Delivery is a key challenge in novel protein therapeutic development. Even though numerous proteins are potential therapeutic candidates, a lack of delivery method limits the development of protein therapeutics. I have studied protein delivery and designed three novel delivery systems: gold nanoparticle based polymer particle system, multifunctional oligonucleotide, and nanostructured microparticles. Each of delivery system was designed to deliver CRISPR/Cas9 ribonucleoprotein, transcription factors, and oral protein drugs.

First of all, CRISPR/Cas9 is a great tool in genome editing field. Moreover, CRISPR/Cas9-mediated genome editing has the potential to revolutionize the treatment of genetic diseases and the development of cell-based therapies. However, precise gene editing with Cas9 is still challenging in vivo because it requires simultaneous and efficient delivery of Cas9, guide RNA, and donor DNA into cells. We designed a gold nanoparticle-based delivery vehicle, CRISPR-Gold, which can directly deliver Cas9 protein, guide RNA (gRNA), and donor DNA in vitro and in vivo and efficiently induce homology directed repair (HDR). CRISPR-Gold is composed of gold nanoparticles assembled with the Cas9-gRNA ribonucleoprotein (RNP) complex, donor DNA, and an endosomal disruptive polymer. We demonstrate that CRISPR-Gold can induce HDR in human stem cells and mouse primary cells with an efficiency that is significantly higher than conventional transfection methods. Notably, we show that CRISPR-Gold can correct a nonsense mutation in the dystrophin gene that causes Duchenne muscular dystrophy in mdx mice, and restore dystrophin protein expression in mouse muscle after a single injection.

Another potential protein therapeutic group is the transcription factor, which can have broad effects in gene regulation. We designed a novel multifunctional oligonucleotide, termed DARTs, which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contain a transcription factor binding sequence and hydrophobic membrane disruptive chains that are masked by acid cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal escape. The DARTs target hepatocytes as a result of the galactose residues and can disrupt endosomes efficiently with minimal toxicity because the unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. DARTs showed efficient delivery of the transcription factor Nrf2 to the liver, catalyzed the transcription of Nrf2 downstream genes, and rescued mice from acetaminophen induced liver injury.

Another method of delivery that continues to be in the process of improvement is the oral drug delivery system for protein therapeutics. Oral drug delivery faces challenges including harsh acidic environment, rapid clearance of drug, and limited paracellular transport of therapeutic molecules. We studied a nanostructured microparticle system to overcome the challenges with an engineering approach. We showed that planar silica particles coated with silicon nanowires loaded proteins efficiently. The planar particles increased the transepithelial permeation of the protein drug as a result of a larger surface area in contact with the cell layer.

The emergence and rapid spread of the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2 (SCoV-2) caused catastrophic levels of morbidity in the world and presented the unmet need for treatments and drugs urgently. Small molecule therapeutics have tremen- dous potential to develop into antivirals and prevent the spread of infection. SCoV-2 genome encodes for 16 non-structural proteins (Nsps) that have been proven as potential target can- didates. Two vital proteins, Papain-like protease (PLpro) (Papain-like protease, from Nsp3) and Non-structural protein 15 (Nsp15) were chosen as therapeutic targets to combat the SCoV-2 virus. PLpro is an essential protein that cleaves the polyprotein into its individual proteins to form a replication-transcription complex for viral replication and synthesis. On another hand, Nsp15 is a crucial protein that evades the host immune response by cleaving the viral RNA, therefore, Nsp15 inhibition stimulates the protective response. Both nsps are highly conserved proteins and the development of drugs against them could also act as an initial step for future coronavirus pandemics. To find the covalent inhibitors of PLpro and Nsp15, the electrophile library was screened against these two proteins using their re- spective fluorescent-based, high-throughput screening assay. To find the inhibitors against the PLpro, a chemical library was screened against the recombinantly purified PLpro using a fluorescent-based high throughput screening assay. One compound, based on mercaptopy- rimidine inhibited PLpro invitro and SARS-CoV-2 viral replication in Vero E6 cells. This compound presented the first example of a thiol-targeted reversible covalent inhibitor of PLpro. In the case of Nsp15, an acrylamide-based electrophile library was screened to find cysteine-binding inhibitors. This discovered a new class of Nsp15 covalent inhibitors. This study not only discovered new covalent lead hits against crucial proteins from SCoV-2, but also a new platform to develop new potent drugs against coronaviruses for future pandemics.

The following dissertation presents work done towards the development of delivery strategies for proteins and nucleic acids to mammalian cells, as well as targeted imaging materials to bacteria. Delivery of large molecules such as proteins, nucleic acids, and large synthetic molecules remains a major bottleneck in the field of drug delivery, and despite an abundance of research into the topic, the delivery of intracellular proteins has still not seen clinical significance. To address this, we sought to develop new delivery vehicles in an endeavor to address current challenges in drug delivery.

Chapter 1 discusses the development of acid-sensitive surfactants for the delivery of nucleic acids. This class of molecules, termed caged surfactants, consists of a membrane disruptive surfactant that has been masked with two PEG chains through an acetal linker. This prevents it from entering the membrane in its intact state; however, after acetal hydrolysis in the acidic endosomal compartment, release of free surfactant causes endosomal release of any co-delivered cargo. The simultaneous delivery of caged surfactant with nucleic acid was achieved using two strategies: (i) inclusion of two primary amines on the caged surfactant to allow complexation through electrostatic interactions, and (ii) inclusion of the RNA-binding dye thiazole orange (TO) to allow tight binding through intercalation. In this study we show that amine-containing caged surfactant (PCS) and TO-containing caged surfactant (TCS) increase the delivery of mRNA and siRNA, respectively.

Chapter 2 discusses the development of delivery vehicles for the delivery of proteins to mammalian cells. The chapter consists of two separate studies. The first study utilized a fluorescent acid-sensitive membrane disruptor (FEDS) as an adjuvant to Lipofectamine-based delivery of CRISPR-Cas9. FEDS successfully increased the delivery efficacy of Cas9 to HEK cells. In addition, due to its fluorescent properties the cells with Cas9-triggered gene editing could be sorted through a fluorescence cell sorter. The second study describes the development of a polyethylene glycol-Eosin Y block copolymer (PEG-pEosin) for protein delivery. The Eosin Y moieties bind a wide range of proteins, and PEG-pEosin can therefore be used as a general protein delivery tool. Using PEG-pEosin we successfully delivered Cre recombinase to Ai9 cells through a PEG-pEosin/Cre/Listeriolysin O complex, wherein the Lysteriolysin O serves as an endosomal disruptive component.

Chapter 3 presents a maltohexaose-indocyanine green conjugate for the detection of infective endocarditis. Maltohexaose (MH) is a sugar that is specifically taken up by bacteria, and it is relatively tolerant of modifications to its reducing end. The FDA-approved dye indocyanine green (ICG) was conjugated to MH to yield a low toxicity, high wavelength probe that was shown to accumulate in bacterial vegetations in an endocarditis rat model.