RECOLECCIÓN Y TRANSPORTE

7.1 INTRODUCTION

In a recent study, approximately two thirds of affected families were found to have the X-Iinked form o f CGD, arising from an abnormality of the P-subunit of the flavocytochrome (Casimir et al. 1991). The remaining patients had autosomal recessive defects, with those exhibiting deficiency o f p47^*‘^^ the most prevalent. Defects in the a - subunit of the flavocytochrome, and in made up the remaining 10% of cases. This observed genetic distribution of CGD is in close agreement with a similar study of 82 American families (Clark et al. 1989). Molecular lesions at CYBB and CYBA, the genetic loci for gp91^^^^ and p22^^°^ respectively, are particularly heterogeneous, and unique to individual families in over 90% o f cases described. In contrast, a GT dinucleotide deletion at a GTGT repeat at the boundary between the first intron and second exon o f the gene accounts for over 90% of mutant alleles in p47^*°^ deficient CGD (Casimir et al. 1991).

The advent of treatment by somatic gene therapy necessitates the identification of the defective component in all patients, and the demonstration that gene transfer restores function to cells derived from individual patients. Immunoblotting for components of the NADPH-oxidase identifies the defective protein in the majority o f cases. However, molecular lesions in either component of flavocytochrome bg^g usually results in absence o f the entire heterodimer, and although the affected component may be inferred from inheritance, it remains difficult to distinguish between males with normal maternal NBT patterns, who may have acquired a new mutation at CYBB, and those with autosomal recessive deficiency of p22^^^^. Identification o f the defective component may also be difficult in rare individuals who express normal levels o f functionally deficient protein. In these cases, complementation studies in a cell-free system using recombinant protein (Rotrosen et al. 1993), or in whole cells by monocyte fusion (Roos et al. 1992), may be helpful but can be technically difficult.

The inability of the adenoviral genome to transmit to progeny cells in the absence of continuing infection limits practical application as a vehicle for transduction of haematopoietic progenitor cells. However, in contrast to current retroviral gene delivery systems, adenoviral vectors efficiently transduce cells which are not actively in cell- cycle, and are therefore ideal for transduction of peripheral blood monocytes which undergo terminal maturation and differentiation in fixed tissues, but do not readily enter the cell-cycle. This application of adenovirus-mediated gene delivery provides a functional assay for molecular diagnosis of CGD by genetic complementation of primary monocytes, and may have therapeutic potential in the short term.

7.2 TRANSDUCTION OF MONOCYTES 7.2.1 Transduction of CGD monocytes

Production of the adenoviral supernatant p47AD2, and the ability of this virus to complement the genetic defect in p47^^‘’^-deficient B lymphoblastoid cells has been described previously. In contrast, transduction of similar cells derived from patients with known deficiencies of the flavocytochrome or p67^^‘’^ failed to restore function (not shown). Peripheral blood monocytes prepared from three CGD patients were transduced with p47AD2 at a m.o.i. of 100-500. To control for non-specific effects arising from adenoviral infection, equal numbers of monocytes were subjected to sham infection with PBS alone, and to infection with a parallel adenoviral vector encoding LacZ (pLacZAD), at a similar m.o.i. Monocytes recovered from a normal individual were treated in the same way, and were used as a positive control.

7.2.2 Functional analysis

After 18-24 hours, monocytes were tested for their ability to reduce nitroblue tétrazolium (NBT) to insoluble and dark blue staining formazan (Fig 7.1, Fig 7.2).

[A]

[B]

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Fig 7.1 NBT staining of transduced monocytes. Adherent mononuclear cells purified from 5 mis o f peripheral blood from one patient known to be p47^*'’^-deficient, were incubated separately with [A] PBS, or [B] p47AD2 recombinant adenoviral particles, for two hours. 18-24 hours post transduction, medium was removed and cells washed three times with PBS, NBT assay mixture was added directly to the adherent cells, and positive staining scored after 30 minutes incubation at 37°C by light microscopy. A significant proportion o f transduced cells show a positive NBT reaction, determined by precipitation o f insoluble blue formazan onto the cells. In a control experiment, 100% o f monocytes prepared from a normal individual were NBT positive.

Mock

P47AD2

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Fig 7.2 NBT staining of transduced monocytes. Adherent mononuclear cells purified from 5 mis o f peripheral blood from three patients ( 1 , 2 and 3) with CGD o f whom patient 1 was known to be p47^^°'-deficient, were incubated separately with p47AD2 or pLacZAD (Mock) recombinant adenoviral particles for two hours. 18-24 hours post transduction, medium was removed and cells washed three times with PBS. NBT assay mixture was added directly to the adherent cells, and positive staining scored after 30 minutes incubation at 37°C by light microscopy. Cells from patient 1 and patient 2, transduced by p47AD2, both show positive staining in a proportion o f cells. In contrast, cells from patient 3 under all transduction conditions remained negative. All CGD cells transduced by the LacZ encoding adenovirus (Mock) remained NBT negative. Control cells (C) were derived from a normal individual.

100^ 80- 60- B c o 40 O 2 0- Oh Control - O - P47A D 2.2 - X - P47AD2.3 - A - nil CO o o o o

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Time after activation (mins)

Fig 7.3 Functional reconstitution of monocytes. Adherent mononuclear cells purified from 5 mis o f peripheral blood from two patients (patients 2 and 3) with CGD o f undetermined molecular pathology, were incubated separately with PBS alone, p47AD2 or pLacZAD recombinant adenoviral particles for two hours. 18-24 hours post transduction, cells were harvested by scraping and assayed by chemiluminescence for production o f superoxide follow ing stimulation with PMA. Only cells from patient 2 transduced by adenovirus encoding p47^*“* (p47A D 2.2) showed significant restoration of function. Similar cells which underwent sham transduction with PBS, or transduction by adenovirus encoding LacZ remained inactive (not shown). No activity was detected in cells obtained from patient 3 under all conditions, (p47A D 2.3). Control and nil represent unmanipulated cells derived from a normal individual and a patient respectively.

gp91phox

p67phox

p47phox