Peptiidse tranfektsioonivektori disain nukleiinhapete transpordiks in vivo tingimustes

Väitekirja elektrooniline versioon ei sisalda publikatsiooneNukleiinhappeid saab kasutada geeniteraapiaks, et reguleerida haigust esilekutsuvaid geene. Nõnda on võimalik parandada mitmesuguseid raskeid geneetilisi haigusi, nagu näiteks tsüstiline fibroosi, Duchenne'i lihasdüstroofiat ning pahaloomulisi kasvajaid. Kahjuks takistab nukleiinhapete sisenemist rakku, ning seega ka nende efektiivset kasutamist geeniteraapias, nukleiinhapete laeng ning suurus. Selleks, et suurendada nukleiinhapete terapeutilist efekti on vajalik kasutada nende transpordiks (transfektsiooniks) rakku tranfektsioonivektoreid. Üheks tranfektsioonivektoriks on rakku sisenevad peptiidid (RSP). RSP-d on tavaliselt kuni 30 aminohappe pikkused katioonsed ja/või amfipaatsed peptiidid, mis suudavad viia rakku mitmesuguseid biomolekule. Peamiseks probleemiks RSP kasutamisel on nende peptiidide vähene transfektsiooni efektiivsus in vivo. Lisaks on RSP tavaliselt vaja modifitseerida, et muuta neid selektiivseks konkreetse koe ja rakutüübi suhtes, et vähendada võimalike kõrvalmõjude teket. Samuti on oluline kontrollida moodustatud RSP-nukleiinhappe osakeste füsiko-keemilisi omadusi, sest ka sellest sõltub osakeste distributsioon organismis. Antud töö eesmärgiks oli tegeleda peamiste RSP kasutamist takistavate probleemidega. Käesolevas töös disainiti uus NF55 peptiid efektiivsemaks in vivo DNA transfekstiooniks. Seejärel kasutati uut formulatsiooni meetodi, et valmistada väiksema läbimõõdu ning kindla suurusjaotusega RSP-DNA osakesi selektiivsemaks ning efektiivsemaks in vivo manustamiseks. Viimaks lisati RSP-nukleiinhappe osakestele magnetilisi rauaoksiidi partikleid, et muuta RSP-nukleiinhapete osakeste bioloogilist aktiivsust veelgi selektiivsemaks. Kokkuvõttes arendati antud töös formulatsiooni meetod, millega on võimalik valmistada palju efektiivsemaid nanopartikleid spetsiifiliselt in vivo manustamiseks.Various serious genetic diseases, such as cystic fibrosis, Duchenne muscular dystrophy and cancer can be treated with gene therapy. Gene therapy can be utilized to regulate the expression of disease causing genes by using therapeutic nucleic acids. Due to their size and cationic nature, these nucleic acids need vectors to enhance their delivery into the diseased tissue. Although impressive results have been accomplished with virus-derived gene delivery vectors their utilization is not without risks and increasingly more effort has been applied into the research of non-viral vectors. Cell penetrating peptides are one group of non-viral delivery vectors. Cell penetrating peptides (CPP) are short cationic and/or amphipathic peptides than are shown to significantly improve the delivery of various biomolecules in vitro and in vivo. The main hindrances of using CPPs are the lack of transfection efficacy and selectivity in vivo. In addition, the size and heterogenous size distribution of prepared CPP-DNA particles also alters the bio-distribution of particles and can cause side-effects. In this thesis we aimed to tackle these previously mentioned main problems of utilization of CPPs. We designed a novel effective CPP NF55 for the systemic delivery of DNA. Thereafter we developed a novel method to prepare stable and uniformly sized particles for systemic gene delivery in vivo. Subsequently, we used incorporation of magnetic iron oxide particles to further increase the efficacy and specificity of the peptide vectors. Taken together, we designed more efficient and safer formulations of CPP/nucleic acid particles for the systemic in vivo delivery of nucleic acids

Galanin pathogenic mutations in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatmen

Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo

Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo

Galanin pathogenic mutations in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment