NFP37 SOMATIC GENE THERAPY Texts for Laypersons

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Prof Heidi Diggelmann;	Inst. de Microbiologie de l'Université de Lausanne; R. du Bugnon 44; 1011 Lausanne; CH; Tel 021 314 40 97; Fax 021 314 40 95; e-mail:
Title:	Development of retroviral vectors targeted to cells of the hematopoietic lineage.
Co-applicants:	Dr. E. Buetti (Lausanne); Dr. P. Meylan (Lausanne); Dr. M. Thali (Lausanne)
Collaborators:
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ABSTRACT FOR LAYPERSONS 1997:

The goal of this project is to use recent insights into the retroviral life cycle to improve the characteristics of retroviruses for use as agents of gene delivery in humans. In particular, we sought to generate retroviral vectors with the following features:

1. stable and complement-resistant virions with tissue tropism for cells of the haematopoietic lineage and HlV-target cells

2. vectors capable of infecting resting cells

3. expression systems which allow the development of efficient packaging cells

Since HIV possesses some of the above characteristics e.g., ability to infect resting cells and inherently, the ability to infect HIV-, cells it seemed appropriate to focus our attention on HIV rather than on the murine retroviruses. The safest protocol currently used for generation of retroviral vectors involves the expression of retroviral components from separate plasmids. Thus, the structural components which form the particle core and enzymes are expressed from one plasmid (the packaging construction), the envelope is expressed from another and the genome to be packaged from a third. In this way, two separate recombination events are required to produce replication competent virus.

We have generated packaging constructs by cloning various parts of the HIV genome into a eucaryotic expression vector. This was carried out in multiple steps and involved modifying the wild-type HIV proviral DNA (clone pNL43) as follow.s: The entire 5' Long Terminal Repeat (LTR) and sequences in the 3' LTR not overlapping with the nef gene were removed. Thus eventual recombination with LTRs of the vector is minimised. To preclude packaging of HIV genes contained in this construction, a 39 bp region between the major splice donor and the start site of the gag gene, the proposed packaging signal, was deleted by site directed mutagenesis. To prevent expression of functional envelope, approximately 580 bp was removed from the env gene. The HIV genes were placed under the control of the human cytomegalovirus (CMV) immediate-early enhancer/promoter in a plasmid which contains a neomycin phosphotransferase gene. Three distinct packaging constructs

were made. They differ with respect to one HIV gene, Vpr. At the time this work was commenced, the Vpr gene product had been shown to play an important role in the nuclear import of the HIV pre-integration complex and hence in the infection of resting cells. However, expression in cells was also shown to cause a G2/M block, a property which is incompatible with continued expression cell lines. We have therefore introduced on two different packaging constructions, different mutations in the Vpr gene which have been reported (Di Marzio et al., 1995) to selectively prevent Vpr causing cell cycle arrest but have no effect on its other functions. On one construction the Vpr gene was left unchanged.

These constructions were tested for HIV structural protein expression by transient transfection in a human embryonic cell line (293T) and a human fibrosarcoma cell line (HT1080). Western analysis revealed that proteins of the size of the Gag precursor and p24 matrix were made Actual levels of p24 protein were measured and found to be high in 293T cell supernatant and somewhat lower in HT1080 cell supernatant. To test the functionality of these constructions 293T cells were cotransfected with an envelope expressing plasmid (VSV-G, kindly provided by Dr. D. Trono), and a 'HIV' vector expressing green fluorescence protein. Cell supernatant was harvested 42 hours later and used to infect NIH3T3 fibroblasts. 72 h post infection cells were viewed using a fluorescence microscope and a number of cells were observed to exhibit green fluorescence, an indication that the above described packaging constructs can give rise to mature viral particles which are capable of packaging an appropriate genome and budding correctly from cells following transient transfection. Currently the titres obtained using our packaging constructs in the transient system described above are being assessed. Once high titres can be reproducibly obtained we will examine transducibility of a variety of cells lines. In parallel with working with the transient system, attempts have been made to generate stable packaging cell lines with both of the Vpr mutant packaging constructs. To do this, advantage was taken of the neomycin phosphotransferase gene contained on these plasmids which confers resistance to neomycin. In all, a total of 16 neomycin resistant colonies have been isolated, grown up and tested for HIV protein expression to date. In each case, p24 expression was either extremely low or absent. We are currently examining reasons for this result.

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