A ‘poly-transfection’ method for rapid, one-pot characterization and optimization of genetic systems

Biological research is relying on increasingly complex genetic systems and circuits to perform sophisticated operations in living cells. Performing these operations often requires simultaneous delivery of many genes, and optimizing the stoichiometry of these genes can yield drastic improvements in performance. However, sufficiently sampling the large design space of gene expression stoichiometries in mammalian cells using current methods is cumbersome, complex, or expensive. We present a 'poly-transfection' method as a simple yet high-throughput alternative that enables comprehensive evaluation of genetic systems in a single, readily-prepared transfection sample. Each cell in a poly-transfection represents an independent measurement at a distinct gene expression stoichiometry, fully leveraging the single-cell nature of transfection experiments. We first benchmark poly-transfection against co-transfection, showing that titration curves for commonly-used regulators agree between the two methods. We then use poly-transfections to efficiently generate new insights, for example in CRISPRa and synthetic miRNA systems. Finally, we use poly-transfection to rapidly engineer a difficult-to-optimize miRNA-based cell classifier for discriminating cancerous cells. One-pot evaluation enabled by poly-transfection accelerates and simplifies the design of genetic systems, providing a new high-information strategy for interrogating biology. ©2019National Institutes of Health (no. R01CA173712)National Institutes of Health (no. R01CA207029)National Institutes of Health (no. P50GM098792)National Science Foundation (no. 1745645)Cancer Center Support Grant (no. P30CCA14051

Biophysical Characterization of a Riboflavin-Conjugated Dendrimer Platform for Targeted Drug Delivery

The present study describes the biophysical characterization of generation-five poly­(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have <i>K</i>_D values of ≥465 nM on a riboflavin basis, an affinity ∼93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein–conjugate stability is achieved with the N-3 series and at lower ligand valency

Bifunctional PAMAM Dendrimer Conjugates of Folic Acid and Methotrexate with Defined Ratio

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules

Extensive identification of genes involved in congenital and structural heart disorders and cardiomyopathy

Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease