The identification of target proteins and the design of antisense oligodeoxynucleotides

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History and overview of malaria

Malaria has plagued mankind since at least 4 BC when Hippocrates first described the manifestations of the disease (Goodwin, 1992). He also linked these to seasonal changes and to the regions where people lived. Since then man has been searching for the cause, treatment and preventative measures. It was however, not until 1880 that Laveran who was working in Algeria at the time, discovered the protozoal nature of the causative agent of malaria. The mode of transmission of the disease was unknown until 1897 when Ronald Ross, a British scientist, identified the mosquito as being the vector for this protozoan parasite (Krettli and Miller, 2001; McKenzie, 2000). Later, workers in Italy specifically linked mosquitoes of the genus Anopheles, to human malaria (Krettli and Miller, 2001). It is interesting to note that the first recorded treatment of malaria dates back to 1600 in Peru, where the Peruvian Indians utilized the bitter bark of the cinchona tree. This is the natural source of quinine, a drug still in use today for the treatment of malaria (Krettli and Miller, 2001; McKenzie, 2000). The malaria parasite belongs to the phylum Apicomplexa, which includes Plasmodia, Toxoplasma gondii and Cryptosporidium sarcocystis. Four different species of Plasmodia infect humans, namely, falciparum, vivax, ovale and malariae. The most virulent of the four is Plasmodium falciparum and its life cycle… There are three distinct parasite stages that invade host cells, namely the merozoites (erythrocytes), ookinetes (mosquito gut) and sporozoites (liver). The cycle begins with sporozoites, being transmitted to the human host from the mosquito salivary gland. The sporozoites enter hepatocytes where they develop into schizonts. Merozoites are then released from the infected hepatocytes from where they enter the circulating blood and infect host erythrocytes, within 1 –2 minutes. Once inside the erythrocyte the parasite develops into the ring phase and then into the feeding trophozoite stage. The trophozoite- phase parasites then undergo division to form schizonts. The schizont-containing erythrocytes rupture, releasing new merozoites. Some of the merozoites develop into micro – and macro – gametocytes, while others go on to re-infect new erythrocytes. Gametocytes are ingested by feeding female Anopheles mosquitoes and develop into zygotes after fertilization in the mosquito’s gut. The zygote develops into an ookinete, which then permeates the midgut lining and forms an oocyst on the outer gut wall. The oocytes eventually rupture, releasing sporozoites. These then migrate to the mosquitoe’s salivary glands from where the human host is infected when the mosquitoes ingest a blood meal. The approximate number of merozoites produced per liver schizont in P. falciparum is 30 000, compared with over 10 000 for P. vivax, 15 000 for P. ovale and 15 000 for P. malariae (Gilles, 1993). The maximum parasitaemia (per 1 mm3 blood) for P. falciparum is 2 000 000, compared with 50 000 for P. vivax, 30 000 for P. ovale and 20 000 for P. malariae. Invasion of erythrocytes by merozoites is assisted by specialized secretory organelles at their apical ends namely, rhoptry organelles, micronemes and dense granules which all function at specific steps in the invasion process as illustrated in Figure 1.2 (Bannister et. al., 2000). Microneme content is released first and occurs with the initial contact between the parasite and host cell. The content of the rhoptry organelles is released directly afterwards and is associated with the formation of the parasitophorous vacuole. Dense granule content is the next to be released and is associated with modifications to the host cell membrane. This is evidenced by the release of proteins such as RESA (ring-infected erythrocyte surface antigen), which is located in the dense granules in merozoites and is transported to the erythrocyte membrane shortly after parasite invasion.

Chapter 1: Literature Overview :

• 1.1 History and overview of malaria
• 1.2 Reasons for the current malaria problem
• 1.3 Anti-malaria drugs and parasite resistance
• 1.4 Malaria vaccine development
• 1.5 Additional malaria-combative strategies under investigation
• 1.5.1 Alternative Medicine (Traditional Medicine)
• 1.5.2 Genetically modified mosquitoes
• 1.5.3 Inhibitor design
• 1.5.4 Small interfering RNAs (siRNAs)
• 1.6 Background and overview of antisense technology
• 1.6.1 Antisense target protein
• 1.6.2 Phosphodiester oligodeoxynucleotide stability
  • 1.6.2.1 Backbone modifications
  • 1.6.2.2 Nitrogenous base modifications
  • 1.6.2.3 Sugar modifications
  • 1.6.2.4 Self-stabilizing loops
• 1.7 Cellular ODN uptake
• 1.8 Aims

• Chapter 2: The identification of target proteins and the design of antisense oligodeoxynucleotides
  • 2.1 Introduction
  • 2.1.1 General
  • 2.1.2 Target protein selection
  • 2.1.3 Target sequence selection
    • 2.1.3.1 Random shotgun approach or gene walk
    • 2.1.3.2 Predictions of mRNA secondary structure
    • 2.1.3.3 Oligonucleotide scanning arrays
    • 2.1.3.4 Oligomer library/ribonuclease-H-digestion
  • 2.1.4 Oligonucleotide modification selection
  • 2.2 Materials and Methods
  • 2.2.1 Materials
  • 2.2.2 Methods
    • 2.2.2.1 α-I-tubulin sequences
    • 2.2.2.2 The RNA folding program, Mfold
    • 2.2.2.2.1 Drawing and evaluation of mRNA structures
  • i RNA Structure
  • ii RNAdraw
  • 2.3 Results
  • 2.3.1 Selection of target mRNA sequences
  • 2.3.1.1 Secondary structure prediction of P. falciparum mature α-I-tubulin mRNA
  • 2.3.1.2 Identification of accessible hybridization sites and design of antisense ODN
  • 2.3.1.3 Design of an antisense ODN targeted against the translation initiation codon
  • 2.4 Discussion
• Chapter 3: Stability of modified antisense phosphodiester ODN under culture conditions
• • 3.1 Introduction
  • 3.1.1 Capillary electrophoresis
  • 3.1.2 High Performance Liquid Chromatography
    • 3.1.2.1 Anion exchange HPLC
    • 3.1.2.2 Reversed Phase HPLC
  • 3.2 Materials and Methods
  • 3.2.1 Materials
  • 3.2.2 Methods
  • 3.2.2.1 Optimization of separation and elution conditions
  • 3.2.2.2 HPLC analysis of ODN samples
  • 3.2.2.3 Method used for extraction of ODNs from parasite culture medium
  • 3.2.2.3.1 Fluid phase extraction
  • 3.3 Results
  • 3.3.1 Extraction of ASL-1 and IS from serum containing medium and subsequent
  • elution conditions from C18 reversed phase HPLC column
  • 3.3.2 Determination of ODN stability
  • 3.4 Discussion
• Chapter 4: Uptake and efficacy of modified phosphodiester ODNs to inhibit in vitro erythrocytic cultures of P. falciparum
• • 4.1 Introduction
  • 4.2 Materials and Methods
  • 4.2.1 Materials
  • 4.2.2 Methods
    • 4.2.2.1 In vitro culturing of malaria parasites
    • 4.2.2.2 Giemsa-stained blood smear preparation
    • 4.2.2.3 In vitro synchronization of malaria parasites
    • 4.2.2.4 Determination of antisense ODN efficacy to inhibit parasite
    • proliferation
    • 4.2.2.5 Flow cytometric (FC) measurement of parasitaemia in fixed parasite
    • cultures
    • 4.2.2.6 Uptake of FITC-labeled ODNs
    • 4.2.2.7 Determination of hybridization site availability
  • 4.3 Results
  • 4.3.1 In vitro ODN inhibitory efficacy
  • 4.3.2 Cellular ODN uptake
  • 4.3.3 Retrospective analysis
  • 4.4 Discussion
• Chapter 5: Concluding discussion
  • Summary
  • References

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