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Thesis (M.S.)--Rochester Institute of Technology, 2008. / Typescript. Includes bibliographical references (leaf 77).

Correction of blood genetic disorders requires permanent gene transfer into self renewing, hematopoietic stem cells (HSC), and regulation of transgene expression in specific cell lineages. HIV-derived lentiviral vectors are very effective in transducing rare, non-dividing stem cell populations without altering their long-term repopulation and differentiation capacity. We developed a strategy for transcriptional targeting' of lentiviral vectors based on replacing the viral LTR control elements with cell lineage specific, genomic control elements. An upstream enhancer (HS2) of the erythroidspecific GATA-l gene was cloned in a second-generation lentiviral vector to replace most of the U3 region of the LTR, immediately upstream of the HIV-l promoter. The modified LTR was used to drive the expression of a reporter gene (GFP), while a second gene (~LNGFR) was placed under the control of an internal, constitutive promoter to monitor cell transduction, or immunoselect transduced cells, independently from the expression of the targeted promoter. The vector was used to transduce cell lines, human CD34⁺ hematopoietic stem/progenitor cells, and murine bone marrow HSCs. Gene expression was analyzed in the differentiated progeny of transduced stem cells in vitro and in vivo, after transplantation into lethally irradiated co-isogenic (for murine cells) or NOD/SCID (for human cells) mice. The transcriptionally targeted HIV LTR allowed very high level of transgene expression specifically in mature erythroblasts, in a <i>tat</i>-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the targeted LTR was higher, better restricted, and showed significantly less position effect variegation than that obtained by the same combination of enhancer/promoter elements placed in the conventional, internal position. Cloning of the woodchuck hepatitis virus post transcriptional regulatory element (WPRE) at a defined position in the targeted vector, allowed selective accumulation of the genomic with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of the spliced, major viral transcript to express a therapeutic gene, and that of an internal, independently regulated promoter to express an additional gene for either cell marking or <i>in vivo</i> selection purposes.

Enzyme enhancement therapy based on pharmacological chaperones (PCs) represents a promising new therapeutic strategy for the treatment of rare genetic disorders associated with protein misfolding. PCs are small extrinsic molecules that activate, stabilize and promote folding as a way to rescue mutant enzymes from endoplasmic reticulum-associated protein degradation. To date, high throughput drug screening has relied on fluorescence-based inhibition and/or thermal stability assays for putative PC selection from large chemical libraries with confirmatory testing on patient-derived cell-based assays or animal models. However, conventional primary screening methods do not directly measure for chaperone activity that may contribute to high attrition rates in drug discovery. The major aim of this thesis is to develop and validate a high quality screening strategy for the discovery of novel PCs that restore the activity of denatured/mutant enzymes associated with Gaucher disease (GD) and phenylketonuria (PKU). Chapter II introduces a simple yet selective capillary electrophoresis (CE)-based inhibition assay for improved characterization of previously-approved FDA drugs that function as putative PCs for β-glucocerebrosidase (GCase), a lysosomal enzyme associated with GD. A novel in-vitro assay based on restoration of enzyme activity via denaturation (READ) was developed in Chapter III for unambiguous characterization of the chaperone activity of previously identified PCs for GCase when using CE with UV detection. Chapter IV subsequently adapted READ to a fluorescence-based high throughput screening platform to discover novel stilbene derivatives as PCs from a chemical library comprising structural unique compounds after in silico assessment of drug-like activity. Chapter V then used this two-tiered screening strategy to discover plant-derived natural products that enhance the activity of phenylalanine hydroxylase (PAH), the enzyme associated with PKU. In summary, an integrated two-tiered strategy for high quality screening of PCs has been developed in this thesis, which is anticipated to enhance drug discovery while reducing false discoveries for treatment of various human diseases associated with deleterious protein misfolding. / Thesis / Candidate in Philosophy

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The purpose of this dissertation is to examine the use, role and function of medical pedigrees as part of extended networks of genetic practices. Integral to my argument is a description of geneticisation (i.e., the redefinition of family problems as genetic in origin), grounded in a set of detailed case studies of the development and use of visual tools in genetic practices. / In recent years, medical sociologists have tended to link geneticisation to medicalisation (i.e., the social control by doctors over patients accompanied by the translation of social problems into medical issues). I argue that the twin notions of geneticisation and medicalisation are problematic, insofar as they embody a simplistic and negative understanding of medical activities and they prevent a sociological inquiry into the technical content of genetic practices. / Medical pedigrees are visual tools used to translate family problems into visual inscriptions, in order to show the genetic nature of a given disease. The use of medical pedigrees in genetic counselling and research rests on a chain of genetic practices including the inscription of family trees, the standardisation of medical pedigrees, the combination of specialised forms of medical pedigrees with other diagnostic inscriptions, and the circulation of published pedigrees. The analysis is based on a genealogical approach, as built on a combination of historical and ethnographic methods. The genealogical approach was applied to the analysis of a long network of genetic practices centred on Huntington's disease. The analysis spans over 120 years and compares two different international settings (North America and Japan). / The thesis examines how lay support group members and family members collect family narratives, family inscriptions and family trees, which were first translated by genetic counsellors into various forms of medical pedigrees, and then circulated as educational material among lay and medical practitioners. On the basis of these case studies, the conclusion is reached that the notion of geneticisation should be understood as a specific process resulting from an emerging cooperative practice between medical practitioners and lay support group members, rather than as a process of medicalisation.

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