Development of a Treg Lineage Specific Lentiviral Vector for the Treatment of IPEX Syndrome

Abstract Autologous hematopoietic stem cell (HSC) transplant with gene therapy has recently demonstrated remarkable clinical success in the correction of genetic blood cell disorders. One excellent candidate disease for HSC gene therapy is Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. IPEX is a devastating autoimmune disease caused by mutations in FOXP3, a transcription factor required for the proper development and function of regulatory T (Treg) cells. Tregs are critical for providing dominant suppression of autoreactive conventional T (Tconv) cells, and thus their absence in IPEX patients causes severe and often fatal multi-organ autoimmunity. Allogeneic HSC transplant has been shown to be curative for IPEX patients, but is limited due to a lack of suitable donors and immunologic transplant complications. In order to explore an autologous HSC gene therapy strategy for IPEX, we sought to design an endogenously regulated lentiviral vector (LV) which confers targeted and developmentally appropriate FoxP3 expression. Here, we utilized three intronic regulatory regions of the FoxP3 gene which have verified enhancer/regulatory function: conserved noncoding sequence (CNS) 1, CNS2, and CNS3. These sequences were added upstream from the FoxP3 promoter and cloned into the CCL vector backbone to generate two LV expressing either mStrawberry (FoxP3 reporter vector), or a human FoxP3 cDNA and mStrawberry (FoxP3 cDNA vector). A primary screen of the FoxP3 reporter vector in human cell lines from different hematopoietic lineages (K562 [erythromyeloid, FoxP3-], Jurkat [Tconv, FoxP3-] and MT-2 [Treg-like, FoxP3+]) revealed mStrawberry expression restricted to the FoxP3+ cell line with low inherent “leakiness” of expression in FoxP3- cell types. In order to evaluate in vivo lineage specificity of the FoxP3 reporter vector, we utilized both murine congenic transplants and humanized NSG xenografts to quantify LV expression in transduced HSPC and their progeny throughout hematopoietic development. In both models, we observed high levels of mStrawberry expression in the Treg lineage with low expression in CD4 Tconv cells and CD8 T cells, and negligible expression in HSPC, B cells, myeloid cells, suggesting that the regulatory elements included in this vector confer developmentally appropriate FoxP3 expression. We next evaluated the capacity of this vector to restore Treg development when introduced into FoxP3-deficient HSC from the scurfy mouse. Scurfy HSC were transduced with the FoxP3 cDNA vector and transplanted into WT CD45.1 congenic recipients to generate corrected scurfy Tregs (cSf-Tregs). Thymic cSf-Tregs phenotypically resembled WT-Treg showing equally high expression of the Treg surface markers CD25, GITR, and CTLA-4. Furthermore, FACS-sorted cSf-Tregs were equivalent to WT-Tregs in their ability to suppress Tconv cell proliferation in vitro. We next evaluated the in vivo functionality of cSf-Tregs by assessing their ability to reverse clinical disease signs in neonatal scurfy mice. Donor CD45.2+CD4+ cells (containing either WT-Tregs, cSf-Tregs, or uncorrected Sf T cells) were purified from the spleens of WT CD45.1 transplant recipients and administered to scurfy neonates by i.p. injection. Treated scurfy mice were evaluated for disease progression at 21 days post-injection. In comparison to WT littermate controls, untreated scurfy mice showed typical signs of disease progression including scaly skin, malformed ears, splenomegaly, and a high percentage of activated (CD62L-CD44+) CD4 T cells. Administration of WT-Tregs or cSf-Tregs resulted in complete correction of all measures of the scurfy phenotype, such that these mice were indistinguishable from WT littermate controls. In contrast, scurfy mice receiving uncorrected Sf T cells showed no signs of disease amelioration, confirming that disease correction was not an artifact of the transplant/CD4 isolation procedure. These results demonstrate that correction of scurfy HSC with a lineage-specific FoxP3 cDNA LV produces CD4+ Tregs capable of controlling autoimmune T cell responses in vivo. These findings pave the way for the treatment of IPEX patients by autologous HSC transplant and may further provide valuable insights into new treatments for patients with autoimmune disorders of different origin. Disclosures Kohn: Orchard Therapeutics Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Research Funding; Biogen IDEC: Research Funding.