Characterization and applications in muscle of a Minicircle vector for Nonviral gene therapy

In gene therapy, the aim is to change the behaviour of a cell by introduction of genetic material, often DNA encoding a protein or a therapeutic RNA. The purpose can be to replace a malfunctioning copy of a gene, as in clinical trials for treatment of X-linked severe combined immunodeficiency, or introduce a new gene into the body to help fight a disease, as has been done in clinical trials for e.g. leukaemia and lymphoma where immune cells has been modified to recognize and destroy cancer. In order to alter the behaviour, the genetic material must be transported into the cell and reach the nucleus. The two main ways to achieve this is either using viral vectors, where engineered viruses carry the therapeutic DNA, or nonviral vectors, which are commonly based on plasmids produced in bacteria. This thesis focuses on nonviral vectors. Nonviral vectors are generally considered safer and more easily produced than viral vectors, but are less efficient in delivery and long term expression. This is thought to be partly due to the plasmid backbone, i.e. sequences needed only for propagation in the bacteria such as origin of replication and selection markers, commonly antibiotics resistance genes. Bacterially produced DNA sequences have a different methylation pattern than eukaryotic DNA. It has been shown that this can induce an immune response, especially in combination with the use of lipids for transfection. Also for naked delivery of plasmids, the expression is transient, which could be due to epigenetic phenomenon. A way to optimize the plasmid vector is to remove the bacterial backbone by recombination in the production bacteria. The resulting vector is called the minicircle (MC). In one of studies included in this thesis, we investigate how the size of the MC vector affects coiling and relate these findings to analysis of other aspects such as robustness, expression efficiency and transfection. We find that reducing the size of the MC affects the configuration of the vector, causing an increased frequency of dimer and trimer formation during production. We also find that there seems to be a lower size limit for efficient expression. However, the smaller sizes also result in a vector which is more robust than conventional plasmids when exposed to shearing forces, and shows extended expression in vivo. In the two other studies, we evaluate the vector for use in muscle. A comparison of the MC to a conventional plasmid for expression of a growth factor in heart and skeletal muscle in the mouse shows that the smaller size allows for a higher effective dose, and thus, higher gene expression. The third study demonstrates that it is possible to use the MC to express small regulatory RNAs for splice-switching, targeting Duchenne muscular dystrophy, and that treatment with these MCs in mouse muscle results in increased dystrophin levels. However, development of suitable delivery methods is required to realize the full potential of MCs in vivo. Thus, the smaller size enabling a higher dose, prolonged expression and increased robustness, and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene. However, for use where systemic treatment is needed, delivery must be enhanced. Consequently, the vector might be more suitable for treatments where only local expression is required, such as single organ treatment, DNA vaccination or ex vivo treatments

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo

Immunization with HIV-1 envelope T20-encoding DNA vaccines elicits cross-clade neutralizing antibody responses

Background: Genetic immunization is expected to induce the expression of antigens in a native form. The encoded peptide epitopes are presented on endogenous MHC molecules, mimicking antigen presentation during a viral infection. We have explored the potential of enfuvirtide (T20), a short HIV peptide with antiviral properties, to enhance immune response to HIV antigens. To generate an expression vector, the T20 sequence was cloned into a conventional plasmid, the novel minicircle construct, and a replicon plasmid. In addition, 3 conventional plasmids that express the envelope of HIV-1 subtypes A, B and C and contain T20 in their gp41 sequences were also tested. Results: All combinations induced HIV-specific antibodies and cellular responses. The addition of T20 as a peptide and as an expression cassette in the 3 DNA vectors enhanced antibody responses. The highest anti-HIV-1 Env titers were obtained by the replicon T20 construct. This demonstrates that besides its known antiviral activity, T20 promotes immune responses. We also confirm that the combination of slightly divergent antigens improves immune responses. Conclusions: The antiretroviral T20 HIV-1 sequence can be used as an immunogen to elicit binding and neutralizing antibodies against HIV-1. These, or similarly modified gp41 genes/peptides, can be used as priming or boosting components for induction of broadly neutralizing anti-HIV antibodies. Future comparative studies will reveal the optimal mode of T20 administration

Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™”) to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized