Pharmaceutical insights into improving oligonucleotide delivery and efficacy

Sammanfattning: The power of gene therapy lies in their capacity to target a disease on a genetic level. Nucleic acid containing therapeutics can be utilized to treat, diagnose or prevent a possible disease or condition. However, the successful delivery of such molecules constitutes a major limitation. The field of gene therapy is revolving around finding the ideal balance between efficacious and safe delivery of gene therapeutics and oligonucleotides (ONs). Different chemistries and delivery vectors were investigated for such purpose. In paper I, the synergism between low generation peptide dendrimers and lipids demonstrated high transfection efficiency of splice-switching ONs compared to peptide dendrimers alone. 20 peptide dendrimers/lipid mixtures were tested to deliver a splice-switching ON in a HeLa Luc/705 reporter cell line (a cervical cancer cell line). Under serum-free conditions optimal complexes displayed 30-fold higher correction levels compared to untreated cells. However, their activity was abolished under serum condition. Surprisingly, sucrose addition to the formulation completely restored complexes efficacy in the presence of serum. Optimal sucrose-containing formulation altered the biodistribution profile more towards the liver and less towards the kidneys following systemic delivery in mice. The in vitro and in vivo results obtained with sucrose addition demonstrated the potential positive effect of excipient addition on the formulation efficacy and biodistribution behavior. Paper II was based on our findings in paper I aiming to optimize the transfection efficacy under serum conditions. We screened different sugar and polymer excipients with the optimal peptide dendrimer and buffer from our previous study. Using this approach, splicing correction levels increased to 95-fold higher in a HeLa Luc/705 reporter cell line compared to untreated cells. We also noticed the enhancement effects of excipients in other reporter cell lines derived from osteosarcoma, hepatocyte-carcinoma and neuroblastoma cells. Cytotoxicity assays highlighted the safety of the formulations which encouraged us to check their biodistribution in mice. We noticed that biodistribution profiles of the ON were influenced by the type of formulations suggesting that excipients can preferentially direct ON therapeutics to certain organs. Despite the success of the approaches used in papers I and II, the presence of many components in the formulation can make them difficult to reproduce. We therefore synthesized another class of peptide dendrimers with lipophilic components (fatty acids or hydrophobic amino acids) conjugated to their core. In paper III, we screened different lipophilic dendrimers and evaluated the efficacy of the transfection of splice-switching ONs in reporter cell models. Factors as composition, stereochemistry, and formulation buffer were investigated. We noticed that 3rd generation peptide dendrimers were more efficient than 2nd generation peptide dendrimers. The effect of stereochemistry was more evident in 3rd generation peptide dendrimers favoring D-amino acid composed dendrimers. Variable results were obtained in different cell lines and levels of correction with the optimal formulation reached 93-fold over untreated in HeLa Luc/705 cells. Toxicity levels were minimal, yet Polyvinyl alcohol 18 (PVA18) addition increased the toxicity of the best candidates. The potential efficacy of the lipophilic peptide dendrimers is inspiring for future optimization and evaluation of different conjugates and /or cargos. Paper IV addressed the possibility to apply a new strategy in which ONs are targeting the DNA structure (anti-gene ONs). Such strategy was tested in a Huntington disease (HD) cell model. HD is a neurodegenerative disorder caused by CAG•CTG repeat expansion at exon 1 of Huntingtin gene (HTT) leading to formation of mutant HTT (mHTT) transcript and protein. The utilized anti-gene ONs were composed of Locked nucleic acids (LNA)/DNA mixmer and are complementary to the template strand of HTT. We were able to down-regulate HTT mRNA using various anti-gene ONs lengths, LNA content, and fatty acid modifications. We observed that high LNA content was necessary for activity. However, ON length and phosphorothioate (PS) content had minimal impact. Additionally, ON containing palmitoyl-modified LNA were superior to conventional LNA/DNA mixmers in serum starvation conditions. Finally, it was apparent that the number and positioning of palmitoyl-modified LNA nucleotides greatly affected activity. Overall, our screening highlighted optimal designs of anti-gene ONs targeting HTT DNA. Further investigation may include other chemistries and other classes of trinucleotide repeat (TNR) disorders.