Brian R. McNaughton

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

Nucleic acid reagents, including small interfering RNA (siRNA) and plasmid DNA, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. We recently described the extensive surface mutagenesis of proteins in a manner that dramatically increases their net charge. Here, we report that superpositively charged green fluorescent proteins, including a variant with a theoretical net charge of +36 (+36 GFP), can penetrate a variety of mammalian cell lines. Internalization of +36 GFP depends on nonspecific electrostatic interactions with sulfated proteoglycans present on the surface of most mammalian cells. When +36 GFP is mixed with siRNA, protein–siRNA complexes ≈1.7 μm in diameter are formed. Addition of these complexes to five mammalian cell lines, including four that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by +36 GFP suppresses target gene expression. We show that +36 GFP is resistant to proteolysis, is stable in the presence of serum, and extends the serum half-life of siRNA and plasmid DNA with which it is complexed. A variant of +36 GFP can mediate DNA transfection, enabling plasmid-based gene expression. These findings indicate that superpositively charged proteins can overcome some of the key limitations of currently used transfection agents.

Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1).

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. We have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)(109)-GGG RNA with MBNL1 in vitro, with K(i) values in the low micromolar range.

Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein

The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to ∼100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo.

Engineered M13 Bacteriophage Nanocarriers for Intracellular Delivery of Exogenous Proteins to Human Prostate Cancer Cells

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

A Nanobody Activation Immunotherapeutic that Selectively Destroys HER2-Positive Breast Cancer Cells

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti‐dinitrophenyl (anti‐DNP) antibodies to the surface of HER2‐positive breast cancer cells, resulting in their targeted destruction by antibody‐dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease‐relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.

Resurfaced cell-penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery.

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease‐relevant macromolecules that present a challenge to small‐molecule reagents. Additionally, high‐throughput screening and evolution‐based methods often make the discovery of new protein binders simpler than the analogous small‐molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease‐relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies—a small and stable protein that can be evolved to recognize virtually any disease‐relevant receptor—are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP‐binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell‐penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.

Programmed cell adhesion and growth on cell-imprinted polyacrylamide hydrogels

Gaining control over cell adhesion and growth is a critical step in microscale tissue engineering, as well as biosensor fabrication, applied cell biology, and high-throughput cell-based screening. Control over cell adhesion and growth is typically achieved by patterning small molecule or macromolecule reagents with affinity for a cell surface receptor onto a non-adhesive surface. These reagents are often susceptible to environmental and/or enzymatic degradation and their preparation and purification increase the overall cost and complexity of surface fabrication. Surface topology can influence cell adhesion and growth; however, engineering a surface with well-defined topology typically requires expensive and/or specialized equipment and/or multi-step processes such as microcontact printing. In this Paper we show that cell-imprinted features on the surface of a polyacrylamide hydrogel act as surface contact cues that promote cell adhesion and growth. In some cases the shape of cell-imprints dramatically affect cell adhesion. Collectively, we demonstrate that cell-imprinting polyacrylamide hydrogels is an inexpensive and straightforward method for programming cell adhesion and growth.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

The immune system utilizes antibodies to recognize foreign or disease‐relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small‐molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post‐translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics.

Resurfaced shape complementary proteins that selectively bind the oncoprotein gankyrin.

Increased cellular levels of protein–protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein–protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin’s putative binding face and large surface areas involved in gankyrin-dependent protein–protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5 × 109 library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.

Inside Job: Methods for Delivering Proteins to the Interior of Mammalian Cells

Currently, 7 of the top 10 selling drugs are biologics, and all of them are proteins. Their large size, structural complexity, and molecular diversity often results in surfaces capable of potent and selective recognition of receptors that challenge, or evade, traditional small molecules. However, most proteins do not penetrate the lipid bilayer exterior of mammalian cells. This severe limitation dramatically limits the number of disease-relevant receptors that proteins can target and modulate. Given the major role proteins play in modern medicine, and the magnitude of this limitation, it is unsurprising that an enormous amount of effort has been dedicated to overcoming this pesky impediment. In this article, we summarize and evaluate current approaches for intracellular delivery of exogenous proteins to mammalian cells and, in doing so, aim to illuminate fertile ground for future discovery in this critical area of research.