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Delivery of mRNA for the augmentation of proteins and enzymes in human genetic diseases

a technology for enhancing proteins and enzymes, applied in the direction of non-active genetic ingredients, genetic material ingredients, drug compositions, etc., can solve the problems of affecting the expression of proteins or enzymes, and reducing the synthesis of proteins, so as to facilitate the transfection and enhance the affinity of compositions

Inactive Publication Date: 2011-10-06
TRANSLATE BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]Also provided are compositions and methods useful for facilitating the transfection and delivery of one or more nucleic acids (e.g., mRNA) to target cells. For example, the compositions and methods of the present invention contemplate the use of targeting ligands capable of enhancing the affinity of the composition to one or more target cells. In one embodiment, the targeting ligand is apolipoprotein-B or apolipoprotein-E and corresponding target cells express low-density lipoprotein receptors, thereby facilitating recognition of the targeting ligand. The methods and compositions of the present invention may be used to preferentially target a vast number of target cells. For example, contemplated target cells include, but are not limited to, 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 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.

Problems solved by technology

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 protein, the reduced synthesis of the protein, or synthesis of a protein lacking or having diminished biological activity.

Method used

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  • Delivery of mRNA for the augmentation of proteins and enzymes in human genetic diseases
  • Delivery of mRNA for the augmentation of proteins and enzymes in human genetic diseases
  • Delivery of mRNA for the augmentation of proteins and enzymes in human genetic diseases

Examples

Experimental program
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Effect test

example 1

General Preparation of Transfer Vehicles by Solvent Dilution Technique

[0098]This example generally illustrates a process for the manufacture of small (<100 nm) liposomal formulations containing mRNA and the means to evaluate the amount of mRNA encapsulated. Parameters which may be modified to further optimize transfection efficiency include, but are not limited to, the selection of lipid, the ratio of lipids, the molar ratio of the PEG-containing lipid, the length of the lipid anchor of the PEG-containing lipid and the sizing of the liposomal transfer vehicles.

[0099]Appropriate quantities of lipids (e.g., DSPC / CHOL / DODAP / C8-PEG2000-Cer) to construct a transfer vehicle of a desired lipid ratio (e.g., a molar ratio of 31:40:25:4) were weighed and dissolved in absolute ethanol at 70° C. to obtain the desired lipid ratios and concentrations. In order to monitor the lipid, a small amount (typically 0.05 mole %) of rhodamine-dioleoylphosphatidylethanolamine (Rh-PE) was routinely added to ...

example 2

Preparation of DSPC / CHOL / DODAP / C8-PEG-2000 Ceramide (Molar Ratio of 31:40:25:4) / Renilla Luciferase mRNA (Formulation I)

[0108]Formulation 1 was prepared by dissolving the appropriate masses of DSPC, CHOL, DODAP and C8-PEG-2000 ceramide in absolute ethanol, then adding this to a solution of Renilla Luciferase mRNA in buffer to produce MLVs at 10.8 mg / mL lipid, 250 μg / mL mRNA, 20% solvent. The MLVs were extruded to produce LUVs, and then passed through a 0.2 μm filter. The pH was increased by combining with an equal volume of 100 mM NaCl-50 mM borate pH 8.0 and the external mRNA removed by anion exchange using the DEAE-Sephacel centrifugation method, as described in Example 1. The solvent was removed, the external buffer exchanged and the sample concentrated by diafiltration / ultrafiltration. The concentrated sample was then passed through a 0.2 μm filter and aliquots were transferred to vials and stored at 2-8° C.

example 3

Preparation of DSPC / CHOL / DOTAP / C8-PEG-2000 Ceramide (Molar Ratio of 31:40:25:4) / Renilla Luciferase mRNA (Formulation 2)

[0109]Formulation 2 was prepared using a similar methodology as Formulation 1 with minor changes. In brief, the appropriate masses of DSPC, CHOL, DOTAP and C8-PEG-2000 ceramide were dissolved in absolute ethanol and then added to a solution of Renilla Luciferase mRNA in buffer to produce MLVs at 10.8 mg / mL lipid, 250 μg / mL mRNA, 20% solvent. The MLVs were extruded to produce LUVs. As DOTAP was used in this formulation, the external mRNA could not be effectively removed by anion exchange and therefore this step was omitted. The solvent was removed, the external buffer exchanged and the sample concentrated by diafiltration / ultrafiltration. The concentrated sample was then passed through a 0.2 μm filter and aliquots were transferred to vials and stored at 2-8° C.

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Abstract

Disclosed herein are compositions and methods of modulating the expression of gene or the production of a protein by transfecting target cells with nucleic acids. The compositions disclosed herein demonstrate a high transfection efficacy and are capable of ameliorating diseases associated with protein or enzyme deficiencies.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 61 / 265,653, filed Dec. 1, 2009 (Attorney Docket No. SHIR-004-001), the entire teachings of which are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Novel approaches and therapies are still needed for the treatment of protein and enzyme deficiencies, particularly strategies and therapies which overcome the challenges and limitations associated with the administration of nucleic acids and the transfection of target cells. Additional approaches which modulate or supplement the expression of a deficient protein or enzyme and thus ameliorate the underlying deficiency would be useful in the development of appropriate therapies for associated disorders.[0003]For example, the urea cycle metabolic disorders represent protein and enzyme deficiencies for which there are no currently available cures. The urea cycle is a series of biochemical reactions which occurs in many an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00A61K9/127A61P1/16
CPCA61K31/7105A61K9/0019A61K9/1271A61K48/0008A61K48/00C12N15/67A61K9/1272A61K48/005A61P1/16A61K9/5123A61K9/1075A61K48/0033C07J43/003
Inventor GUILD, BRAYDON CHARLESDEROSA, FRANKHEARTLEIN, MICHAEL
Owner TRANSLATE BIO INC
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