Insertion of Sequences into Safe-Harbor Loci

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Safe-harbor loci are regions of the genome that can be disrupted in a wide variety of cell types without causing adverse effects. These safe-harbor loci are thus ideal sites for inserting exogenous nucleic acid sequences.

Our scientists developed a method for inserting sequences in safe-harbor loci using mRNA encoding geneediting proteins.

Insertion of Sequences into Safe-Harbor Loci is protected by two U.S. patents, as well as patents in Canada, Korea, and Mexico (with additional patents pending in the U.S. and in other countries). Of note, certain granted patents include claims that are not limited by type of nucleic acid, or formulation.

Example Applications

  • High-efficiency insertion into the AAVS1 safe-harbor locus
  • Achieve insertion of longer sequences than are possible with AAV donor vectors
  • Combine with Factor’s Chromatin Context-Sensitive Gene-Editing Endonuclease for high-specificity ex vivo or in vivo gene editing
  • Combine with Factor’s ToRNAdoTM Nucleic Acid Delivery System for high efficiency in vivo delivery – proven delivery to various cells and tissues ex vivo and in vivo

Representative Data

Figure 1. Biallelic insertion of a fusion protein and a self-cleaving protein into the AAVS1 safe-harbor locus of iPS cells.
Figure 2. Expression of an inserted fusion-protein sequence.
Figure 3. Expression of proteins encoded by an inserted self-cleaving protein sequence.

Representative Claim

U.S. Pat. No.11,332,758

An in vitro method for inserting a nucleic-acid sequence into a safe-harbor location of a genome of a cell comprising transfecting in vitro a cell comprising a safe-harbor location with

(i) a first nucleic acid comprising a nucleic-acid sequence for insertion and

(ii) a second nucleic acid comprising a synthetic ribonucleic acid (RNA) molecule encoding an artificial transcription activator-like (TAL) effector targeting the safe-harbor location, the artificial TAL effector comprising:

(a) a DNA-binding domain and

(b) a nuclease domain,

wherein the DNA-binding domain comprises a repeat domain consisting of a plurality of TAL effector repeat sequences and at least one of the TAL effector repeat sequences consists of the 36-amino-acid sequence LTPvQVVAIAwxyzGHGG (SEQ ID NO: 74), wherein:

“v” is Q, D or E,

“w” is S or N,

“x” is N, H or I,

“y” is any amino acid, and


wherein the cell is a human cell and the safe-harbor location is the AAVS1 locus, to result in insertion of the nucleicacid sequence into the safe-harbor location.