Project Gene_Transfer_Tolerance (Molecular basis of immune tolerance after gene transfer) | ANR - Agence Nationale de la Recherche ANR funded project | ANR - Agence Nationale de la Recherche

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ANR funded project

JCJC - SVSE 3 - Microbiologie, immunologie, infectiologie (JCJC SVSE 3)
Edition 2011


Gene_Transfer_Tolerance


Molecular basis of immune tolerance after gene transfer

Molecular basis of immune tolerance after gene transfer
Clear clinical success have now been obtained by gene therapy approaches. The patient's immune system may, however, reject the injected product perceived as foreign, and thus limit its effectiveness. This project aims to understand the basis of this rejection and determine how to overcome it to reach a state of immune tolerance.

Adverse immune response after gene transfer
Gene therapy treatments of various metabolic diseases and genetic disorders consist of vectorization of therapeutic transgenes in specific target tissues, such as microdystrophine in muscles of patients with Duchenne Muscular Dystrophy. This approach is based on de novo expression of a functional protein that is either absent or deficient in the patient. This implies that, often, no immune tolerance against the transgene product does exist and that a deleterious immune response is induced, limiting the effectiveness of the treatment. One of the hypothesis explaining the cellular immune response initiation is direct presentation of transgene epitopes by the transduced antigen presenting cells (APC). To test this hypothesis, vectors were modified to abolish transgene expression in cells of hematopoietic origin. Thus the sole cross-presentation was committed, i.e. presentation by APCs of exogenous antigens, coming from others cells. This strategy allowed us to characterize the molecular and cellular mechanisms leading to cross-presentation-induced tolerance.

Tools to dissect the direct and cross-presentation pathways
In order to study the transgene-specific T-cell response, a new chimeric transgene was constructed with known epitopes compatible with the C57Bl/6 genetic background, on which most of the transgenic and knockout animals are. Thus, epitopes of the HY male antigen were added to the Ovalbumin (OVA) transgene. In addition, recombinant adeno-associated vectors (rAAV) encoding cytosolic, membrane or secreted forms of the OVA were produced. Finally, were inserted into these vectors the target sequence of miR142-3p to prevent transgene expression in cells expressing this particular miR, i.e. cells of hematopoietic origin. Regarding the mouse models, we used various TCR transgenic mice directed against OVA (OT-I, OT-II) or the male HY antigen (Mata Hari, Marilyn) and knockout mice for some genes coding for molecules involved in antigen presentation (TAP, ERAP and IRAP KO). Thus, we set up a unique system in which only the direct presentation of the transgene product happened (WT vector into IRAP KO mice) or only the cross-presentation of this transgenic protein occured (mir-regulated vector into WT mice).

Results

By trying first to determine whether the cross-presentation of the rAAV-vectorized transgene was able to induce a state of tolerance against the transgene, we characterized several parameters related to the transgene and injection site that could modulate the decision immune response/tolerance. For example, cross-tolerance could be obtained only against cytosolic forms of the transgene, or the intradermal route seemed much more immunogenic than the intramuscular one. Dissection of the molecular components controlling transgene cross-presentation and the initiation of the cytotoxic immune response led to a totally unexpected observation, currently under investigations. Finally, we have been able to demonstrate that aging prevented the conversion of conventional T cells into regulatory T cells (Treg) and we started to characterize the role of these Treg in our cross-tolerance system.

Outlook

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Scientific outputs and patents

A number of results were validated and finalized in the form of international publications. Thus, three articles were published on the transgene characteristics allowing to induce or not cross-tolerance, as well as on the importance of aging. Moreover, a fourth article on the role of the injection route is currently under review and a fifth on the importance of Treg in these processes is in preparation.

Partners

INSERM UMR_S 1013 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION DE PARIS V

ANR grant: 280 000 euros
Beginning and duration: décembre 2011 - 36 mois

Submission abstract

Summary

Acquisition of immune tolerance to gene transfer products is a major issue for the success of novel therapeutic modalities in the field of gene therapy of rare monogenic disorders. This is of particularly high importance for therapeutic transgenes delivered in non-lymphoid tissues and detailed knowledge of the molecular and cellular pathways that control the initiation of cytotoxic immune responses (CD8+ T cell responses) is unknown. I wish here to dissect the molecular components which control the presentation of transgene product delivered in muscle tissue as well as the mechanisms leading to recruitment of regulatory T cell activities and sustained immune tolerance.

Using defined cell-associated antigens delivered with clinically relevant adeno-associated viral vector (AAV) systems, I will assess the contribution of the direct and indirect MHC class I presentation pathways leading to cross-tolerisation of CD8 T cell responses. For this, I will use engineered AAV vectors in which was inserted in 3’ of the transgene the target of microRNA142.3p (miRNA strongly expressed in the hematopoietic system) to restrict transgene expression in muscle tissues, avoiding direct expression in antigen presenting cells. This system allows us to characterize for the first time the contributions of the direct and indirect pathway of transgene product presentation in vivo. Using 1) mice genetically invalidated for different components of the direct and indirect MHC class I presentation pathways, 2) cellular ablation or transfer of key players such as regulatory T cells, and 3) novel immune-modulation approaches such as the anti-T cell mAb (anti-CD3) used in the clinics to reset tolerance in autoimmune type 1 diabetes, we will delineate the rules that govern the induction of tolerance to foreign transgenes delivered in peripheral tissues.

I believe that the fundamental knowledge acquired through this program will be important to develop novel tolerance induction modalities for both gene therapy and autoimmunity applications.


Publications of the team

Chappert P, et al. Antigen-specific Tregs impair CD8 T cell priming by blocking early T cell expansion. Eur J Immunol. (2010) 40:339-50
Saveanu L, et al. IRAP identifies an endosomal compartment required for MHC class I cross-presentation. Science. (2009). 325:213-7
Chappert P, et al. Antigen-driven interactions with dendritic cells and expansion of foxp3+ regulatory T cells occur in the absence of inflammatory signals. J Immunol. (2008) 180:327-34
Lorain S, Gross DA, et al. Transient immunomodulation allows repeated injections of AAV1 and correction of muscular dystrophy in multiple muscles. Mol Ther. (2008) 16:541-7
Firat E, et al. The role of endoplasmic reticulum-associated aminopeptidase 1 in immunity to infection and in cross-presentation. J Immunol. (2007) 178:2241
Gross DA, et al. Simple conditioning with mono-specific CD4+CD25+ regulatory T cells for bone marrow engraftment and tolerance to multiple gene products. Blood. (2006) 108:1841-8
Saveanu L, et al. Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat Immunol. (2005) 6:689
Saveanu L, et al. Dendritic cells: open for presentation business Nat Immunol. (2005) 6:7 Review
Gross DA, et al. High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy. J Clin Invest. (2004) 113:425-33
Gross DA, et al. CD4+CD25+ regulatory T cells inhibit immune-mediated transgene rejection. Blood. (2003) 102:4326-8

 

ANR Programme: JCJC - SVSE 3 - Microbiologie, immunologie, infectiologie (JCJC SVSE 3) 2011

Project ID: ANR-11-JSV3-0005

Project coordinator:
Monsieur David A. GROSS (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION DE PARIS V)
david.gross@nullinserm.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.