920. Assessing the Risk from Transfer of an Integrin β3 Gene That Encodes a Naturally Occurring Polymorphism Associated with Acute Coronary Thrombosis
Authors: Juan Fang, Bryon D. Johnson, David A. Wilcox
Journal: Molecular Therapy (2004)

Glanzmann thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular defects in either the αIIb- or β3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. When planning genetic transfer strategies for hematological disorders, it may be important to consider thrombotic risk factors associated with some naturally occurring polymorphisms of the transgene before choosing a cDNA cassette for gene therapy. This is exemplified in the debate concerning the increased risk of acute coronary thrombosis in association with inheritance of the platelet alloantigen (PlA2) form of the integrin β3 subunit encoding a proline at amino acid 33 versus PlA1 that has a leucine at residue 33. As a model to examine the suitability for expressing alternate PlA forms of β3 as gene therapy for GT, hematopoietic stem cells were transduced with either form of β3 (PlA1, PlA2 or PlA1/2) and then transplanted into a homogenous population of mice deficient in β3. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector. The 3'-LTR viral enhancer/promoter was absent from this construct to produce a self-inactivating vector and the human αIIb gene promoter (nucleotides -889 to +35) was used to direct megakaryocyte-specific synthesis of β3. The vector utilized the woodchuck hepatitis virus postregulatory element, and the central polypurine tract to enhance the efficiency of transgene expression. Mononuclear cells isolated from bone marrow of β3 deficient mice were prestimulated for 2 days in medium containing cytokines, transduced with β3 virions and transplanted into lethally-irradiated littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of 30–50% circulating platelets from 2 weeks post-transplant. Immuno-analysis also indicated that expression of β3 was confined exclusively in platelets. Cytometric analysis using monoclonal antibodies and human serum that reacts specifically with the PlA1 or PlA2 were used to confirm expression of each subunit. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates in vitro upon stimulation with a cocktail of physiological platelet activation agonists (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide). In contrast, platelets isolated from β3 deficient mice were unable to aggregate upon stimulation with physiological agonists. The results from this study demonstrate feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine the effect of these differences on platelet function.