The challenges of evolution in a complex biochemical environment—coupling genotype to phenotype and protecting the genetic material—are solved elegantly in biological systems by nucleic acid encapsulation. In the simplest examples, viruses use capsids to surround their genomes. While these naturally occurring systems have been modified to change their tropism1 and to display proteins or peptides2–4, billions of years of evolution have favored efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a “blank slate” to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids—computationally designed icosahedral protein assemblies5, 6 with positively charged inner surfaces capable of packaging their own full-length mRNA genomes—and explore their ability to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in drastically improved genome packaging (>133-fold), stability in whole murine blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus (AAV) vectors7, 8. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at “top-down” modification of viruses to be safe and effective for drug delivery and vaccine applications1, 9, 10; the ability to computationally design synthetic nanomaterials and to optimize them through evolution now enables a complementary “bottom-up” approach with considerable advantages in programmability and control.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, DPYD, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify DPYD variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydrofluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wildtype DPD (120%, P=0.025; 116%, P=0.049; 130%, P=0.0077; 138%, P=0.048; respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (P=0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had <25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations, but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient DPYD variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent.

Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) is the rate-limiting enzyme in the uracil catabolic pathway and has a pivotal role in the pharmacokinetics of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). Deficiency of DPD, whether due to inadequate expression or deleterious variants in DPYD, has been linked to severe toxic responses to 5-FU. Little is known about the mechanisms governing DPD expression in the liver. In this report, we show increased accumulation of RNA induced silencing complex (RISC) proteins on DPYD mRNA in cells overexpressing the highly homologous microRNAs miR-27a and miR-27b. These microRNAs were shown to repress DPD expression through two conserved recognition sites in DPYD. The IC50 of 5-FU for HCT116 cells over-expressing miR-27a or miR-27b was 4.4 μM (both), significantly lower than that for cells expressing a non-targeting (scramble) control microRNA (14.3 μM; P=3.3×10−5 and P=1.5×10−7, respectively). Mouse liver DPD enzyme activity was inversely correlated with expression levels of miR-27a (R2=0.49, P=0.0012) and miR-27b (R2=0.29, P=0.022). A common variant in the hairpin loop region of hsa-mir-27a (rs895819) was also shown to be associated with elevated expression of the miR-27a in a panel of cell lines (P=0.029) and in a transgenic overexpression model (P=0.0011). Furthermore, rs895819 was associated with reduced DPD enzyme activity (P=0.028) in a cohort of 40 healthy volunteers. Taken together, these results suggest that miR-27a and miR-27b expression may be pharmacologically relevant modulators of DPD enzyme function in the liver. Furthermore, our data suggest that rs895819 may be a potential risk allele for 5-FU sensitivity.

<p>PDF file - 93KB, DPD enzyme kinetics.</p>

<p>PDF file - 138KB, Supplementary Table S1. Primers used for site directed mutagenesis of DPYD expression plasmids. Supplementary Table S2. List of missense DPYD variants identified in dbSNP, ESP, and 1000 Genomes databases. Supplementary Table S3. Comparison of in silico predictions of damaging variants and measured enzyme activity of DPD variants.</p>

<div>Abstract<p>Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, <i>DPYD</i>, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify <i>DPYD</i> variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydro-fluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wild-type DPD (120%, <i>P</i> = 0.025; 116%, <i>P</i> = 0.049; 130%, <i>P</i> = 0.0077; 138%, <i>P</i> = 0.048, respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (<i>P</i> = 0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had <25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient <i>DPYD</i> variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent. <i>Cancer Res; 74(9); 2545–54. ©2014 AACR</i>.</p></div>

<p>PDF file - 138KB, Supplementary Table S1. Primers used for site directed mutagenesis of DPYD expression plasmids. Supplementary Table S2. List of missense DPYD variants identified in dbSNP, ESP, and 1000 Genomes databases. Supplementary Table S3. Comparison of in silico predictions of damaging variants and measured enzyme activity of DPD variants.</p>

<p>PDF file - 93KB, DPD enzyme kinetics.</p>