Engineering of chitosan-based derivatives for non-viral gene delivery

Gene therapy offers the possibility to treat or even cure diseases originating from genetic defects by introducing a therapeutic gene in target cells or correcting the initial defective gene. Amongst different options, non-viral vectors rely on the delivery of such genes using vehicles composed of lipids, inorganic nanoparticles or polymers. These systems are designed to protect the genetic material, while efficiently delivering it to the cells of interest. The initial backbone can be complemented with various components to help it overcome the many physiological barriers it may encounter during its journey. Amongst polymers, chitosan is a polysaccharide presenting many advantages for such purpose. Besides its biocompatibility and biodegradability, its potential for functionalization, thanks to numerous primary amines and hydroxyl groups, make it a backbone of choice for the preparation of a polymeric delivery vehicle. This work describes the covalent functionalization of chitosan with other polymers, peptides, and small molecules to later form polymeric nanoparticles condensing therapeutic DNA. Each component brings additional features to the initial system, thus finely tuning it for biological applications. The final system aims at treating liver inherited diseases such as ornithine transcarbamylase deficiency and phenylketonuria.This manuscript first exposes the preparation of a chitosan-based cationic system able to properly condense anionic DNA through electrostatic interactions. Due to its excellent DNA condensation properties, polyethylenimine was grafted to chitosan via a succinyl linker. The first in vitro investigations confirmed the potential of the resulting core system, but in vivo experiments highlighted toxicity coming from the remaining cationic charge on the nanoparticles. In order to better shield this cationic charge, bifunctional polyethylene glycol chains terminated by carboxylic acid moieties were grafted to chitosan amines. The resulting polymer was used to coat the first polymeric system through electrostatic interactions between the carboxylate groups and the remaining free amines of the chitosan-polyethylenimine derivative. The newly formed core-shell complex showed sustained cell viability in vitro and induced higher transfection efficiency of the gene as compared to the core system alone. However, its in vivo evaluation via retrograde intrabiliary infusion did not confirm such results: although transfection efficiency was retained, the core-shell complex did not significantly decrease the toxicity previously reported.The last part of this work includes the decoration of the core-shell system with targeting ligands making possible to perform in vivo assays via intravenous injection, for which the developed polymeric systems did not induce toxicity in preliminary experiments. So far, only a proof-of-concept with folic acid, an anti-cancer targeting ligand, was achieved. To do so, the shell polymer was further functionalized with folic acid-derived polyethylene glycol chains through strain-promoted azide-alkyne cycloaddition. The same strategy shall be applied in the near future with a liver-specific targeting ligand. Lastly, the improvement of the system's cell-penetration features was tackled by the grafting asparagusic acid-derived polyethylene glycol chains following the same strategy than for folic acid.

AAV based gene therapy for rare diseases

Cylia Cloé Rochat

Of the seven thousand diseases that are described as rare, 80% of them have an identified genetic cause. With this in the mind, the development of technologies, such as gene therapy, to address the genetic factors involved in these pathologies, might be a game-changing therapeutic approach. Viral delivery of transgenes for RNA interference, DNA editing, or protein overexpression may offer opportunities for novel treatments. In this thesis, we developed adeno-associated virus (AAV) vector systems to tackle two rare diseases: Amyotrophic lateral sclerosis (ALS) caused by gain-of-function mutations in the superoxide dismutase 1 (SOD1) gene and inherited hearing loss disorder caused by mutation in the transmembrane channel-like 1 (TMC1) gene. ALS is a fatal neurodegenerative disease caused by the progressive degeneration of motoneurons (MN). Glial cells have been shown to importantly contribute to the disease. Silencing of the human pathogenic mutated SOD1 protein (SOD1G93A) in MN and/or astrocytes by AAV-mediated expression of microRNA targeting SOD1 has been shown to provide therapeutic benefits in a mouse model which is overexpressing SOD1G93A. However, in order to have an effective treatment, it is important to understand the influence of the therapy on several relevant ALS cell types. In this work, we determined the effects of lowering SOD1G93A expression in astrocytes using an AAV serotype 9, in combination with an astrocyte-specific promoter, in order to express a microRNA targeting SOD1. In the treated mice, we observed a significant improvement of the neuromuscular function and a partial protection of the vulnerable fast-fatigable MN. SOD1 silencing also induced a significant re-innervation of the neuromuscular junctions (NMJ) in the gastrocnemius muscle, observed after the initial phase of denervation, occurring around the age of 60 days. Re-innervation was associated with a grouping of the muscle fibers from the same type, consistent with enhanced axonal plasticity in the treated SOD1G93A mice. Overall, we demonstrated that silencing of SOD1 in astrocytes has an impact on MN plasticity at the level of NMJ, which translates into improved neuromuscular function towards the end stage of the disease. Mutations in the TMC1 gene lead to either complete deafness at birth, or to progressive hearing loss beginning in childhood, depending on the mode of inheritance (autosomal recessive or dominant, respectively). TMC1 is likely a component of the mechanotransduction channel, which is responsible for the transformation of mechanical sound-induced stimuli into electrical signals. This protein has been found to be expressed in the stereociliae of hair cells. In the second part of the thesis, we report on the development of viral tools to deliver a functional copy of the TMC1 gene, or its related paralog TMC2, into hair cells. We engineered several AAV vectors, in combination with different promoters, to identify a suitable combination able to restore TMC expression in the mouse cochlear hair cells, both in vitro and in vivo. We demonstrated, in collaboration with the lab of J. Holt, that when injected in TMC-null mice at birth, these vectors could partially restore loss of auditory function in this mouse model otherwise completely deaf. Overall we showed that AAV-based gene therapy is a promising approach to treat rare genetic disease.

EPFL2017

Gene Delivery with Hyperbranched Polylysine

Zuzana Kadlecova

The delivery of exogenous nucleic acids into mammalian cells is a valuable technique for basic research studies on the expression, regulation and function of genes and proteins. At the same time, gene delivery to cultivated mammalian cells has become fundamentally important as a technology for the production of therapeutic proteins for preclinical and clinical applications. Alongside with the applications of gene delivery in biotechnology, gene transfer emerged in the early 1990s as a potential therapeutical method in which a functional copy of the defective gene is introduced to provide the missing function. Polymeric cations are widely used as nonviral transfection agents. The efficiency of these vectors, however, is lower than that of viral vectors and has until now precluded their extensive use in applications where stable and high level gene expression is required. Therefore a better understanding of the parameters that govern transfection efficiency of polycationic vectors is crucial for rational design of novel gene carriers. Thus, the main motivation behind this research was to investigate the role of the architecture and molecular weight of the polycation on the efficiency of transfection. The efficiency of transfection is determined by the cytotoxicity of the gene carrier, its reversible complexation with DNA, and proper intracellular trafficking of the resulting complex. For this purpose the 17 member library was synthesized, containing linear (LPL), hyperbranched (HBPL) and dendritic polylysine (DPL) analogues covering a broad range of molecular weights. HBPL, a new class of cationic polymers prepared by polycondensation of L-lysine, was developed to explore the influence of very high molecular weights and randomly branched structures on cytotoxicity, transfection efficiency, and internalization. The thesis is divided into four chapters. Chapter 1 reviews the current state-of-the-art on gene carriers and the future prospects of the field. Additionally, the current use of polycationic gene carriers for the production of recombinant proteins (r-proteins) via a large-scale transient gene expression (TGE) is discussed. The main goal of the Chapter 2 was to determine the influence of the architecture and molecular weight of the polycations on their in vitro biocompatibility. The extent of acute cell death after short exposure was found to be molecular weight dependent. The acute cytotoxicity of high molecular weight polycations was triggered by the permeabilization of the cell membrane. At high concentrations of small molecular weight polycations, membrane destabilization was caused by increased osmotic pressure. In contrast, delayed cell death was correlated to the collapse of mitochondrial transmembrane potential, triggering caspase-dependent apoptosis after intracellular accumulation of the polycation. The onset and extent of the delayed mode of cell death was shown to be dependent on the molecular weight and degree of branching of the polylysine analogues. Based on these results, the differential long term cytotoxicity reflects the resistance of branched polylysine analogues to proteolytic degradation. Thus the degradability and cumulative cytotoxicty can be predetermined by fine-tuning the frequency of branching points in the architecture of peptidic polycation. Chapter 3 demonstrates the evolution of transfection activity with the molecular weight and degree of branching of polylysine analogues. In summary, the results show that under identical cell culture and transfection conditions, the most critical parameter for high gene expression is the structure of the gene carrier. HBPL is the most efficient gene carrier in comparison to LPL and DPL and results in gene expression levels comparable to polyethyleneimine (PEI). Physico-chemical characterization of complexes revealed that those derived from plasmid DNA and HBPL contained a high quantity of the free polycation in comparison to those formed by LPL and DPL. The presence of the free polycation in the course of endocytosis of the complex may provide an explanation for the high transfection efficiency of HBPL. Chapter 4 describes a feasibility study aimed at proving whether HBPL is potentially applicable to large-scale r-protein production via TGE. Gene delivery efficiency and yield of r-protein were evaluated in serum-free, suspension-adapted CHO-DG44 cells under conditions mimicking the industrial manufacture of r-protein by TGE. High molecular weight HBPL mediates transfection and r-protein expression at levels comparable to the ones obtained with PEI. The main advantage of HBPL is its partial enzymatic degradability, potentially decreasing its cumulative cytotoxicity in the course of TGE. Also, HBPL efficiently transfects cell lines and primary cells in the presence of serum, thus broadening the potential applications of HBPL as a novel gene carrier for basic research applications in molecular and cellular biology. In conclusion, it is possible to obtain an efficient gene carrier by simple rearrangement of the topology of poly-L-lysine.

EPFL2011

Improved biocompatibility of polyethylenimine (PEI) as a gene carrier by conjugating urocanic acid: In vitro and in vivo

Jeffrey Alan Hubbell, Jong Eun Ihm, Irina Krier

Polyethylenimine (PEI) is a promising gene carrier among polymeric vectors because of its high transfection efficiency, buffering capacity, which is responsible for its escape from endosomes, facility of modification, reasonable price and wide range of molecular weight. However, toxicity of PEI is an important obstacle to overcome for using it as a gene carrier in gene therapy protocols. In our study, PEI (10 kDa) was modified with urocanic acid, which has an imidazole ring (PEIU). This modification of PEI played an important role by improving transfection efficiency and reducing toxicity. The PEIU was condensed with DNA in different nitrogen of polycation/phosphate of DNA (N/P) ratios starting from 1.25. The PEIU/DNA complexes formed smaller (60-80 versus 100-400 nm) and less dispersible particles than PEI/DNA complexes. Cell viability test showed that in all the transfection experiments PEIU was always found less toxic than the PEI on three different cell-lines, Cos-7, HeLa, and 293T. The PEIU demonstrated improved biocompatibility as compared to the PEI. The in vitro transfection experiments showed that the PEIU displayed similar or lower transfection efficiencies than the PEI in the absence of serum. In medium complemented with serum, the PEIU had higher transfection efficiencies than the PEI. In vivo transfection experiments were carried out with intravenous and subcutaneous injections into mice. Five different organs were taken and lysed for quantification of expressed GFP. The quantification of the fluorescence of these organs exhibited that the PEIU has better transfection efficiency in in vivo experiments compared to the PEI. This study generated a strong evidence indicating that PEIU can be considered as a promising versatile gene carrier because of its biocompatibility and good transfection efficiency.

Polymer Soc Korea2015