Methods And Products For Transfecting Cells

What is this patent about?

’229 is related to the field of nucleic acid transfection , specifically addressing methods and reagents for inducing cells to express proteins, transfecting, gene editing, and reprogramming cells. The background acknowledges the existing methods for delivering nucleic acids to cells, including the use of transfection reagents. However, it highlights the limitations of these methods, such as the risks associated with introducing exogenous DNA and the inefficiencies of protein-based reprogramming.

The underlying idea behind ’229 is to use synthetic RNA molecules containing modified nucleotides to improve transfection efficiency and reduce toxicity. The key insight is that by incorporating non-canonical nucleotides with specific substitutions at certain positions, the RNA molecules can evade cellular defense mechanisms, leading to enhanced protein expression and reduced cell death. This approach aims to provide a safer and more effective alternative to traditional DNA-based methods for gene editing and cell reprogramming.

The claims of ’229 focus on methods for producing gene-edited cells using synthetic RNA molecules encoding TALENs (transcription activator-like effector nucleases). Claim 1 covers the basic method of transfecting a cell with two synthetic RNA molecules, each encoding a TALEN, to create a double-strand break in a target DNA sequence. Claim 10 expands on this by including a DNA repair template to facilitate the insertion of a specific DNA sequence at the site of the double-strand break.

The invention works by first synthesizing RNA molecules with modified nucleotides that encode TALEN proteins. These TALENs are designed to bind to specific DNA sequences and create a double-strand break. When a cell is transfected with these RNA molecules, the cell's ribosomes translate the RNA into TALEN proteins, which then target and cleave the DNA at the desired location. If a DNA repair template is also provided, the cell's natural repair mechanisms use the template to insert a new DNA sequence at the break site, effectively editing the gene.

This approach differentiates itself from prior methods by avoiding the use of DNA vectors, which can lead to uncontrolled mutagenesis. By using RNA, the gene-editing process is transient and less likely to cause permanent changes to the cell's genome. Furthermore, the use of modified nucleotides reduces the cell's immune response, improving cell viability and transfection efficiency. The combination of RNA-based TALENs and DNA repair templates provides a precise and controlled method for gene editing with reduced off-target effects .