Forum 08/20/2009

“Development of tools to study a Microsporidian parasite’s life cycle and to disrupt HIV-1 co-receptor CCR5 in hematopoietic stem cells“

Vanessa Taupin – Children’s Hospital Los Angeles

Microsporidia are ubiquitous obligate intracellular parasites, and some species are opportunistic pathogens in immune-deficient humans. They show a complex life cycle including four stages where the infective phase is mediated by small resistant spores (1-2 um). In order to study the biogenesis of its structural components we developed a serial ultrathin-cuts method using transmission electron microscopy (TEM) combined with image analysis that allowed the 3-dimensional reconstruction of all of the different developmental stages of the parasite.We then investigated the differential expression of cell wall proteins during the intracellular development of Microsporidia. We adapted an in situ hybridization method for mRNA localization via ultrathin frozen sections coupled to immunolocalization of the corresponding proteins in TEM. One previous limitation of studying Microsporidian development was the inability to purify the various stages. To overcome this, and to enable the use of biochemical methods to study the metabolism and development of these parasites, we developed a method to isolate sporogonial stages using TEM, based on density gradient centrifugation. We also looked for the first time at the post-translational modifications of Microsporidia to identify all glycosylated motifs possibly implicated in attachment and invasion of host cells. We demonstrated the absence of N-glycosylation, characterized an unusual O-mannosylation and localized fucosylated proteins.

CCR5 is the major cellular co-receptor for HIV-1 entry and an important target for HIV therapeutics. Therefore, we developed a gene therapy approach using Zinc Finger Nucleases (ZFNs) to knock out the CCR5 gene in hematopoietic stem cells (HSCs). We optimized several parameters, first in human cell lines and then in HSCs, including the delivery of ZFNs. By comparing nucleofection of plasmid DNA versus transduction by non-integrating lentiviral (NIL) vectors, we have achieved 4 to 15% disruption in HSCs. We further evaluated the effect of this procedure on HSC function by transplanting ZFN-treated HSCs into humanized NOD/SCID/IL2-R ^-/- mice. Following HIV-1 challenge, we observed a strong selection for cells carrying CCR5-disrupted alleles.