Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Growth factor engineering by degenerate homoduplex gene family recombination.

There is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of "top-strand" oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic "scaffold" oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human-mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.

Salt-induced aggregation of a monoclonal human immunoglobulin G1.

Physical stability is critical for any therapeutic protein's efficacy and economic viability. No reliable theory exists to predict stability de novo, and modeling aggregation is challenging as this phenomenon can involve orientation effects, unfolding, and the rearrangement of noncovalent bonds inter- and intramolecularly in a complex sequence of poorly understood events. Despite this complexity, the simple observation of protein concentration-dependent diffusivity in stable, low ionic-strength solutions can provide valuable information about a protein's propensity to aggregate at higher salt concentrations and over longer times. We recently verified this notion using two model proteins, and others have shown that this strategy may be applicable to antibodies as well. Here, we expand our previous study to a monoclonal human immunoglobulin G1 antibody and discuss both merits and limitations of stability assessments based on the diffusional virial coefficient k(D). We find this parameter to be a good predictor of relative protein stability in solutions of different chaotropic salts, and a telling heuristic for the effect of kosmotropes. Both temperature and glycosylation are seen to have a strong influence on k(D), and we examine how these factors affect stability assessments. Protein unfolding is monitored with a fluorescence assay to assist in interpreting the observed aggregation rates.

Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display.

In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.