Lipid nanoparticle Fas gene therapy suppresses autoimmune lymphoproliferative syndrome in mice Open Access

Autoimmune lymphoproliferative syndrome (ALPS) is a genetic disease of immune dysregulation. Throughout their lives, patients continue to experience continuous and chronic nonmalignant lymphoproliferation.¹ Accompanying autoimmune manifestations, such as hemolytic anemia or immune thrombocytopenia, may wax and wane over a patient’s life span.^1-3 Current treatments include methylprednisone, sirolimus, rituximab, mycophenolate mofetil, or splenectomy.⁴ 

The most common form of ALPS is due to autosomal dominant transmission of germ line FAS mutations, causing 80% of ALPS.⁵ Fas is a membrane-bound cell death receptor of the tumor necrosis factor superfamily of proteins that initiates the apoptosis signaling pathway once engaged by its physiological ligand FasL.^6,7 FasL is expressed on the surface of activated T cells and natural killer cells under physiological conditions.^7,8 The best-known function of the Fas-FasL pathway is apoptosis under physiological and pathological conditions, preventing autoimmunity.^9,10 Because Fas mutations and resultant Fas loss of function are the primary cause of ALPS, restoring wild type (WT) Fas expression therefore represents an effective approach for treating and potentially curing ALPS. However, this remains a hypothesis to be tested.

Protein-encoding DNA can be encapsulated into cationic lipid nanoparticle and delivered into mammalian cells to express a functional protein.^11-14 DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate)-cholesterol is a cationic lipid nanoparticle that has been shown to be effective in encapsulating and delivering plasmid DNA to tumor cells in human cancer patients without significant toxicity.¹² We have created a Fas complementary DNA (cDNA)–encoding mini-plasmid and formulated the plasmid DNA with cationic lipid nanoparticle DOTAP-cholesterol to generate Fas cDNA cationic lipid nanoparticle (DOTAP-Chol-Fas). DOTAP-Chol-Fas therapy is effective in transfecting tumor cells to express functional Fas on the tumor-cell surface to restore tumor-cell sensitivity to FasL-induced apoptosis in vitro and tumor growth suppression in vivo.^11,15 We therefore, aimed to test the hypothesis that DOTAP-Chol-Fas is effective in suppression of ALPS in vivo.

Human Jurkat, K562, U937, and MOLT-4 cell lines were purchased from American Type Culture Collection (Manassas, VA).

Balb/c and FAS-LPR mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Animal use protocol was approved by the Augusta University Institutional Animal Care and Use Committee (protocol: 20080162).

DOTAP-Chol (1:1 molar ratio) was produced in T&T Scientific Corp (Knoxville, TN). Codon usage–optimized mouse Fas and human Fas cDNA were cloned to the plasmid NTC9385R by Nature Technology Corp (Lincoln, NE). Lipid nanoparticle–encapsulated DNA, named mCOFas01 and hCOFAS01, were formulated as previously described. Mice received 100 μL encapsulated DNA or lipid nanoparticle (LNP) control via IV injection.^11,15 

Fas ligand (MegaFasL/APO010) was provided by Peter Buhl Jensen at Oncology Venture A/S, Denmark. Cells were collected from the culture medium and resuspended in annexin V-binding buffer,¹⁵ then incubated with allophycocyanin (APC)-annexin V (BioLegend, San Diego, CA) for 30 minutes, followed by incubation with propidium iodide. The stained cells were then diluted in annexin V-binding buffer and analyzed by flow cytometry.

Cells were incubated with fluorescent dye–conjugated anti-mouse FAS, CD45, CD8 or anti-human Fas, CD45, CD3, CD4, CD8 antibodies (BioLegend). Samples were then washed and analyzed on a FACSCalibur with CellQuestPro or LSRFortessa with BD Diva 8.01 (BD Biosciences). All flow cytometry data analyses were conducted with FlowJo v10.6.0 (BD Biosciences).

Cells were washed in serum-free medium. hCOFAS01 was then added to the hematopoietic cells. Cells were incubated in serum-free RPMI medium for 3 hours. All cells were then cultured in complete RPMI medium for 24 hours. Cells were then harvested and stained with Fas-specific monoclonal antibody and analyzed by flow cytometry.

Tissues were embedded in paraffin and sectioned, then stained with hematoxylin and eosin by the Electron Microscopy and Histology Core. Images were acquired using LAS4.1 software (Leica). Samples were analyzed by a board-certified pathologist.

Cell were lysed in total protein lysis buffer and separated in 4% to 20% sodium dodecyl sulfate polyacrylamide gels.¹⁵ The blots were probed with anti-human cleaved poly (ADP-ribose) polymerase (PARP) (catalog no. 9544s, 1:750 dilution) (Cell Signaling Tech, Danvers, MA), cleaved caspase 3 (catalog no. 9661s, 1:750 dilution), and β-actin (catalog no. A 5441, 1:5000 dilution) (Sigma-Aldrich, St Louis, MO).

The P values were determined by a 2-tailed Student t test using GraphPad Prism 10.5.0.

Codon usage–optimized mouse Fas cDNA sequence is as previously described.^11,15 

Human lymphoma and leukemia cell lines are similar in terms of proliferative ability to ALPS associated blast cells and are therefore, relevant models for determining Fas function. Cells were stained for cell surface Fas protein level. Among the 4 cell lines, 3 cell lines (Jurkat, MOLT4, and U937) express Fas and 1 cell line (K562) does not express Fas (Figure 1A). Treatment of the cells with recombinant FasL–induced cell death in all 3 Fas⁺ cell lines in a FasL concentration–dependent manner but not in the Fas^– K562 cell line. These cells die through apoptosis (Figure 1B-C). Analysis of caspase activation determined that FasL induces caspase activation and PARP cleavage (Figure 1D). These findings indicate that Fas mediates FasL-induced caspase activation and apoptosis in human T and myeloid cells in vitro.

Although a major phenotype of ALPS is mass accumulation of CD4^–CD8^– double-negative immature T lymphocytes, patients with later disease onset often exhibit autoimmune manifestations.^1,2 Patients with ALPS also exhibit deregulation of myeloid cells in the late stage.¹⁶ Fas^lpr mice resembles human ALPS with accumulation of CD4^–CD8^– double-negative immature T lymphocytes.^17-19 We therefore, sought to determine whether loss of Fas expression and function leads to dysregulation of other types of immune cells. We compared 6-month old WT and Fas^lpr mice. The aged Fas^lpr mice developed lymphadenopathy (Figure 2A). Analysis of spleens of WT and Fas^lpr mice revealed that loss of Fas leads to decreased spontaneous cell death (Figure 2B-C) due to decreased apoptosis (Figure 2D-E). In the thymus, CD4⁺CD8⁺ double-positive T cells decreased significantly (Figure 2F). In the spleens and lymph nodes, the γδ T cell receptor (γδTCR)⁺ T cells increased. Strikingly, CD11b⁺ cells increased significantly in the Faslpr mice as compared to WT mice (Figure 2F).

Our above findings suggest that restoring Fas expression might be an effective approach to suppress ALPS. Recent literature indicates that lipid nanoparticles are effective in transfecting hemetopoietic cells in vivo.^20,21 Specifically, DOTAP-cholesterol lipid nanoparticle can efficiently transfect spleen and lympho node cells to deliver DNA to the cells to express the coded protein.²² We therefore, hypothesize that lipid nanoparticle delivery of Fas-encoding plasmid should restore Fas expression and function to suppress ALPS. To test this hypothesis, we used cationic lipid nanoparticle DOTAP-Chol^11,15 (Figure 3A) to encapsulate mouse Fas cDNA–encoding plasmid (Figure 3B) to generate lipid nanoparticle–encapsulated Fas-encoding plasmid (LNP-mFas) (Figure 3C). Fas^lpr mice were then treated with LNP-mFas. LNP-mFas therapy significantly decreased the lymph node size (Figure 3D). Histological analysis of the excised lymph nodes from control mice revealed enlarged lymph nodes with a paracortical expansion consisting of small lymphocytes, plasma cells (Figure 3E), and few immunoblasts (Figure 3E). The lymph nodes from the LNP-mFas–treated mice were smaller in size. However, in all lymph nodes, plasma cells aggregated and included Mott cells (Figure 3E). Flow cytometry analysis determined that LNP-mFas therapy significantly decreased the CD4^–CD8^– double-negative T cells, the hallmark of human ALPS (Figure 3F). LNP-mFas therapy also increased CD3⁺ T cells (Figure 3F). The percentages of myeloid cell and B cells were not changed by LNP-mFas therapy. Taken together, we determine that LNP-mFas gene therapy effectively suppresses ALPS in mice.

A hallmark of patients with ALPS is the accumulation of CD4^–CD8^– double-negative immature T cells.²³ Patients with ALPS exhibit expansion of other subsets of T cells.²⁴ Patients with later ALPS disease onset frequently presented with autoimmune manifestations rather than lymphoproliferative disease^1,2 and a patient with ALPS was observed to have myeloid cell accumulation that progresses to acute myeloid leukemia.¹⁶ Consistent with this observation in the human patient, we observed that Fas^lpr ALPS mice exhibit mass accumulation of myeloid cells. Fas is known to be essential for myeloid cell differentiation and loss of Fas expression and function leads to dysregulation of myeloid cell differentiation and accumulation of immature myeloid cells in mice.²⁵ 

In this study, we showed that LNP-mFas therapy effectively suppressed the CD4^–CD8^– double-negative T cells and ALPS phenotype in mice. LNP-mFas is potentially an effective therapy for ALPS. Fas expression and function in normal T cells are tightly regulated.²⁶ A potential side effect might be overexpressed Fas after LNP-mFas therapy promote normal functional T-cell apoptosis. However, effector T cells are protected from FasL-induced apoptosis under physiological conditions.²⁷ ALPS T cells are highly proliferative and thus are selective targets of lipid nanoparticles. Consistent with this phenomenon, we observed in this study decreased CD4^–CD8^– immature T cells, but an increased CD3⁺ mature T-cell population. LNP-Fas therapy therefore, may not interrupt normal T-cell function and is potentially an effective therapy for ALPS (see the visual abstract).

A limitation of this study is the lack of toxicity profiles. However, DOTAP-cholesterol and encapsulated plasmid is a US Food and Drug Administration–approved agent and has been tested in clinical trials involving human patients with cancer²⁸ (ClinicalTrials.gov identifiers: NCT0570397, and NCT04486833). The next aim is to move this agent to test its efficacy in suppression of ALPS blasts in humanized NSG mice.

The authors thank Donna Kuminski in the Augusta University Electron Microscopy and Histology Core.

The study received grant support from the National Cancer Institute, National Institutes of Health (grant R01CA278852) and the US Department of Veterans Affairs (grant I01CX001962)

Contribution: D.B.P. and K.L. conceptualized the study; D.B.P., Z.T., P.R., A.M., N.S., and K.L. designed the methodology; D.B.P., Z.T., P.R., and A.M. conducted the investigation; K.L. acquired funding and provided project administration and supervision; and D.B.P. and K.L. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Kebin Liu, Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1410 Laney Walker Blvd, Augusta, GA 30912; email: kliu@augusta.edu.