CRISPR gene editing

CRISPR gene editing (pronounced ˈkrɪspər "crisper") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo. The technique is considered highly significant in biotechnology and medicine as it enables editing genomes in vivo very precisely, cheaply, and easily. It can be used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also has possibilities in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations such as cancer. However, its use in human germline genetic modification is highly controversial

Core-shell Structured Chitosan-polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency

Sandrine Gerber, Laura Camille Louise Nicolle, Perrine Agnes Edith Robin

Gene therapy emerged as a promising treatment option for acquired and inherited diseases. The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially non-viral gene delivery systems are of increasing interest. However, accomplishing therapeutic transgene expression levels and limited tolerability of these systems remain a challenge. Therefore, we investigate in the improvement of nucleic acid delivery using depolymerized chitosan – polyethylenimine DNA complexes (dCS-PEI/DNA). These core complexes are further entrapped into chitosan-based shells, functionalized with polyethylene glycol and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physico-chemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-LPEI/DNA complex with a shell consisting of dCS-PEG-COOH, which resulted in improved nucleic acid encapsulation, cellular uptake, transfection efficiency, and transfection potency in human hepatoma HuH-7 cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induce a high reporter gene signal for three days. We conclude that ternary core-shell structured particles comprising functionalized chitosan are a promising gene delivery technology for both in vitro as well as in vivo applications.

2022

Towards automating de novo protein design for novel functionalities: controlling protein folds and protein-protein interactions

Zander Harteveld

The sheer size of the protein sequence space is massive: a protein of 100 residues can have 20^100 possible sequence combinations; and knowing that this exceeds the number of atoms in the universe, the chance of randomly discovering a stable new sequence with the desired characteristics is infinitesimally small. Therefore, computational methodologies that can search through the sequence space and expand beyond naturally occurring functional protein sequence variants hold enormous potential in biomedicine and nanotechnology.My thesis work leverages machine learning, physics-based, and data-driven techniques to design new protein molecules with distinct shapes (folds) so that they can precisely interact with other molecules to perform biological functions.The first part of my thesis is dedicated to the design of functional proteins. The re-designed of an anti-CRISPR protein that can be controlled via blue light (optogenetic control) to regulate the genome editing activity of the enzyme CRISPRâCas9 is presented. A surface-based design of a broad-spectrum inhibitory Acr towards another natural target SauCas9) exemplifies the re-purposing of existing inhibitory molecules against other related targets. This ultimately led to the development of a general surface-centric design method for generating specific protein-protein interactions from scratch and exemplified by the successful design of novel PD-L1 inhibitors, an immune checkpoint that can halt the immune system from attacking the cancer cells.The successful design of protein-protein interactions heavily relies on the underlying protein fold and structure stabilizing the functional motif in a protein. Because nature has only evolved a small set of protein folds, generated protein-interaction motifs can rarely be incorporated into existing protein structures. To address this problem, the second part of my thesis is dedicated to the development of computational de novo protein design methods for the crafting of proteins with customized folds. To this end, the TopoBuilder framework utilizes a large collection of native proteins to transform a literal description of a protein fold into a physically-realistic protein. Finally, Genesis, a deep neural networks approach for the tailored de novo protein design is presented. Employing both, the TopoBuilder and Genesis, proteins completely absent from the natural repertoire were designed and experimentally validated.My thesis sets the path to explore possibilities of jointly optimizing the protein's shape and its surface geometry to master biological functions. We are now at entering a new era where newly designed protein-based drugs and materials with the potential to solve a vast array of technical challenges and open new avenues for next-generation precision drugs and advanced nanomaterials.

EPFL2022

Towards automating de novo protein design for novel functionalities: controlling protein folds and protein-protein interactions

Zander Harteveld

EPFL2022

Core-shell Structured Chitosan-polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency

Cas9/gRNA selective targeting of the Beethoven tmc-1 mutant allele for treating progressive hearing loss by AAV-based delivery

Towards automating de novo protein design for novel functionalities: controlling protein folds and protein-protein interactions

Towards automating de novo protein design for novel functionalities: controlling protein folds and protein-protein interactions

Core-shell Structured Chitosan-polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency

Cas9/gRNA selective targeting of the Beethoven tmc-1 mutant allele for treating progressive hearing loss by AAV-based delivery