Platelet-Targeted Gene Transfer Induces Antigen-Specific Immune Tolerance

Abstract Induction of antigen-specific immune tolerance is desirable in autoimmune diseases, transplantation, and gene therapy. Our previous studies have demonstrated that FVIII or FIX expression ectopically targeted to platelets under control of the platelet-specific αIIb promoter results in transgene protein storage in platelet α-granules. Further studies have demonstrated that lentivirus-mediated platelet-specific gene delivery to hematopoietic stem cells (HSCs) not only restores hemostasis but also induces antigen-specific immune tolerance in hemophilic mice. We wanted to explore whether platelet-specific gene transfer can be used as a means of immune tolerance induction. In the current study, we used ovalbumin (OVA) as a non-coagulant protein to further examine the potential of a platelet gene therapy-based immune tolerance induction approach. We constructed a lentiviral vector (LV) in which OVA is driven by the αIIb promoter (2bOVA). Evidence suggests that VWF propeptide can reroute unrelated secreting proteins to a storage pathway. Thus, we designed another vector, 2bVpOVA, which contains VWF propeptide to secure OVA storage in platelet granules. HSCs from wild type B6/CD45.2 mice were transduced with 2bOVA or 2bVpOVA LV and transplanted into B6/CD45.1 recipients preconditioned with 660 cGy total body irradiation. We found that 96% of OVA expression in whole blood was stored in platelets with a level of 51.3 ± 22.5 ng/108 platelets (n = 5) while 4% was detectable in plasma in 2bOVA-transduced recipients at 12-week after transplantation. This distribution is very similar to the results we obtained from the FIX study. In contrast, 98% of OVA was stored in platelets with a level of 3.9 ± 3.3 ng/108 platelets (n = 5) in 2bVpOVA-transduced recipients. The lower total OVA expression level in the 2bVpOVA group could be due to the size effect of transgene expression cassette as the 2bVpOVA cassette is 3-fold larger than the 2bOVA cassette. To investigate whether anti-OVA immune tolerance was established in recipients after platelet-specific OVA gene transfer, 16-weeks post-transplantation, animals were challenged with OVA. The titer of anti-OVA total IgG determined by ELISA assay was 640 ± 101 in the 2bOVA group and 320 ± 0 in the 2bVpOVA group. These titers were significantly lower than that obtained from the untransduced control group (10210 ± 3636), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. Of note, the titer of anti-OVA total IgG in the 2bVpOVA group was significantly lower than in the 2bOVA group although the total OVA expression levels in the 2bOVA group is 13-fold higher than in the 2bVpOVA group. The percentage of regulatory T cells in peripheral blood in 2bOVA and 2bVpOVA-transduced recipients was significantly higher than in untransduced control animals. In summary, our data demonstrate that targeting transgene expression and storage in platelet a-granules is a potentially promising approach for inducing immune tolerance. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Partial phenotypic correction and immune tolerance induction to enzyme replacement therapy after hematopoietic stem cell gene transfer of α-glucosidase in Pompe disease

The Journal of Gene Medicine ◽

10.1002/jgm.1305 ◽

2009 ◽

Vol 11 (4) ◽

pp. 279-287 ◽

Cited By ~ 15

Author(s):

Gaëlle Douillard-Guilloux ◽

Emmanuel Richard ◽

Lionel Batista ◽

Catherine Caillaud

Keyword(s):

Stem Cell ◽

Gene Transfer ◽

Enzyme Replacement Therapy ◽

Immune Tolerance ◽

Tolerance Induction ◽

Hematopoietic Stem ◽

Immune Tolerance Induction ◽

Enzyme Replacement ◽

Cell Gene ◽

Stem Cell Gene

Download Full-text

Light-induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy

Cancer Gene Therapy ◽

10.1038/sj.cgt.7700447 ◽

2002 ◽

Vol 9 (4) ◽

pp. 365-371 ◽

Cited By ~ 56

Author(s):

Anders Høgset ◽

Birgit Øvstebø Engesæter ◽

Lina Prasmickaite ◽

Kristian Berg ◽

Øystein Fodstad ◽

...

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Gene Delivery ◽

Cancer Gene Therapy ◽

Specific Gene ◽

Cancer Gene ◽

Delivery Technology

Download Full-text

Liver-specific Factor VIII gene therapy for immune tolerance induction in hemophilia

Science-Business eXchange ◽

10.1038/scibx.2010.1222 ◽

2010 ◽

Vol 3 (40) ◽

pp. 1222-1222

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Immune Tolerance ◽

Tolerance Induction ◽

Specific Factor ◽

Factor Viii Gene ◽

Immune Tolerance Induction

Download Full-text

177. rAAV9-Mediated Microdystrophin Gene Transfer with Immune Tolerance Induction Improves Dystrophic Phenotype of Canine X-Linked Muscular Dystrophy

Molecular Therapy ◽

10.1016/s1525-0016(16)35981-0 ◽

2012 ◽

Vol 20 ◽

pp. S71

Keyword(s):

Muscular Dystrophy ◽

Gene Transfer ◽

Immune Tolerance ◽

Tolerance Induction ◽

Immune Tolerance Induction

Download Full-text

Lentivirus-Mediated Platelet Gene Therapy Corrects Bleeding Diathesis and Induces Immune Tolerance in Murine Hemophilia B Mice

Blood ◽

10.1182/blood.v120.21.1101.1101 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1101-1101

Author(s):

Yingyu Chen ◽

Jocelyn A. Schroeder ◽

Erin L. Kuether ◽

Guowei Zhang ◽

Robert R. Montgomery ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Immune Tolerance ◽

Whole Blood ◽

Tolerance Induction ◽

Hemophilia B ◽

Hematopoietic Stem ◽

B Mice ◽

Inhibitory Antibodies ◽

Tail Clip

Abstract Abstract 1101 While data from the clinical trials using AAV vector expression FIX in hemophilia B gene therapy in humans are very encouraging, for individuals with severe liver disease or neutralizing antibodies to AAV, an alternative gene therapy approach might be desired. Our previous studies have demonstrated that lentivirus-mediated platelet gene therapy can correct murine hemophilia A phenotype, but this approach has not been explored for hemophilia B. In the current study, we developed a clinical translatable approach for platelet gene therapy of hemophilia B. Platelet-FIX (2bF9) expression in hemophilia B (FIXnull) mice was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). The recipients were analyzed beginning at 3 weeks after bone marrow (BM) transplantation. Expression of the 2bF9 product was detected by PCR in all recipients that received 2bF9 LV-transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF9 LV transfer vector. The expression of the hFIX transgene protein in the transduced platelets was confirmed by immunofluorescent confocal microscopy. Flow cytometry showed that there were 20.8 ± 12.1% (n = 7) and 14.8 ± 10.7% (n = 6) 2bF9 LV-transduced platelets respectively in the recipients preconditioned with 1100 cGy or 660 cGy. The antigen levels of FIX (FIX:Ag) were 2.89 ± 1.75 mU/108 platelets (n = 9) in the recipients preconditioned with 1100 cGy and 1.87 ± 1.30 mU/108 platelets (n = 7) in the 660 cGy group, while the activity (FIX:C) levels were 1.67 ± 1.15 and 1.13 ± 0.85 mU/108 platelets respectively. There was a small amount of FIX detected in the 2bF9 LV-transduced recipient plasma with the average levels of 2.22 mU/ml in 1100 cGy group and 1.44 mU/ml in 660 cGy group. To analyze the distribution of the FIX between platelets and plasma, we normalized FIX levels to total whole blood FIX content. The results demonstrated that 90% to 95% of whole blood FIX was stored in platelets. The tail clip survival test demonstrated that 15 out of 16 mice that received 2bF9 LV-transduced HSCs survived the tail clip challenge, while 8 out of 10 FIXnull control mice died after tail clipping. Nine months after transplantation, sequential transplantation was performed on some of the primary recipients. Platelet-hFIX expression in the secondary recipients was sustained, leading to phenotypic correction and confirming that long-term engrafting HSCs were successfully transduced with 2bF9 LV. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. To investigate whether immune tolerance was induced in 2bF9 LV-transduced recipients, we challenged the recipients with recombinant human FIX (rhFIX) in the presence of adjuvant. Only 1 out of 9 2bF9 LV-transduced recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) as measured by a modified Bethesda assay. In contrast, all of the FIXnull controls developed inhibitory antibodies ranging from 17 – 37 BU/ml after the same challenge (n = 5). To ensure that the immune system was not defective in the 2bF9 LV-transduced recipients and that the tolerance induction is FIX antigen-specific, we further challenged the animals with ovalbumin (OVA) absorbed on Alum. Both the 2bF9 LV-transduced and FIXnull control mice developed high-titer of anti-OVA antibodies. The levels of anti-OVA IgG in the 2bF9 transduced recipients were not significantly different from FIXnull mice after the OVA immunization, confirming that tolerance induction in 2bF9 LV-transduced mice is FIX-specific. Taken together, our data suggest that lentivirus-mediated bone marrow transduction and transplantation can not only provide sustained phenotypic correction, but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Targeting FVIII Expression to Platelets Induces Immune Tolerance in Hemophilia A Mice with or without Pre-Existing Anti-FVIII Immunity,

Blood ◽

10.1182/blood.v118.21.4170.4170 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4170-4170

Author(s):

Yingyu Chen ◽

Erin L. Kuether ◽

Jocelyn A. Schroeder ◽

Robert R. Montgomery ◽

David W. Scott ◽

...

Keyword(s):

Gene Therapy ◽

Immune Response ◽

Immune Tolerance ◽

Hemophilia A ◽

Conflicts Of Interest ◽

High Titer ◽

Control Group ◽

Hematopoietic Stem ◽

Therapy Strategy ◽

Inhibitory Antibodies

Abstract Abstract 4170 Our previous studies have shown that targeting FVIII expression to platelets (2bF8) can correct murine hemophilia A phenotype even in the presence of inhibitory antibodies. In the present study, we wanted to explore 1) whether platelets containing FVIII can act as an immunogen; and 2) whether platelet-derived FVIII can induce immune tolerance in a hemophilia A mouse model. To investigate whether platelets containing FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets to naïve FVIIInull mice weekly for 8 weeks. These platelets were between 30 to 50% of total platelets upon infusion and the levels of platelet-FVIII in the infused animals were 0.11 ± 0.01 mU/108 platelets (n = 6) one week after infusion. No anti-FVIII inhibitory antibodies were detected in the infused mice during the study course. All animals developed inhibitors following further challenged with recombinant human FVIII (rhFVIII) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger immune response in hemophilia A mice. To explore whether platelet-derived FVIII will act as an immunogen in the presence of primed spleen cells (from mice already producing inhibitory antibody), we co-transplanted splenocytes from highly immunized FVIIInull mice and bone marrow (BM) cells from 2bF8 transgenic mice into 400 cGy sub-lethal irradiated FVIIInull recipients. We monitored the levels of inhibitory antibodies in recipients for up to 8 weeks and found that inhibitor titers declined with time after transplantation. We then challenged co-transplantation recipients with rhFVIII and found that inhibitor titers in the control group co-transplantat of FVIIInull BM cells increased 103.55 ± 64.83 fold (n = 4), which was significantly more than the group receiving 2bF8 transgenic BM cells (14.34 ± 18.48, n = 5) (P <.05). The inhibitor titers decreased to undetectable in 40% of 2bF8 transgenic BM cells co-transplantation recipients even after rhFVIII challenge, indicating immune tolerance was induced in these recipients. To further explore the immune response in the lentivirus-mediated platelet-derived FVIII gene therapy of hemophilia A mice, we transduced hematopoietic stem cells from pre-immunized FVIIInull mice with 2bF8 lentivirus (LV) followed by syngeneic transplantation into pre-immunized lethally irradiated FVIIInull recipients and monitored the levels of inhibitor titers in recipients. After full BM reconstitution, platelet-FVIII expression was sustained (1.56 ± 0.56 mU/108platelets, n = 10), but inhibitor titers declined with time, indicating that platelet-derived FVIII does not provoke a memory response in FVIIInullmice that had previously mounted an immune response to rhFVIII. The t1/2 of inhibitor disappearance in 2bF8 LV-transduced recipients (33.65 ± 11.12 days, n = 10) was significantly shorter than in untransduced controls (66.43 ± 22.24 days, n = 4) (P <.01). We also transplanted 2bF8 LV-transduced pre-immunized HSCs into 660 cGy sub-lethal irradiated naïve FVIIInull mice. After BM reconstituted, recipients were assessed by platelet lysate FVIII:C assay and tail clip survival test to confirm the success of genetic therapy. Animals were then challenged with rhFVIII. Only 2 of 7 2bF8 LV-transduced recipients developed inhibitory antibodies (55 and 87 BU/ml), while all untransduced control developed high titer of inhibitors (735.50 ± 94.65 BU/ml, n = 4). In conclusion, our results demonstrate that 1) platelets containing FVIII are not immunogenic in hemophilia A mice; and 2) platelet-derived FVIII may induce immune tolerance in hemophilia A mice with or without pre-existing inhibitory antibodies. This tolerance induction would add an additional significant benefit to patients with platelet-derived FVIII gene therapy strategy because protein infusion could be administered in some special situations (e.g. surgery in which a greater levels of FVIII may be required) with minimized risk of inhibitor development. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Platelet Gene Therapy by Lentiviral Gene Delivery to Hematopoietic Stem Cells Restores Hemostasis and Induces Humoral Immune Tolerance in FIXnull Mice

Molecular Therapy ◽

10.1038/mt.2013.197 ◽

2014 ◽

Vol 22 (1) ◽

pp. 169-177 ◽

Cited By ~ 33

Author(s):

Yingyu Chen ◽

Jocelyn A Schroeder ◽

Erin L Kuether ◽

Guowei Zhang ◽

Qizhen Shi

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Gene Delivery ◽

Hematopoietic Stem Cells ◽

Immune Tolerance ◽

Hematopoietic Stem ◽

Humoral Immune

Download Full-text

Congenital Bleeding Disorders

Hematology ◽

10.1182/asheducation-2003.1.559 ◽

2003 ◽

Vol 2003 (1) ◽

pp. 559-574 ◽

Cited By ~ 7

Author(s):

Margaret E. Rick ◽

Christopher E. Walsh ◽

Nigel S. Key

Keyword(s):

Gene Transfer ◽

Immune Tolerance ◽

Hemophilia A ◽

Tolerance Induction ◽

Von Willebrand Disease ◽

Bleeding Disorders ◽

Immune Tolerance Induction ◽

Inhibitor Development ◽

History Of ◽

Von Willebrand

Abstract Both clinical and basic problems related to the congenital bleeding disorders continue to confront hematologists. On the forefront are efforts to bring genetic correction of the more common bleeding disorders such as hemophilia A to the clinic in a safe and accessible manner. A second issue, particularly for patients with hemophilia, is the development of inhibitors—questions of how they arise and how to prevent and treat these problems that confound otherwise very successful replacement therapy and allow patients to maintain normal lifestyles. A third issue is the continuing question of diagnosis and management of von Willebrand disease, the most common congenital bleeding disorder, especially in individuals who have borderline laboratory values, but have a history of clinical bleeding. In Section I, Dr. Christopher Walsh discusses general principles of effective gene transfer for the hemophilias, specific information about viral vectors and non-viral gene transfer, and alternative target tissues for factor VIII and factor IX production. He highlights information about the immune response to gene transfer and reviews data from the hemophilia gene transfer trials to date. The future prospects for newer methods of therapy such as RNA repair and the use of gene-modified circulating endothelial progenitors are presented as possible alternatives to the more traditional gene therapy approaches. In Section II, Dr. Nigel Key focuses on inhibitor development in patients with hemophilia A. He reviews the progress in our understanding of the risk factors and presents newer information about the immunobiology of inhibitor development. He discusses the natural history of these inhibitors and the screening, laboratory diagnosis, and treatment, including the use of different modalities for the treatment of acute bleeding episodes. Dr. Key also presents information about the eradication of inhibitors by immune tolerance induction and reviews recent information from the international registries regarding the status and success of immune tolerance induction. In Section III, Dr. Margaret Rick discusses the diagnosis, classification, and management of von Willebrand disease. Attention is given to the difficulty of diagnosis in patients with mild bleeding histories and borderline laboratory test results for von Willebrand factor. She presents the value of different laboratory assays for both diagnosis and classification, and she relates the classification of von Willebrand disease to the choice of treatment and to the known genetic mutations. Practical issues of diagnosis and treatment, including clinical cases, will be presented.

Download Full-text