Methods and products for transfecting cells

Abstract

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation of U.S. patent application Ser. No. 14 / 296,220, filed Jun. 4, 2014, which is a continuation of International Patent Application No. PCT / US2012 / 067966, filed on Dec. 5, 2012, which claims priority to U.S. Provisional Application No. 61 / 566,948, filed on Dec. 5, 2011, U.S. Provisional Application No. 61 / 569,595, filed on Dec. 12, 2011, U.S. Provisional Application No. 61 / 637,570, filed on Apr. 24, 2012, and U.S. Provisional Application No. 61 / 664,494, filed on Jun. 26, 2012, which are all hereby incorporated by reference in their entireties.FIELD OF THE INVENTION[0002]The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms,...

AI Technical Summary

Benefits of technology

The patent text describes the development of synthetic RNA molecules that can be used for therapeutic purposes, including the treatment of various diseases such as diabetes, heart disease, and cancer. The invention also provides methods for producing and delivering these molecules to cells, as well as a cell-culture medium for efficient transfection, reprogramming, and gene editing. The patent also mentions the use of cells for therapeutic purposes, such as for the treatment of type 1 diabetes and heart disease. Overall, the invention provides a valuable tool for research and development of new treatments for various diseases.

Problems solved by technology

However, the undefined nature of serum makes cells that are contacted with this component undesirable for both research and therapeutic applications.

However, recombinant serum albumin does not benefit from such treatment, even in the same sensitive stem-cell-culture applications (See Ng et al.

In addition, the effect of such treatment on other cell types such as human fibroblasts, and the effect of such treatment on transfection efficiency and transfection-associated toxicity have not been previously explored.

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.

DNA-free reprogramming methods based on direct delivery of reprogramming proteins have been reported, however these methods are too inefficient and unreliable for commercial use.

In addition, RNA-based reprogramming methods have been described, 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.

The many drawbacks of previously disclosed cell-reprogramming methods make them undesirable for both research and therapeutic use.

As a result, these gene-editing methods are inefficient, and carry a risk of uncontrolled mutagenesis, making them undesirable for both research and therapeutic use.

Methods for DNA-free gene editing of somatic cells have not been previously explored, nor have methods for simultaneous or sequential gene editing and reprogramming of somatic cells.

Finally, the use of gene editing in an anti-bacterial, anti-viral, or anti-cancer treatment has not been previously explored.

However, the use of gene-edited reprogrammed cells to generate genetically modified organisms, including mice and rats has not been previously explored.

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