Droplette: A Platform Technology to Directly Deliver Nucleic Acid Therapeutics and Other Molecules into Cells and Deep into Tissue Without Transfection Reagents.

Droplette (US 9,700,686 B2 and PCT/US2016/035695) is the first portable and contact-free transdermal technology comprising the unique combination of a piezoelectric transducer and a pneumatic diaphragm pump to deliver large biomolecules including nucleic acid therapeutics (NATs) deep into cells, and into skin and soft tissue for effective delivery over short timescales. The droplets that come out of the device are 10-50× smaller upon impact than what is created through other commercial atomizers, such as the piezoelectric transducer alone. This device has been tested extensively in vitro, in vivo and in IRB approved human studies. The Droplette device delivers metered doses using a water droplet dispersal technology already commonly used in humidifier devices, by utilizing a piezoelectric material. Three key innovations make this device technically novel and tailored specifically for both field and lab use: (1) The combination of the piezo and pump to generate sub-micron drug-loaded droplets that penetrate cells, skin, and soft tissue to effectively deliver a range of large molecules, proteins, and nucleic acids. (2) Their assembly in a modular manner which enables portability, safe sterilization, and ejection without direct device-surface contact or significant force, allowing for improved safety and ease of use in both research and clinical settings. (3) The integration of a single-use, sterile cartridge that contains a therapeutic formulation and allows easy integration of a large number of molecules. The platform has broad applications across multiple fields, such as delivery of drugs for inflammatory skin diseases, antibiotics for skin infections, and gene delivery for gene therapy and biomedical research. In this chapter, we briefly introduce the Droplette delivery technology, instructions for building your own setup (note that fully built devices are available for purchase from Droplette, Inc), and provide protocols for directly transfecting adherent cell cultures and for direct in vivo transfection using the Droplette system without the need for traditional transfection reagents or methods.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway.

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

Retroviral strategy to stabilize viral RNA.

Unspliced Rous sarcoma virus (RSV) retroviral mRNA undergoes nonsense-mediated RNA decay (NMD) if it has premature termination codons in the gag gene. However, its normal gag termination codon is not subject to NMD despite being 7kb from the 3' poly(A) sequence. An RNA stability element (RSE) has been identified immediately downstream of gag in the RSV genome. It appears to determine the proper context for translation termination and protects the RNA from NMD. The viral stability element may prevent Up-frameshift 1 (Upf1) protein from interacting with the terminating ribosome and release factors to initiate NMD.