Deliverables

D1

Selection of engineered nucleases pairs and cognate donor-IDLVs targeting the IL2RG gene with high efficiency and specificity. Validate the gene targeting protocol in xenotransplant repopulation studies and establish the efficacy and safety profile of gene correction versus gene replacement in a humanized SCID-X1 mouse model. Provide proof-of-efficacy of disease correction in SCID-X1 HSPCs  by IL2RG through an efficient gene-targeting protocol on HSPC.

D1.1: Schiroli et al. manuscript under revision

D1.2: Schiroli et al. manuscript under revision

D1.3: Genovese et al., Nature 2014

D1.4: Pyrimidoindole derivatives increases HSPC expansion and repopulation in NSG mice

D1.5: Conditioning regimen before HSPC infusion required to avoid the risk of thymic lymphoma.

D1.6: IL2RG corrected cells persist long term in mice, sustain lymphopoiesis and partially correct the disease.

D2

Development of TCR addition vectors against human tumor antigens and identification of the optimal T-cell subset for TCR editing. Development of a TCR gene editing protocol compatible with clinical testing and preclinical validation of TCR gene edited cells. Analysis of TCR repertoire in donor cells and gene edited cells with database for TCR repertoire.

D2.1: Retroviral vectors expressing melanoma and leukemia specific TCR and Becattini et al . Science 2015

D2.2: Oliveira et al., Sci Transl Med. 2015

D2.3: Mastaglio et al, manuscript under revision

D2.4: Mastaglio et al, manuscript under revision

D2.5: RNA-based TCR deep sequencing approach and Oliveira et al., Sci Transl Med. 2015

D3

E. Coli Pre-Master Cell Banks (Pre-MCB) and plasmid production for the plasmids encoding the nucleases for WP1 and for WP2. Optimized protocols for mRNA production and RD-MolPack for IDLV-IL2RG production.Large scale optimized gene-targeting protocols in HSPC and T-cells for the two proposed gene therapy applications.one full-scale lot of gene-modified cells for each proposed gene therapy application and GLP studies of toxicity and biodistribution of IL2RG and TCR in NSG mice.

D3.1: Production of large stocks of plasmids encoding the specific ZFN pairs

D3.2: Production and purification of research- and GMP-grade plasmids and IL2RG IDLV at medium-large scale

D3.3: In vitro transcription and dHPLC purification of medium-large scale ZFN mRNAs targeting human IL2RG gene

D3.4: Production of IL2RG IDLV in small/medium scale

D3.6: Large-scale lot of CD34+ IL2RG gene-targeting

D3.8: Preclinical bio-distribution study in a xenotransplantation NSG mouse model of IL2RG corrected HSPCs and Schiroli et al., under revision.

D4

Project website, promotion of SUPERSIST results to the general public. Production of intellectual and industrial knowledge management report. Two projects for exploitation & business plan and dissemination & marketing material. SUPERSIST sponsored session at ESGCT Annual meeting European Workshop.

D5

Project Management Manual.