and constant regions) or antibody fragments (e.g., Fab, Fv or a single chain Fv (scFv) to confer immunity to a subject. Although an embodiment of the present invention relates to methods and compositions useful for conferring immunity to a subject (for example, through the translation of mRNA encoding functional antibodies), the inventions disclosed herein and contemplated herein are broadly applicable. In an alternative embodiment, the compositions of the present invention encode antibodies that can be used to produce a functional response in subjects chronically or transiently. For example, the mRNA of the present invention can encode a functional monoclonal or polyclonal antibody, which after translation and secretion from the target cell, may be useful for addressing and / or deactivating a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the mRNA nucleic acids of the present invention may encode, for example, functional anti-nephritic factor antibodies useful for the treatment of type II membranoproliferative glomerulonephritis or acute hemolytic uremic syndrome, or alternatively they may encode an anti-factor antibody. of vascular endothelial growth (VEGF) useful for the treatment of diseases mediated by VEGF, such as cancer. In other embodiments, the secreted protein is a cytokine and another secreted protein that comprises more than one subunit (for example, IL-12 or IL-23).

The compositions of the invention can be administered to a subject. In some embodiments, the composition is formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmaceutical compositions where it is mixed with suitable excipients. For example, in one embodiment, the compositions of the invention can be prepared to administer mRNA encoding two or more different proteins or enzymes. Techniques for drug formulation and administration are found in Remington's Pharmaceutical Sciences, ”Mack Publishing Co., Easton, Pa., Latest edition.

A wide range of molecules that can exert pharmaceutical or therapeutic effects can be administered to target cells using compositions and methods of the invention. The molecules can be organic or inorganic. The organic molecules can be peptides, proteins, carbohydrates, lipids, sterols, nucleic acids (including peptide nucleic acids) or any combination thereof. A formulation for administration to target cells may comprise more than one type of molecule, for example, two different nucleotide sequences, or a protein, an enzyme or a steroid.

The compositions of the present invention can be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the regimen of administration, age, sex, body weight and other relevant factors. of the patient for doctors skilled in the art. The "effective amount" for the purposes of this may be determined by such relevant considerations as are known to those skilled in the art of experimental clinical, pharmacological, clinical and medical research. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indications such as are selected as appropriate measures of evolution, regression or improvement of a disease by those skilled in the art. For example, a suitable amount and dosage regimen is one that causes at least one production of transient protein.

Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary administration including intratracheal or inhaled, or intestinal; parenteral administration, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections.

Alternatively, the compositions of the invention can be administered in a local rather than systemically, for example, by injecting the pharmaceutical composition directly into a target tissue, preferably in a sustained release formulation. Local administration can be affected in several ways, depending on the target tissue. For example, aerosols containing compositions of the present invention (for nasal, tracheal or bronchial administration) can be inhaled; Compositions of the present invention can be injected at the site of injury, manifestation of the disease or pain, for example; Dragee compositions may be provided for oral, tracheal or esophageal application; they can be supplied as a liquid, tablet or capsule for administration in the stomach or intestines, it can be supplied as a suppository for rectal or vaginal application; or they can even be administered in the eye by using creams, drops or even injection. Formulations containing compositions of the present invention forming complexes with therapeutic molecules or ligands can even be administered surgically, for example, in association with a polymer or other structure or substance that may allow the compositions to be dispersed from the implantation site to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.

In one embodiment, the compositions of the invention are formulated so that they are suitable for extended release of the mRNA contained therein. Such extended release compositions can conveniently be administered to a subject at extended dosage intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice daily, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks or more preferably every four weeks, once a month , every six weeks, every eight weeks, every two months, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions and liposomal vehicles that are formulated for depot administration (eg, intramuscularly, subcutaneously, intravitreally) to administer or release an mRNA for extended periods of time. Preferably, the extended release means employed are combined with modifications made to the mRNA to enhance stability.

Lyophilized pharmaceutical compositions comprising one or more of the liposomal nanoparticles disclosed herein and related methods for the use of such lyophilized compositions such as are disclosed, for example, in US Provisional Application No. 61/494,882 are also contemplated herein. , filed on June 8, 2011, whose disclosures are incorporated herein by reference in their entirety. For example, lyophilized pharmaceutical compositions according to the invention can be reconstituted before administration or can be reconstituted in vivo. For example, a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (for example, an intradermal dosage form such as a disk, rod or membrane) and administered in such a way that the dosage form is rehydrated over time in vivo. through the individual's body fluids.

Although certain compounds, compositions and methods of the present invention have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention are not intended to limit it. Each of the publications, reference materials, access numbers and the like mentioned herein to describe the background of the invention and to provide additional details in relation to its practice are incorporated herein by reference in its entirety.

The articles "el / la" and "un / una" as used in the specification and the claims, unless clearly stated otherwise, should be understood as including plural referents. Claims or descriptions that include "or" among one or more members of a group are considered satisfied if one, more than one or all members of the group are present, employed or otherwise relevant to a product or process given to unless otherwise indicated or unless it is evident from the context. The invention includes embodiments in which exactly one member the group is present, employed, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one or all of the members the group is present, employed, or otherwise relevant to a given product or process. Additionally, it will be understood that the invention encompasses all variations, combinations and permutations in which one or more limitations, elements, conditions, descriptive terms, etc., of one or more of the stated claims are introduced into another dependent claim therein. base claim (or, as relevant, any other claim) unless otherwise indicated or unless it is apparent to a person skilled in the art that a contradiction or inconsistency will arise. When the elements are presented as lists, (for example, in Markush's group or similar format) it will be understood that each subgroup of the elements is also disclosed, and any element (s) can be removed from the group. It will be understood that, in general, when it is mentioned that the invention or aspects of the invention comprise / n specific elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially, of said elements, features, etc. For the purposes of simplification, said embodiments have not been established in each case specifically in so many words herein. It will also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is mentioned in the specification. Publications and other reference materials mentioned herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference in its entirety.

EXAMPLES

EXAMPLE 1

Deposit of protein production through intravenous administration of polynucleotide compositions

Messenger RNA

Human erythropoietin (EPO) (SEQ ID NO: 3; FIG. 3), human alphagalactosidase (GLA) (SEQ ID NO: 4; FIG. 4), human alpha-1 antitrypsin (A1AT) (SEQ ID NO: 5; FIG 5), human factor IX (FIX) (SEQ ID NO: 6; FIG. 6) were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, and then the structure was added 5 'cap (Capí) (Fechter &amp; Brownlee, J. Gen. Virology 86: 1239-1249 (2005)) and a 3 'poly (A) end of approximately 200 nucleotides in length as determined by gel electrophoresis. The 5 'and 3' non-translated regions were present in each mRNA product in the following examples and are defined by SEQ ID NOs: 1 and 2 (FIG. 1 and FIG. 2) respectively.

Lipid nanoparticle formulations

Formulation 1: 50 mg / mL aliquots of C12-200 ethanol solutions, DOPE, Col and DMG-PEG2K (40: 30: 25: 5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of mRNA was prepared from 1 mg / mL of concentrated solution. The lipid solution was rapidly injected into an aqueous mRNA solution and stirred to provide a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

Formulation 2: 50 mg / mL aliquots of ethanol solutions of DODAP, DOPE, cholesterol and DMG-PEG2K (18: 56: 20: 6) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of EPO mRNA was prepared from 1 mg / mL of concentrated solution. The lipid solution was rapidly injected into a solution of

Aqueous mRNA and stirred to provide a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration = 1.35 mg / mL EPO mRNA (encapsulated). Z_bird = 75.9 nm (Dv₍₅₀) = 57.3 nm; Dv₍90) = 92.1 nm).

Formulation 3: 50 mg / mL aliquots of ethanolic solutions of HGT4003, DOPE, cholesterol and DMG-PEG2K (50: 25: 20: 5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of mRNA was prepared from 1 mg / mL of concentrated solution. The lipid solution was rapidly injected into an aqueous mRNA solution and stirred to provide a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

Formulation 4: 50 mg / mL aliquots of ethanol solutions of ICE, DOPE and DMG-PEG2K (70: 25: 5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of mRNA was prepared from 1 mg / mL of concentrated solution. The lipid solution was rapidly injected into an aqueous mRNA solution and stirred to provide a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

Formulation 5: 50 mg / mL aliquots of ethanolic solutions of HGT5000, DOPE, cholesterol and DMG-PEG2K (40: 20: 35: 5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of EPO mRNA was prepared from 1 mg / mL of concentrated solution. The lipid solution was rapidly injected into an aqueous mRNA solution and stirred to provide a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration = 1.82 mg / mL EPO mRNA (encapsulated). Z_bird = 105.6 nm (Dv₍₅₀) = 53.7 nm; Dv₍90) = 157 nm).

Formulation 6: 50 mg / mL aliquots of ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (40: 20: 35: 5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of EPO mRNA was prepared from 1 mg / mL of concentrated solution. The lipid solution was rapidly injected into an aqueous mRNA solution and stirred to provide a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

Protein analysis produced through intravenously administered mRNA loaded nanoparticles

Injection protocol

Study was carried out using male CD-1 mice approximately 6-8 weeks at the start of each experiment, unless otherwise indicated. Samples were introduced by an injection into the tail vein of a single bolus of an equivalent total dose of 30-200 micrograms of encapsulated mRNA. Mice were sacrificed and perfused with saline at the indicated time points.

Isolation of organic tissues for analysis

The liver and spleen of each mouse were collected, split into three parts and stored in 10% neutral buffered formalin or frozen immediately and stored at -80 ° C for analysis.

Isolation of serum for analysis

All animals were sacrificed by asphyxiation with CO₂ 48 hours after the administration of the dose (± 5%), a thoracotomy and extraction of the terminal cardiac blood were subsequently carried out. Whole blood (maximum volume obtainable) was extracted through cardiac puncture in the animals sacrificed in serum separator tubes, allowed to coagulate at room temperature for at least 30 minutes, centrifuged at 22 ° C ±

5 ° C at 9300 g for 10 minutes and then the serum was extracted. For provisional blood draws, approximately 4050pL of whole blood was extracted through puncture of the facial vein or tail cut. Samples taken from untreated animals were used as a reference value for comparison with study animals.

Enzyme-linked immunosorbent assay (ELISA) analysis

EPO ELISA: The quantification of the EPO protein was carried out following the procedures indicated for the ELISA kit for human EPO (Quantikine IVD, R&amp;D Systems, Catalog No. Dep-00). The positive controls used consisted of recombinant human erythropoietin protein of ultrapure grade and tissue culture (R&amp;D Systems, Catalog No. 286-EP and 287-TC, respectively). Detection was monitored through absorption (450 nm) in a Molecular Device Flex Station instrument.

GLA ELISA: Standard ELISA procedures were performed using sheep anti-Alpha-galactosidase G-188 IgG as the capture antibody with rabbit anti-alpha-galactosidase TK-88 IgG as the secondary antibody (detection) (Shire Human Genetic Therapies). Goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) was used to activate the substrate solution of 3,3 ', 5,5'tetramethylbenzidine (TMB). The reaction was quenched using 2SO H2SO4 after 20 minutes. Detection was monitored through absorption (450 nm) in a Molecular Device Flex Station instrument. Untreated mouse serum and the alpha-galactosides to human protein were used as negative and positive controls, respectively.

FIX ELISA: The quantification of the FIX protein was carried out following the procedures indicated for the ELISA kit for human FIX (AssayMax, Assay Pro, Catalog No. EF1009-1).

A1AT ELISA: The quantification of the A1AT protein was carried out following the procedures indicated for the ELISA kit for human A1AT (Innovative Research, Catalog No. IRAPKT015).

Western blot analysis (EPO): Western blot analysis was carried out using an anti-hEPO antibody (R&amp;D Systems No. MAB2871) and ultrapure human EPO protein (R&amp;D Systems No. 286-EP) as a control.

Results

The work described in this example demonstrates the use of lipid nanoparticles with encapsulated mRNA as a reservoir source for protein production. Said deposition effect can be achieved at multiple sites within the body (ie, liver, kidneys, spleen and muscles). Measurements of the desired exogenous base protein derived from messenger RNA administered through liposomal nanoparticles were achieved and quantified, and protein secretion from a depot was demonstrated using

Human erythropoietin mRNA (hEPO), human alpha-galactosidase (hGLA), human alpha-1 antitrypsin (hA1AT) and human Factor IX (hFIX).

1A. Production results of human EPO protein in vivo

The production of the hEPO protein was demonstrated with several formulations of lipid nanoparticles. Of four different cationic lipid systems, C12-200-based lipid nanoparticles produced the highest amount of hEPO protein after four hours of intravenous administration as measured by ELISA (FIG. 7). This formulation (Formulation 1) resulted in 18.3 ug / mL of hEPO protein secreted in the bloodstream. Normal serum hEPO protein levels for humans are 3.3-16.6 mlU / mL (NCCLS Document C28-P; Volume 12, No. 2). Based on a specific activity of 120,000 lU / mg of EPO protein, which provide an amount of 27.5-138 pg / mL of hEPO protein in normal human individuals. Therefore, a single 30 ug dose of a C12-200-based cationic lipid formulation with encapsulated hEPO mRNA provided an increase in the respective protein more than 100,000 times than physiological levels.

Of the lipid systems evaluated, the formulation with lipid nanoparticles based on DODAP was the least effective. However, the observed amount of human EPO protein derived from administration through DODAP-based lipid nanoparticles with

Encapsulated EPO mRNA was 4.1 ng / mL, which is still 30 times higher than the normal physiological levels of the EPO protein (Table 1).

TABLE 1

Crude values ​​of secreted hEPO protein for various cationic lipid-based nanoparticle systems measured through ELISA analysis (as depicted in FIG. 8).

<td>Cationic / ionizable lipid component</td><td>Dose of encapsulated mRNA (ug)</td><td>Secreted human EPO protein (ng / mL)</td><td>Increase in hematocrit (%)</td>

<td>C12-200</td><td> 30</td><td> 18 306</td><td> 15.0</td>

<td>HGT4003</td><td> 150</td><td> 164</td><td> 0.0</td>

<td>ICE</td><td> 100</td><td> 56.2</td><td> 0.0</td>

<td>DODAP</td><td> 200</td><td> 4.1</td><td> 0.0</td>

Doses are based on encapsulated hEPO mRNA. Protein values ​​are represented as nanograms of human EPO protein per milliliter of serum. Changes in hematocrit are based on comparison before extraction (Day -1) and Day 10.

In addition, the resulting protein was evaluated to determine if it was active and functioning properly. In the case of mRNA replacement therapy (MRT) in which hEPO mRNA is used, changes in the hematocrit were monitored for a period of ten days for five different lipid nanoparticle formulations (FIG. 8, Table 1) for Evaluate protein activity. During this period of time, two of the five formulations demonstrated an increase in hematocrit (> 15%), which is indicative of the production of active hEPO protein from these systems.

In another experiment, changes in hematocrit were monitored for a period of 15 days (FIG. 9, Table 2). The lipid nanoparticle formulation {Formulation 1) was administered as a single dose of 30 pg or as three smaller doses of 10 pg injected on day 1, day 3 and day 5. Similarly, Formulation 2 was administered as 3 doses of 50 pg on day 1, day 3 and day 5. C12-200 produced a considerable increase in hematocrit. In general, an increase of up to -25% change was observed, which is indicative of the production of active human EPO protein from such systems.

TABLE 2

Hematocrit levels for each group in an observation period of 15 days (FIG. 9).

<td rowspan="2">Test article</td><td rowspan="2">Dose (pg / animal)</td><td colspan="4">Average levels of Hct (%) ± SEM</td>

<td>Day 4</td><td>Day 7</td><td>Day 10</td><td>Day 15^to</td>

<td>C12-200</td><td>30 (dose only)</td><td> 50.811.8</td><td> 58.313.3</td><td> 62.811.3</td><td> 59.913.3</td>

<td>C12-200</td><td>30 (in 3 doses)</td><td> 52.210.5</td><td> 55.312.3</td><td> 63.311.6</td><td> 62.3±1.9</td>

<td>DODAP</td><td>150 (in 3 doses)</td><td> 54.811.7</td><td> 53.511.6</td><td> 54.213.3</td><td> 54.010.3</td>

Hct = hematocrit; SEM = standard error of the mean.

^toBlood samples were taken in non-heparinized hematocrit tubes.

The mice were dosed with a single injection or three injections, every two days. N = 4 mice per group.

1 B. Production results of human GLA protein in vivo

A second exogenous base protein system was explored to demonstrate the "deposition effect" when lipid nanoparticles loaded with mRNA are employed. The animals were injected intravenously with a single dose of 30 micrograms of human alpha-galactosidase mRNA (hGLA) encapsulated using a C12200-based nanoparticle system and sacrificed six hours later (Formulation 1). The quantification of the hGLA protein was carried out through ELISA. Untreated mouse serum and human Alpha-galactosidase protein were used as controls. The detection of the alpha-galactosidase protein was monitored for a period of 48 hours.

Measurable levels of hGLA protein were observed throughout the experiment with a maximum level of 2.0 ug / mL of hGLA protein at six hours (FIG. 10). Table 3 indicates the specific amounts of hGLA found in the serum. It has been indicated that normal activity in healthy male humans is approximately 3.05 nanomoles / hr / mL. Activity for Alpha-galactosidase, a recombinant human alpha-galactosidase protein, 3.56x10⁶ nanomoles / hr / mg. The analysis of these values ​​provides an amount of approximately 856 pg / mL of hGLA protein in normal healthy male individuals. The amount of 2.0 ug / mL of hGLA protein observed after six when lipid nanoparticles loaded with hGLA mRNA were dosed is more than 2300 times greater than normal physiological levels. In addition, after 48 hours, appreciable levels of hGLA protein (86.2 ng / mL) can still be detected. This level is representative of almost 100 times larger amounts of hGLA protein with respect to the physiological amounts still present at 48 hours.

TABLE 3

Crude values ​​of hGLA protein secreted with respect to time measured by ELISA analysis (as depicted in FIG.

10).

<td>Post-administration time (hr)</td><td>Secreted human GLA protein (ng / mL)</td>

<td> 6</td><td> 2038</td>

<td> 12</td><td> 1815</td>

<td> 24</td><td> 414</td>

<td> 48</td><td> 86.2</td>

Values ​​are represented as nanograms of hGLA protein per milliliter of serum. N = 4 mice per group.

In addition, the half-life of Alpha-galactosidase when administered at 0.2 mg / kg is approximately 108 minutes. The production of the GLA protein through the "deposition effect when administering lipid nanoparticles loaded with GLA mRNA shows a substantial increase in residence time in blood when compared with direct injection of the naked recombinant protein. As described above, significant amounts of the protein are present after 48 hours.

The activity profile of the α-galactosidase protein produced from lipid nanoparticles loaded with GLA mRNA was measured as a function of the metabolism of 4-methylumbelliferyl-aD-galactopyranoside (4MU-a-gal). As shown in FIG. 11, the protein produced from these nanoparticle systems is quite active and reflects the levels of protein available (FIG. 12, Table 3). AUC comparisons of hGLA production based on mRNA therapy with respect to enzyme replacement therapy (ERT) in mice and humans show an increase of 182 times and 30 times, respectively (Table 4).

TABLE 4

C value comparison_max and AUC¡nf in patients with Fabry after IV dosing of 0.2 mq / kq of Alpha-qalactosidase (pharmacological dose) with those of mice after IV dosing with Alpha-qalactosidase and GLA mRNA.

<td></td><td>Test item</td><td>Description</td><td>Dose (mg / kg)</td><td>Cmax (U / mL)</td><td>AUC_{in (}(hr.U / mL)</td><td>n</td>

<td rowspan="3">Patient with Fabry³</td><td rowspan="3">AGAL protein</td><td>Transplant</td><td> 0.2</td><td> 3478</td><td> 3683</td><td> 11</td>

<td>Dialysis</td><td> 0.2</td><td> 3887</td><td> 3600</td><td> 6</td>

<td>Without ESRD^b</td><td> 0.2</td><td> 3710</td><td> 4283</td><td> 18</td>

<td rowspan="2">Mouse</td><td>AGAL protein (MM1)</td><td>athletic hairless</td><td> 0.04</td><td> 3807</td><td> 797</td><td> 3</td>

<td>AGAL protein (MM2)</td><td>athletic hairless</td><td> 0.04</td><td> 3705</td><td> 602</td><td> 3</td>

<td>Mouse</td><td>AGAL mRNA</td><td>Mouse</td><td> 0.95</td><td>5885 (C_tot 6hr)^C</td><td> 109428</td><td> 6</td>

^to Data from a published article (Gregory M. Pastores et al. Safety and Pharmacokinetics of hGLA in patients with Fabry disease and end-stage renal disease. Nephrol Dial Transplant (2007) 22: 1920-1925. ^b non-terminal stage kidney disease. ^c A-Galactosidase activity 6 hours after dosing (the earliest time point evaluated in the study).

The ability of lipid nanoparticles with encapsulated mRNA to target organs that can act as a reservoir for the production of a desired protein has been demonstrated. The levels of secreted protein observed have been above normal physiological levels by several orders of magnitude. This "deposit effect" is repeatable. FIG. 12 again shows that a solid protein production is observed after dosing to natural mice (CD-1) a single 30 ug dose of C12-200-based lipid nanoparticles loaded with hGLA mRNA (Formulation 1). In this experiment, hGLA levels were evaluated for a period of 72 hours. A maximum average of 4.0 ug of human hGLA protein / mL of serum is detected six hours after administration. Based on a value of ~ 1 ng / mL of hGLA protein for normal physiological levels, hGLA MRT provides approximately 4000 times higher protein levels. As before, the hGLA protein could be detected for 48 hr post-administration (FIG. 12).

An analysis of isolated tissues from this same experiment provided an idea of ​​the distribution of hGLA protein in mice treated with hGLA MRT (FIG. 13). Supraphysiological level of hGLA protein was detected in the liver, spleen and kidneys of all mice treated with a maximum observed between 12 and 24 hours post-administration. Detective values ​​of MRT-derived protein could be observed three days after a single injection of lipid nanoparticles loaded with hGLA.

In addition, it was shown that hGLA production after administration of the C12-200 nanoparticles loaded with hGLA mRNA exhibited a dose response in the serum (FIG. 14A), as well as in the liver (FIG. 14B).

An inherent feature of replacement therapy for

Lipid nanoparticle mediated mRNA would be the pharmacokinetic profile of the respective protein produced. For example, ERT-based treatment of mice using Alpha-galactosidase results in a plasma half-life of approximately 100 minutes. In contrast, MRT-derived alpha-galactosidase has a residence time in blood of approximately 72 hrs with a peak time of 6 hours. This allows a much greater exposure for the organs to participate in the possible continuous absorption of the desired protein. A comparison of the PK profiles is shown in FIG. 15 and demonstrates the clear difference in clearance rates and that a significant change in the area under the curve (AUC) can ultimately be achieved through MRT-based rates.

In a separate experiment, the MRT of hGI_A was applied to a mouse disease model, mice with disabled hGl_A (mice with Fabry). A dose of 0.33 mg / kg of C12200-based lipid nanoparticles loaded with hGI_A mRNA (Formulation 1) was administered to inactivated female mice as a single intravenous injection. Substantial amounts of MRT-derived hGLA were produced with a peak at 6 hr (~ 560 ng / mL serum) that is approximately 600 times higher than normal physiological levels. In addition, the hGI_A protein was still detectable 72 hr post-administration (FIG. 16).

Quantification of the GLA protein derived from MRT in vital organs demonstrated the substantial accumulation shown in FIG. 17. A comparison of the MRT derived hGLA protein observed with respect to the indicated normal physiological levels found in the key organs is plotted (normal levels plotted as dotted lines). Although the protein levels at 24 hours are higher than at 72 hours post-administration, the hGLA protein levels detected in the liver, kidneys, spleen and hearts of treated Fabry mice are equivalent to natural levels. For example, 3.1 ng of hGLA protein / mg of tissue were found in the kidneys of mice treated 3 days after a single MRT treatment.

In a subsequent experiment, a comparison of treatment with Alpha-galactosidase based on ERT was performed with respect to treatment with hGLA based on MRT of male Fabry inactivated mice. A single intravenous dose of 1.0 mg / kg was provided for each therapy and the mice were sacrificed one week after administration. The serum levels of the hGLA protein were monitored at 6 hr and 1 week post-injection. The liver, kidneys, spleen and heart were analyzed to determine the accumulation of hGLA protein one week after administration. In addition to biodistribution analyzes, a measure of efficacy was determined through measurements of reductions of globotrioasilceramide (Gb3) and Iiso-Gb3 in the kidneys and heart. FIG. 18 shows serum levels of the hGLA protein after treatment with lipid nanoparticles loaded with Alpha-galactosidase or GLA mRNA (Formulation 1) in male Fabry mice. Serum samples were analyzed at 6 hr and 1 week after administration. A solid signal was detected for mice treated with MRT after 6 hours, with serum levels of hGLA protein of ~ 4.0 ug / mL. In contrast, there was no detectable Alphagalactosidase remaining in the bloodstream at this time.

The mice with Fabry from this experiment were sacrificed one week after the initial injection and the organs were collected and analyzed (liver, kidneys, spleen, heart). FIG. 19 shows a comparison of the human GLA protein found in each respective organ after treatment with MRT of hGLA or with Alphagalactosidase of ERT. The levels correspond to the hGLA present one week after administration. The hGLA protein was detected in all the organs analyzed. For example, mice treated with MRT resulted in an accumulation of hGLA protein in the kidneys of 2.42 ng hGLA / mg protein, while mice treated with Alpha-galactosidase had only residual levels (0.37 ng / mg protein). This corresponds to a ~ 6.5 times higher level of hGLA protein when treated through hGLA MRT. After heart analysis, 11.5 ng of hGLA / mg protein was found in the MRT-treated cohort compared to only 1.0 ng / mg of Alpha-galactosidase protein. This corresponds to an accumulation -11 times greater in the heart for mice treated with MRT of hGLA with respect to therapies based on ERT.

In addition to biodistribution analyzes, evaluations were made to determine efficacy through measurements of globotrioasilceramide (Gb3) and Iiso-Gb3 levels in key organs. A direct comparison of the reduction of Gb3 after a single-dose intravenous GLA MRT treatment of 1.0 mg / kg GLA MRT compared to an alpha-galactosidase ERT-based therapy of an equivalent dose provided a considerable difference in the levels of Gb3 in the kidneys and in the heart. For example, Gb3 levels for MRT of GLA with respect to Alpha-galactosidase provided reductions of 60.2% and 26.8%, respectively (FIG. 20). In addition, Gb3 levels in the heart were reduced by 92.1% with respect to 66.9% for MRT and Alpha-galactosidase, respectively (FIG. 21).

A second relevant biomarker for efficacy measurement is Iiso-Gb3. GLA MRT reduced Iiso-Gb3 more effectively than Alphagalactosidase in the kidneys and heart (FIG. 20 and FIG. 21, respectively). In particular, Fabry mice treated with MRT demonstrated Iiso-Gb3 reductions of 86.1% and 87.9% in the kidneys and heart compared to mice treated with Alpha-galactosidase that provided a decrease of 47.8% and 61.3%, respectively.

The results for nanoparticles based on C12-200 with hGLA were extended to the other formulations of lipid nanoparticles. For example, lipid nanoparticles based on HGT4003 (Formulation

3) or based on HGT5000 (Formulation 5) loaded with hGLA mRNA administered in a single IV dose result in hGLA production 24 hours after administration (FIG. 22). HGLA production exhibited a dose response. Similarly, the production of hGLA was observed 6 hours and 24 hours after the administration of lipid nanoparticles based on HGT5001 (Formulation 6) loaded with hGLA mRNA administered as a single IV dose. The production of hGLA was observed in the serum (FIG. 23A), as well as in the organs (FIG. 23B).

In general, mRNA replacement therapy applied as a reservoir for protein production produces large amounts of functionally therapeutic protein, active at supraphysiological levels. This method was shown to provide a sustained circulation half-life of the desired protein and this MRT-derived protein is highly effective for therapy as demonstrated with the alpha-galactosidase enzyme in mice with Fabry.

1 C. Production results of human FIX protein in vivo

Studies were carried out by administering lipid nanoparticles loaded with Factor IX mRNA (FIX) in natural mice (CD-1) and determining the FIX protein that is secreted in the bloodstream. After intravenous injection of a single dose of 30 ug of C12-200-based lipid nanoparticles loaded with FIX mRNA (C12-200: DOPE: Col: PEG at a ratio of 40: 30: 25: 5) (dose based on Encapsulated mRNA) (Formulation 1), a solid protein production was observed (FIG. 24).

Pharmacokinetic analysis at 72 showed that MRT-derived FIX protein could be detected at all time points evaluated (FIG. 24). The peak serum concentration was observed at 24 h post injection with a value of ~ 3 ug (29951738 ng / mL) FIX protein / mL of serum. This represents another successful example of the deposit effect.

1D. Production results of human A1AT protein in 5 vivo

Studies were conducted by administering lipid nanoparticles loaded with alpha-1-antitrypsin mRNA (A1AT) in natural mice (CD-1) and determining the A1AT protein that is secreted in the bloodstream. After intravenous injection of a single dose of 30 ug 10 of C12-200-based lipid nanoparticles loaded with A1AT mRNA (encapsulated mRNA based dose) (Formulation 1), a solid protein production was observed (FIG. 25).

As shown in FIG. 25, detectable levels of human A1AT protein derived from A1AT MRT could be observed for a 15-hour period of post-administration. A maximum serum level of ~ 48 ug A1AT protein / mL of serum was detected 12 hours after injection.

EXAMPLE 2

Deposit of protein production through pulmonary administration of polynucleotide compositions

Invention Protocol

All studies A study was conducted using female CD-1 or BALB / C mice approximately 7-10 weeks at the start of each experiment. The items tested were introduced through a single intratracheal aerosolized administration. Mice were sacrificed and perfused with saline at the indicated time points. The lungs of each mouse were collected, split into two parts and stored in 10% neutral buffered formalin or frozen immediately and stored at -80 ° C for analysis. Serum was isolated as described in Example 1. EPO ELISA: as described in Example 1.

Results

The deposition effect can be achieved through pulmonary administration (eg, intranasal, intratracheal, nebulization). The measurement of the desired exogenous base protein derived from messenger RNA administered through nanoparticle systems was achieved and quantified.

The production of human EPO protein through lipid nanoparticles loaded with hEPO mRNA was evaluated in mice.

CD-1 through a single intratracheal administration (MicroSprayer®). Several formulations were evaluated using several cationic lipids (Formulations 1, 5, 6). All formulations resulted in high encapsulation of human EPO mRNA. After administration, the 5 animals were sacrificed six hours after administration and the lungs and serum were removed.

Human EPO protein was detected at the site of administration (lungs) after treatment through aerosol administration. Serum analysis six hours after administration exhibited detectable amounts of the circulating hEPO protein. These data (shown in FIG. 26) demonstrate the ability of the lung to act as a "reservoir" for the production (and secretion) of the hEPO protein.