Drug Discovery

Most Recent Inventions

Polyplex Delivery System for Proteins, Nucleic Acids and Complexes

UW-Madison researchers have developed an improved biocompatible polyplex that can successfully deliver various biomolecular payloads (e.g., nucleic acids, RNP, RNP together with a single-stranded oligonucleotide DNA donor template). This polyplex contains two polymeric chains that can interact with negatively charged nucleic acid and/or protein payloads – a cationic polymer (poly-β-amino acid, PBAA) containing glutathione (GSH)-responsive disulfide bonds in the backbone and a second polymer that includes modifiable ends (polyethylene glycol, PEG) . Randomly incorporated imidazole groups in the cationic polymer enhance endosomal escape of the polyplex. To further enhance the stability of the polyplexes, adamantane (AD) and β-cyclodextrin (β-CD) are conjugated to the polymers. The crosslinked polyplexes formed by host–guest interaction between β-CD and AD are more stable than non-crosslinked polyplexes in physiological conditions. Once in the cytosol, the disulfide bonds are cleaved by GSH, thereby releasing the protein/nucleic acid from the polyplex. Importantly, at the PEG terminal ends, simple chemistry can be used to add various cellular or tissue-specific targeting ligands and imaging probes.

When compared to a commercially available transfection reagent (Lipofectamine 2000), the polyplex significantly decreased toxicity and showed equal or better nucleic acid transfection efficiency in HEK 293 and RAW 264.7 cells. Testing both sgRNA/Cas9 complex (i.e., RNP) and RNP together with single-stranded DNA complex, the inventors showed that using the polyplex delivery system yielded superior gene knockout and gene editing results.

Nanocapsule Delivery System for Ribonucleoproteins

UW–Madison researchers have engineered a biodegradable GSH-responsive nanocoating surrounding the sgRNA/Cas9 RNP complex for efficient delivery into cells. The RNP nanocapsule is a polymeric network synthesized from a mixture of (meth)acrylate monomers, acrylate crosslinkers and acrylate polyethylene glycol (PEG), built around the RNP cargo. The interactions between the RNP and the monomers include electrostatic and hydrophobic interactions and hydrogen bonding. Nanocapsule formation is completed by free radical polymerization.

Each component of this nanocapsule is essential for cellular delivery. Inclusion of the imidazole-containing monomer provided a mechanism for endosomal/lysosomal escape. Without this, the sgRNA/Cas9 RNP would be destroyed before exerting its effects. A crosslinker containing a disulfide (–S-S–) bond produced a covalently linked, yet biodegradable, shell around the cargo. And finally, to reduce potential recognition by the immune system and increase the circulation half-life, a PEG outer shell was introduced. The PEG outer shell also provides a chemical handle for attaching fluorescent dyes or targeting ligands onto the RNP nanocapsule.

S1mplex: A New Tool for Precision Gene Editing

UW–Madison researchers have developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to ‘sharpen the scalpels’ used in genome surgery. In the new approach, CRISPR-Cas9 RNPs are complexed with a nucleic acid donor template, as well as other biotinylated molecules (e.g., quantum dots).

In human pluripotent stem cells, tailored S1mplexes increased the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene editing methods, and enriched cell populations containing multiplexed precise edits up to 42-fold.

These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro/ex vivo gene editing applications in precision medicine and drug discovery, and aid in the development of increased and multiple dosing regimens for somatic gene editing in vivo.

Enhanced Endotoxin Detection: New Advantages in Liquid Crystal Assays for Gram-Negative Pathogens

UW–Madison researchers have now demonstrated enhanced endotoxin detection in the presence of masking agents in their previous liquid crystal system.

Unlike the LAL assay, the LC-based method does not suffer from LER or any loss of sensitivity due to the presence of cations (e.g., Ca2+ or Mg2+), buffers (e.g., citrate), surfactants (e.g., SDS), chelating agents (e.g., EDTA), proteins or nucleic acids (e.g., DNA or RNA). Thus, the LC-based method provides faster and cheaper detection of endotoxin when compared to existing methods, such as the LAL assay.

Photoreceptor Scaffold for In Vitro Modeling and Transplantation Therapy

Using state-of-the-art microfabrication techniques, UW–Madison researchers have developed microstructured scaffold systems that can guide the growth of photoreceptor cells and mimic polarized outer retinal tissue. The scaffolds also may be used for transplantation of organized photoreceptor tissue with or without RPE.

Transplantation of photoreceptor-seeded scaffolds may improve grafted cell retention, survival, integration and functional visual rescue as compared to simple bolus injections. By recapitulating in vivo outer retinal architecture, these uniquely fabricated scaffolds also can be used for in vitro developmental and disease studies as well as drug screening.

The microfabrication process for scaffold production is fully compatible with numerous biomaterials, including biodegradable and non-biodegradable materials, thus allowing the scaffolds to be tailored to both in vitro and in vivo applications. The scaffolds feature biocompatible support layers (e.g., PDMS film) patterned with an array of unique through-holes having a curvilinear cell receiver and cell guide channels. The structure enables photoreceptors to be grown in a polarized orientation that mimics their development in vivo.

Most Recent Patents

Lipid-Free, Stabilized Emulsions for Delivering Anesthesia and Other Hydrophobic Drugs

UW–Madison researchers have developed non-lipid nanoemulsions for delivering propofol and other hydrophobic compounds. The formulations contain miniscule droplets of semifluorinated block copolymers and perhalogenated fluorous compounds, such as perfluorooctyl bromide or perfluorodecalin.

These ingredients are capable of forming a stable nanoemulsion without the need for conventional lipid components (e.g., soybean oil) that support bacterial and/or fungal growth. The emulsions have enhanced stability with respect to droplet size due to decreased particle coarsening, coagulation and/or phase separation.

Genetic Testing for Acquired Peripheral Neuropathy in Dogs

UW–Madison researchers have identified a single nucleotide polymorphism (SNP) that is predictive of APN syndrome in dogs, based on a genome-wide association study. Using a population of Labrador retrievers (56 cases and 26 controls), the researchers have shown that a SNP on CFA1 tags the causal variant for APN in the Labrador retriever breed.

Thermogel for Combination Drug Delivery

UW–Madison researchers have developed hydrogels for delivering drug combinations to cancer patients. The gel is made of a solution of heat-sensitive, biodegradable block copolymers (PLGA-PEG-PLGA) that turn semisolid at body temperature.

The gel can contain a combination of therapeutic agents like rapamycin, paclitaxel and 17-AAG. After being administered to a patient, the gel releases the drugs at a controlled rate, and then biodegrades into nontoxic fragments.