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Rational designing of new meganucleases as molecular scissors for genomic tailoring

Periodic Report Summary 1 - SMARTBREAKER (Rational designing of new meganucleases as molecular scissors for genomic tailoring.)

The purpose of SMARTBREAKER is to study the use of new highly specific engineered homoendonucleases (HEs) as a possible therapeutic tool to correct defects in different types of diseases. This requires in-depth understanding of their physicochemical properties to elucidate mechanisms underlying their protein-DNA recognition and their mode of action. The project can be divided in 4 phases that can be used to follow the progression of the proposal. For each phases a main goal is identified.
Phase description
Phase A’: Identification of new native HEs.
The main goal is the identification and determination of the DNA binding target of new HEs. Structural information of the new HE-DNA complexes will be obtained in phase C.
Phase A: Design of new HEs using the native scaffolds (also from A') with new DNA sequence specificity.
The main goal is to generate a library of plasmids expressing the new variants of HEs. Activity information will be obtained in phase B.
Phase B: Cell based screening.
The main goal is the screening of the library of HEs produced in phases As and the selection of the HEs able to cleave the DNA target. Structural information of the new HE-DNA complexes will be obtained in phase C. The new HEs will be delivered and tested for their ability to cleave DNA fragment of clinical interest in IPS cells in phase D.
Phase C: Characterization, crystallization and structural determination .
The main goal is the characterization, crystallization and structural determination of the HE-DNA complexes from phases As.
Phase D: Analysis of HEs repair in induced pluripotent stem cells.
The mail goal is the production of new tools that can be use in gene therapy.
Although a large number of HEs has been identified, the landscape of possible target sequences is still too much limited to cover the complexity of the whole eukaryotic genome. To solve this problem, we have generated HEs redesigned changing its recognition pattern towards a new DNA sequence. Using the I-CreI scaffold as a template we were able to generate a large number of variants recognizing a wide number of DNA sequences. According to that we have characterized 35 new I-CreI variants that recognize sequence targets involved in diseases ranging from sickle cell anaemia to neuromuscular disorders. Furthermore to find new HEs scaffolds to open new possibilities of recognition and cleavage, we have carried out the expression and purification of unknown HEs such as I-CvuI and I-MsoI.
According to the main goals in phases A-A’ of SMARTBREAKER we were able to obtain a Library of HEs with a number of new HEs, large enough to consider these phases of the project fully achieved.
According to phase B, the Library of HEs was tested in a cell based assays and the optimal binding site for each HEs was determined thus this phase can be consider fully achieved.
We performed biochemical and biophysical characterization of all new HEs. We analysed the molecular mass, the secondary structure the binding capacity and the cleavage rate of the HEs for their DNA targets. The high-throughput crystallization of HEs in complex with their DNA targets was performed. We were able to obtain 30 different structures of I-CreI variants in complex with the DNA and the structure of I-CvuI. In order to fully achieve the main goal of Phase C the characterization of the other 5 variants of I-CreI and I-MsoI need to be done.
Phase D involved analysis of the activity of the new HEs in induced pluripotent stem cell. The genes encoding for the I-CreI variants obtained from the previous phases will be introduced and expressed into the iPS cells using DNA transfection or viral vectors. Several I-CreI variants are already cloned into expression vectors in mammalian cells.
The main results obtained :
1) I-CreI Mutants Library :35 different I-CreI variants were generated and tested
2) Identification and partial characterization of I-CvuI and I-MsoI
• Non-specific protein–DNA interactions control I-CreI target binding and cleavage. Molina R, Redondo P, Stella S, Marenchino M, D'Abramo M, Gervasio FL, Epinat JC, Valton J, Grizot S, Duchateau P, Prieto J, Montoya G. Nucleic Acids Res. 2012 Aug;40(14):6936-45.
• Structure of the AvrBs3-DNA complex provides new insights into the initial thymine-recognition mechanism S. Stella, R. Molina, I. Yefimenko, J. Prieto, G. Silva, C. Bertonati, A. Juillerat, P. Duchateau and G. Montoya. Acta D 2013 (accepted )

The expected final results:
After characterizing the new I-CreI variants bound to their redesigned target (phase C) we will analyze all the in vivo, in vitro and structural data to find the trend in the direct pattern recognition between I-CreI and DNA in order to prepare the in silico HEs design software able to

a) provide a tool to know if a certain DNA sequence can be cleaved by an I-CreI HEs
b) provide information about the mutations needed in I-CreI to recognize that sequence
c) validated searching for a gene region that fulfils point.

After cloning all the variants of I-CreI into mammalian cloning vector we will test the efficiency of induces Homologues Recombination in iPS cells. Sensitive and quantitative assays will be performed to evaluate the HEs activity and the genes replacement. Also the current methods used to transfect the cells with the engineered HEs may not be ideal for clinical development. In the latest phase of SMARTBREAKER, we will try to design, produce and characterize novel transfection reagents and hybrid viral vectors able to carry a HE into the cells as a coding DNA or RNA, or as a protein.