P1372: NON-MYELOTOXIC CHEMICALS AS A PREPARATORY REGIMEN FOR HEMATOPOIETIC STEM CELL GENE THERAPY

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Primary immunodeficiencies (PID) are characterized by the absence or dysfunction of lymphocytes and affect both cellular and humoral adaptive immunity. RAG2 (Recombination activating gene 2) deficiency is characterized by a lack of both mature B and T lymphocytes, causing severe viral and bacterial infections that are usually lethal before the first year of life. Currently, the only curative treatment for RAG2 deficiency is Hematopoietic Stem Cell transplantation (HSCT), while gene therapy for RAG2 deficiency is still being developed. However most conditioning regimens consist of a combination of alkylating myelotoxic drugs, such as Busulfan (BU), with or without radiation and have been shown to affect growth and development in the pediatric age and may result in a delay or absence of puberty, infertility, permanent organ damage and even secondary malignancy. Aims: Here, we aimed to develop a non-myelotoxic regimen that can be used as preparatory conditioning before transplantation of genetically corrected HSCs, consisting of G-CSF, VLA-4I or AMD3100. These drugs are known as HSC mobilizers or affect BM permeability and may also support the homing of intravenously infused (transduced) HSCs back to the BM niche, without inducing any harm. Methods: Mice were pre-treated with 25 mg/kg BU i.p, 6 μg/kg/d G-CSF s.c. for 4 days, 1 mg/kg VLA-4I i.v. or 1 mg/kg AMD3100 s.c. c-kit+ BM cells of male RAG2-/- mice were transduced with LV-UCOE-hRAG2co and transplanted into female RAG2 mice Expression of CD45, CD3, CD19, CD11b, CD45R and NK1.1 in peripheral blood was measured at 1 and 3 months after transplantation. Spleen and bone marrow cellularity were assessed at sacrifice of the mice at 6 months post-transplant. Results: Peripheral blood cell counts increased significantly in the groups treated with G-CSF (p<0.05), VLA-4I (p<0.01) and AMD3100 (p<0.05) in comparison to baseline levels, but not in the BU-treated group. Reconstitution of CD3+ and CD19+ PB cells after transplantation of mice with c-Kit+ BM cells was similar in all groups, although mice subjected to BU treatment showed a slight advantage in terms of absolute CD19+ numbers, starting from 1 month after transplantation (p<0.05). At 6 months after transplantation, all mice displayed full immune reconstitution, despite different methods of conditioning. Reconstituted spleen lymphocytes responded to activation signals. Survival of mice treated with BU was significantly lower (32%) than survival of mice in any of the other groups (p<0.0007), with the probability of survival highest in the AMD3100-treated group (87,5%) at 6 months post-transplant followed by G-CSF (71,4%) or VLA-4I (70,4%). However, the overall immune reconstitution was best in mice treated with the latter groups. Summary/Conclusion: Here, we show that the non-myelotoxic chemicals G-CSF, VLA-4I, and AMD3100 are highly effective as conditioning regimen before HSC gene therapy and can be used instead of BU. The most robust immune reconstitution was observed after treatment with G-CSF and VLA-4I. Keywords: Stem cell gene therapy, Hematopoietic stem cell, Non myeloablative conditioning regimen, Stem cell mobilization