Methods and products for nucleic acid production and delivery
Pending Publication Date: 2018-09-27
FACTOR BIOSCI
View PDF1 Cites 0 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Benefits of technology
[0018]In some aspects, synthetic RNA molecules with low toxicity and high translation efficiency are provided. In one aspect, a cell-culture medium for high-efficiency in vivo transfection, reprogramming, and gene editing of cell
Problems solved by technology
However, previously described synthetic RNA molecules are unstable and trigger a potent innate-immune response in human cells.
In addition, methods for efficient non-viral delivery of nucleic acids to patients, organs, tissues, and cells in vivo have not been previously described.
The many drawbacks of existing synthetic RNA technologies and methods for delivery of nucleic acids make them undesirable for therapeutic or cosmetic use.
While several reprogramming methods have been previously described, most that rely on ectopic expression require the introduction of exogenous DNA, which can carry mutation risks.
However, these methods are too inefficient and unreliable for commercial use.
However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require many transfections (resulting in significant expense and opportunity for error), can reprogram only a limited number of cell types, can reprogram cells to only a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeders.
Method used
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
example 1
RNA Synthesis
[0204]RNA encoding green fluorescent protein or the human proteins Elastin, Tyrosinase, Melanocortin 1 receptor, Hyaluronan synthase 1, Hyaluronan synthase 2, Hyaluronan synthase 3, Collagen type III al, Collagen type VII al, Interleukin 10, P-selectin glycoprotein ligand-1, Alpha-(1,3)-fucosyltransferase Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 or TALENs targeting the human genes XPA, CCR5, TERT, MYC, and BIRC5, and comprising various combinations of canonical and non-canonical nucleotides, was synthesized from DNA templates using the T7 High Yield RNA Synthesis Kit and the Vaccinia Capping System kit with mRNA Cap 2′-O-Methyltransferase (all from New England Biolabs, Inc.), according to the manufacturer's instructions and the present inventors' previously disclosed inventions (U.S. application Ser. No. 13 / 465,490 (now U.S. Pat. No. 8,497,124), International Application No. PCT / US12 / 67966, U.S. application Ser. No. 13 / 931,251, and International Application No. PCT / U...
example 2
Transfection of Cells with Synthetic RNA
[0206]For transfection in 6-well plates, 2 μg RNA and 6 μL transfection reagent (Lipofectamine RNAiMAX, Life Technologies Corporation) were first diluted separately in complexation medium (Opti-MEM, Life Technologies Corporation or DMEM / F12+10 μg / mL insulin+5.5 μg / mL transferrin+6.7 ng / mL sodium selenite+2 μg / mL ethanolamine) to a total volume of 60 μL each. Diluted RNA and transfection reagent were then mixed and incubated for 15 min at room temperature, according to the transfection reagent-manufacturer's instructions. Complexes were then added to cells in culture. Between 12 μL and 240 μL of complexes were added to each well of a 6-well plate, which already contained 2 mL of transfection medium per well. Plates were shaken gently to distribute the complexes throughout the well. Cells were incubated with complexes for 4 hours to overnight, before replacing the medium with fresh transfection medium (2 mL / well). Volumes were scaled for transfe...
example 3
Toxicity of and Protein Translation from Synthetic RNA Containing Non-Canonical Nucleotides
[0207]Primary human fibroblasts were transfected according to Example 2, using RNA synthesized according to Example 1. Cells were fixed and stained 20-24 h after transfection using an antibody against Oct4. The relative toxicity of the RNA was determined by assessing cell density at the time of fixation.
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Property
Measurement
Unit
Structure
aaaaa
aaaaa
Login to view more
Abstract
The present invention relates in part to nucleic acids, including nucleic acids encoding proteins, therapeutics and cosmetics comprising nucleic acids, methods for delivering nucleic acids to cells, tissues, organs, and patients, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, therapeutics, and cosmetics produced using these methods, kits, and devices. Methods and products for altering the DNA sequence of a cell are described, as are methods and products for inducing cells to express proteins using synthetic RNA molecules, including cells present in vivo. Therapeutics comprising nucleic acids encoding gene-editing proteins are also described.
Description
PRIORITY[0001]The present application is continuation of U.S. application Ser. No. 14 / 761,461, filed Jul. 16, 2015 and issued as U.S. Pat. No. 9,770,489 on Sep. 26, 2017. U.S. Ser. No. 14 / 761,461 is a U.S. National Phase Application of PCT / US15 / 13949, filed Jan. 30, 2015. PCT / US15 / 13949 claims priority to U.S. Provisional Application No. 61 / 934,397, filed on Jan. 31, 2014, U.S. Provisional Application No. 62 / 038,608, filed on Aug. 18, 2014, and U.S. Provisional Application No. 62 / 069,667, filed on Oct. 28, 2014. The entire contents of the aforementioned applications are hereby incorporated by reference in their entireties.[0002]The present application is related to U.S. application Ser. No. 13 / 465,490, filed on May 7, 2012 and issued as U.S. Pat. No. 8,497,124 on Sep. 30, 2013; International Application No. PCT / US2012 / 067966, filed on Dec. 5, 2012; U.S. application Ser. No. 13 / 931,251, filed on Jun. 28, 2013 and issued as U.S. Pat. No. 9,127,248 on Sep. 8, 2015; and International Ap...
Claims
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more