Platelet-Targeted Gene Transfer Prevents Graft Rejection and Induces Immune Tolerance Even in a Primed Model
Authors: Luo Xiaofeng , Li Jing , Chen Juan , Schroeder Jocelyn A. , Hu Jianda , Shi Qizhen 
Published: October 2019

Abstract
Induction of antigen-specific immune tolerance is desirable in autoimmune diseases, transplantation, gene therapy, and some protein therapies in order to prevent or reverse adverse immune responses. Our previous studies have demonstrated that targeting FVIII or FIX expression to platelets under control of the platelet-specific aIIb promoter can restore hemostasis and induce immune tolerance in hemophilia mice. Here we explored if this approach can be applied to prevent graft rejection and induce immune tolerance to a non-coagulant protein even with pre-existing immunity. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). We designed another vector, 2bVpOVA, which includes the VWF propeptide (Vp) cassette to secure OVA storage in platelet granules. The third construct, 2bGFP, in which the GFP cassette is driven by the same promoter, was used as a control vector.
We confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to hematopoietic stem cells (HSCs) can result in OVA expression with greater than 95% of OVA stored in platelet a-granules. Under a non-myeloablative conditioning 6.6Gy total body irradiation (TBI), platelet-OVA expression levels were 24.22±8.72 ng/108 platelets and 1.41±0.73 ng/108 platelets in 2bOVA and 2bVpOVA transduced wild type (WT) recipients, respectively. When recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in un-transduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. To explore whether platelet targeted gene transfer can be applied to prevent graft rejection, skin grafts from Act-mOVA transgenic mice, in which OVA is expressed on the cell surfaces of all organs, were transplanted onto transduced recipients. Full thickness tail skin successfully grafted onto OVA-immunized 2bOVA- or 2bVpOVA-transduced recipients and was sustained for the rest of the animals' lives or during the study course for up to 6 months. In contrast, skin grafts were rejected in OVA-immunized untransduced WT and 2bGFP-transduced animals within 6 weeks.
To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP and transplanted into CD45.1/B6 WT recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (85.7±4.3%, 84.9±3.9%, and 86.5±2.9%, respectively), but donor-derived CD45.2+CD4+ T cells in the 2bOVA (0.2±0.1%, n=10) and 2bVPOVA (0.8±0.3%, n=11) groups were significantly lower than in the 2bGFP group (2.6±0.5%, n=11) in peripheral blood. Similarly, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy.
To investigate whether platelet-targeted gene transfer can still induce the immune tolerance when immune system is primed, Sca-1+ cells from OTII mice were transduced with 2bOVA lentivirus and transplanted into OVA-primed CD45.1/B6 WT animals preconditioned with 6.6Gy TBI. We found that the platelet-OVA expression level in the OVA-primed group after 2bOVA gene transfer was not significantly different from the unprimed group (26.47±4.47 vs. 29.56±6.29 ng/108 platelets). The engraftments were comparable among the primed, the unprimed, and the primed untransduced control groups. Similar to the unprimed model, OVA-specific CD4+ T cells in 2bOVA-transduced OVA-primed recipients were significantly lower than in the untransduced controls (0.23±0.07% vs. 3.98±0.61%). Importantly, the anti-OVA total IgG declined with time after 2bOVA gene transfer, even when the animals were rechallenged with OVA.
In summary, our data demonstrate that platelet-targeted gene transfer can prevent graft rejection and induce immune tolerance even when the immune system is primed, suggesting that platelet gene therapy can be a promising approach to induce immune tolerance and prevent undesired immune responses.

Disclosures
No relevant conflicts of interest to declare.