In search of therapeutic candidates for tandem repeat disorders

Triplet-repeat disorders, including Friedreich's Ataxia (FRDA) and Huntington's disease (HD), are genetic conditions characterized by the expansion of specific trinucleotide sequences within affected genes. In FRDA, the expansion of GAA•TTC repeats within the FXN gene leads to a reduction in Frataxin expression, causing debilitating symptoms. Conversely, HD is caused by an expansion of CAG•CTG repeats within the HTT gene, resulting in the production of toxic mRNA and protein. This Ph.D. thesis investigated the molecular mechanisms underlying repeat expansions in FRDA and HD, presenting innovative therapeutic strategies.

In paper I, an innovative experimental system was established to analyze largescale repeat expansions in mammalian cells. Using a shuttle plasmid, the study demonstrated that LNA-DNA mixmers and PNA oligomers effectively reduce GAA•TTC repeat expansions. Additionally, it reveals that these repeats block replication fork progression, providing insight into potential therapeutic targets.

Paper II focused on increasing Frataxin expression in patient-derived cells using single-strand Locked Nucleic Acid (LNA)-DNA mixmers. By targeting repeatexpanded chromosomal DNA, we achieved significant increase of FXN mRNA and protein expression. The study emphasizes the impact of oligonucleotide design on treatment effectiveness, implying the potential of chemically modified oligonucleotides (ONs) in upregulating FXN expression.

In paper III, Minicircles (MCs) are introduced as non-viral DNA vectors for Frataxin gene expression. By using oligonucleotides MC production is optimized, demonstrating the potential for Frataxin expression in patient-derived fibroblasts. This research offers insights into novel gene expression methods, highlighting the efficiency and safety of MCs.

In paper IV, the efficacy of Anti-gene Oligonucleotides (AGOs) in Huntington's disease (HD) was investigated. HD is another trinucleotide repeat disorder characterized by CAG•CTG expansions within the HTT gene. Screening different AGO sequences and designs revealed parameters influencing HTT downregulation. High LNA content and palmitoylated lipophilic moieties enhance AGOs' potency. RNA sequencing confirms significant downregulation of HTT and reveals regulatory effects on genes involved in immune response, mRNA processing, and neurogenesis.

Together, these studies advance the understanding of triplet-repeat disorders like FRDA and HD, offering innovative therapeutic strategies and hope for affected individuals.

List of scientific papers

I. Anastasia Rastokina*, Jorge Cebrián*, Negin Mozafari*, Nicholas H Mandel, C I Edvard Smith, Massimo Lopes, Rula Zain#, Sergei M Mirkin#. Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers. Nucleic Acid Research. 2023, 51, 16. *Joint first authors. #Co-senior authors.
https://doi.org/10.1093/nar/gkad441

II. Negin Mozafari, Salomé Milagres, Tea Umek, Cristina S. J. Rocha, Claudia Marina Vargiu, Fiona Freyberger, Osama Saher, Pontus Blomberg, Per T. Jørgensen, C. I. Edvard Smith, Jesper Wengel, Rula Zain. Anti-gene oligonucleotides targeting Friedreich’s ataxia expanded GAA•TTC repeats increase Frataxin expression. [Submitted]

III. Negin Mozafari, Maureen Fredriks, Karin E. Lundin, Jesper Wengel, C. I. Edvard Smith, Pontus Blomberg. and Rula Zain. Triplexmediated purification of Frataxin expressing Minicircle DNA. [Manuscript]

IV. Osama Saher, Eman M. Zaghloul, Tea Umek, Daniel W. Hagey, Negin Mozafari, Mathias B. Danielsen, Alaa S. Gouda, Karin E. Lundin, Per T. Jørgensen, Jesper Wengel, C.I. Edvard Smith and Rula Zain. Chemical modifications and design influence the potency of Huntingtin Anti-Gene oligonucleotides. NUCLEIC ACID THERAPEUTICS. 2023, 33,2.
https://doi.org/10.1089/nat.2022.0046