Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering

<div><p>Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human <i>CCR5</i> and <i>BMPR1A</i> genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.</p></div

TALEN conjugation is reversible and R9 must be removed for TALEN cleavage activity.

<p>(A) Purified TALEN proteins are incubated with Cys-nitropyridyl (Npys) Arg₉ cell-penetrating peptide (R9-CPP) for 1 hr at room temperature. (B) <i>In vitro</i> cleavage analysis of TALEN proteins conjugated at (left) various peptide-to-protein ratios and (right) various protein concentrations at a 30-to-1 peptide-to-protein ratio in the (top) absence or (bottom) presence of 10 mM DTT.</p

Modification of the endogenous <i>BMPR1A</i> gene by cell-permeable TALEN proteins.

<p>(A) Frequency of gene disruption in HEK293 cells treated for 2 hr with 1.0 µM <i>BMPRIA</i>-targeting TALEN proteins conjugated at various peptide-to-protein ratios. All R9-conjugated <i>BMPR1A</i>-targeted TALEN proteins were labeled in the presence of protease inhibitor cocktail. (B) Comparison of the frequency of <i>BMPR1A</i> knockout in HEK293 cells transfected with 200 ng of TALEN expression vectors or treated with 1.0 µM cell-permeable TALEN proteins for 2 hr. Black triangles indicate expected Surveyor nuclease assay cleavage products.</p

TAL effector structure.

<p>(Left) Front view of the PthXo1 DNA-binding domain in the absence of target DNA and (right) side view in the presence of target DNA. Surface-exposed Cys residues depicted as white spheres. TAL effector repeats are colored cyan and purple. DNA is shown as grey sticks. PDB ID: 3UGM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085755#pone.0085755-Mak1" target="_blank">[41]</a>.</p

Human Serum Albumin Domain I Fusion Protein for Antibody Conjugation

Bioorthogonal labeling of antibodies enables the conjugation of compounds, such as small molecules or peptides, which expand targeting capacity or enhance cytotoxicity. Taking advantage of a cyclohexene sulfonamide compound that site-selectively labels Lys64 in human serum albumin (HSA), we demonstrate that domain I of HSA can be used as a fusion protein for the preparation of antibody conjugates. Trastuzumab fusions were expressed at the N-terminus of the light chain or the C-terminus of the heavy chain enabling conjugation to small molecules. Moreover, these conjugates retained HER2 binding and proved to be highly stable in human plasma. Antibody conjugation via HSA domain I fusion should therefore have broad utility for making serum-stable antibody conjugates, particularly for antibody–drug conjugates