- Email: [email protected]
345. Hybrid Flp-TALE and Cre-TALE Recombinases with Designed Target Specificity as Genome Engineering Tools
DNA VECTOROLOGY & GENE TARGETING I 345. Hybrid Flp-TALE and Cre-TALE Recombinases with Designed Target Specificity as Genome Engineering Tools Feng Li,1 Riddhi Shah,1 Eugenia Voziyanova,1 Yuri Voziyanov.1 School of Biosciences, Louisiana Tech University, Ruston, LA.
1
Figure 1- OTP: An oligo (Red) is selected from a comprehensive oligo array to anneal to the 3’ of the growing chain (Green) such that the oligo’s 5’end protrudes and serves as a template for subsequent elongation. Denaturation then allows for a new cycle to begin.
344. Nanoparticle-Mediated Gene Delivery for Rhodopsin-Associated Retinitis Pigmentosa
Zongchao Han,1 Rasha Makkia,1 Junjing Guo,1 Miles J. Merwin,1 Mark J. Cooper,2 Muna I. Naash.1 1 Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Copernicus Therapeutics, Inc., Cleveland, OH. Purpose: Retinitis pigmentosa (RP) due to mutations in the rhodopsin gene is one of the leading causes of inherited blindness affecting over 10% of all cases. In this study, we developed CK30PEG10k-compacted DNA nanoparticles (NPs) as non-viral gene replacement strategies and tested their efficiency in the rhodopsin knockout (RKO) mouse model. Methods: NPs carrying either the 1.1 kbp full length mouse opsin cDNA (7.5 kbp of vector in size, named NP-c) or 5.1 kbp genomic DNA (10.5 kbp of vector in size, named NP-g) containing all exons and introns, and both directed by the 500 bp endogenous opsin promoter (MOP). A matrix-attachment region (S/MAR) was cloned downstream of the termination signal to facilitate long-term expression. NPs were delivered subretinally into RKO pups at postnatal (P) day 3. Phenotypic rescue was evaluated functionally by electroretinogram (ERG), structurally by histology at the light and EM levels and by immunohistochemistry (IHC), and biochemically by Northern blot, qRT-PCR and western blot at postinjection days 3 (PI-3) and PI-30. Results: Compared with un-injected RKO eyes, treated eyes with either NP-c or NP-g demonstrated transgene expression that was evident on northern blot analysis and by IHC using antibody against rhodopsin. There was about 60% of WT levels of both Rho mRNA and protein expression at PI-3 for both vector injected animals, and about 40% in NP-c and 60% in NP-g of WT levels in mRNA at PI-30. However, there was about 10% in NP-c and 30% in NP-g of WT levels in protein expression at PI-30. No transgene expression was detected in uninjected or saline injected RKO mice at any time points. Interestingly, modest improvement of rod function (measured by ERG) was found in NP-g injected but not NP-c injected RKO eyes at PI-30. Further experiments are currently being performed to determine whether these NPs are capable of providing long-term gene expression and phenotypic improvement in RKO. Conclusions: Our results reveal that these NPs have the ability to transfer larger and more complex genetic expression cassettes for treating diseases that currently are not being addressed because of vector limitations. These NPs may be an attractive therapeutic strategy for the treatment of rhodopsin-associated RP.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
Successful gene therapy applications rely, in part, on the efficient, precise, and safe genome engineering approaches. These approaches can be used either to inactivate a gene or to integrate a gene into a desired genome locale or to replace a defective gene with its wildtype allele. The most efficient genome engineering approaches to inactivate genes are those that rely on DNA repair, for example, the ones that are based on zinc finger and TALE nucleases (ZFNs or TALENs). Several genome engineering approaches can be used to add or replace genes. These include the nuclease-based approaches and the approaches that are based on site-specific DNA recombinases. As we recently showed, the latter genome engineering approaches, which are currently utilizing primarily Flp/FRT, Cre/loxP, and phiC31/att recombination systems, can accomplish the task both efficiently and precisely if the dual recombinase-mediated cassette exchange (dual RMCE) is used as a gene delivery/replacement tool. Under optimal conditions, the efficiency of gene replacement by dual RMCE can reach about 50% of the transfected cells. The dual RMCE reaction depends on the pre-introduction of the recombination target sites into a genome locale of interest before the desired genetic manipulations can be carried out. This dependence can be lifted if variants of sitespecific recombinases are evolved to recognize pre-existing target-like sequences that flank a genome region of interest. We present here the results of the engineering of the hybrid Flp-TALE and Cre-TALE recombinases that recognize genomic sequences of interest. These task-specific variants of Flp and Cre recombinases can be paired to be used in the dual RMCE approach to replace the desired genome regions. We show that the engineered hybrid Flp-TALE and CreTALE recombinases can be used to mimic the replacement of the mutation that causes sickle-cell anemia in the model setting of CHO cells.
346. Permanent Genetic Modification of Dividing Cells Using Episomally Maintained S/MAR DNA Vectors and the Correction of a Cancer Phenotype in Renal Tumour Cells
Suet Ping Wong,1 Richard P. Harbottle.1 1 Section of Molecular Medicine, Imperial College London, London, United Kingdom.
The simple, stable and efficient application of episomal DNA vectors to genetically modify dividing cells without the risk of integration-mediated genotoxicity provides a valuable tool in cell biology research. Here, we demonstrate the utility of Scaffold/Matrix Attachment Region (S/MAR) DNA plasmid vectors to rapidly and simply generate novel genetically modified cell lines. In this study we utilize these vectors to model the restoration of a functional wild-type copy of the gene implicated in the renal cancer BirtHogg-Dube (BHD) in a cell-line (UOK257) derived from a BHD patient. Inactivation of the BHD gene, encoding a protein called folliculin (FLCN) has been shown to be involved in the development of sporadic renal neoplasia in BHD. Persistent genetic correction of UOK257 cells with an S/MAR-FLCN plasmid (UOK257-FS) restores FLCN expression and normalises downstream TGF signalling. We demonstrate that UOK257-FS cells show a reduced growth rate in vitro and suppressed xenograft tumour development in vivo, compared to the original FLCN-null UOK257 cell line. We also show that mTOR signaling in serum-starved FLCN-restored cells is differentially regulated compared to the FLCN-deficient cells indicating the complex role of FLCN in separate signalling pathways. The novel UOK257-FS cell line will be a useful tool for studying S133
1
Figure 1- OTP: An oligo (Red) is selected from a comprehensive oligo array to anneal to the 3’ of the growing chain (Green) such that the oligo’s 5’end protrudes and serves as a template for subsequent elongation. Denaturation then allows for a new cycle to begin.
344. Nanoparticle-Mediated Gene Delivery for Rhodopsin-Associated Retinitis Pigmentosa
Zongchao Han,1 Rasha Makkia,1 Junjing Guo,1 Miles J. Merwin,1 Mark J. Cooper,2 Muna I. Naash.1 1 Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Copernicus Therapeutics, Inc., Cleveland, OH. Purpose: Retinitis pigmentosa (RP) due to mutations in the rhodopsin gene is one of the leading causes of inherited blindness affecting over 10% of all cases. In this study, we developed CK30PEG10k-compacted DNA nanoparticles (NPs) as non-viral gene replacement strategies and tested their efficiency in the rhodopsin knockout (RKO) mouse model. Methods: NPs carrying either the 1.1 kbp full length mouse opsin cDNA (7.5 kbp of vector in size, named NP-c) or 5.1 kbp genomic DNA (10.5 kbp of vector in size, named NP-g) containing all exons and introns, and both directed by the 500 bp endogenous opsin promoter (MOP). A matrix-attachment region (S/MAR) was cloned downstream of the termination signal to facilitate long-term expression. NPs were delivered subretinally into RKO pups at postnatal (P) day 3. Phenotypic rescue was evaluated functionally by electroretinogram (ERG), structurally by histology at the light and EM levels and by immunohistochemistry (IHC), and biochemically by Northern blot, qRT-PCR and western blot at postinjection days 3 (PI-3) and PI-30. Results: Compared with un-injected RKO eyes, treated eyes with either NP-c or NP-g demonstrated transgene expression that was evident on northern blot analysis and by IHC using antibody against rhodopsin. There was about 60% of WT levels of both Rho mRNA and protein expression at PI-3 for both vector injected animals, and about 40% in NP-c and 60% in NP-g of WT levels in mRNA at PI-30. However, there was about 10% in NP-c and 30% in NP-g of WT levels in protein expression at PI-30. No transgene expression was detected in uninjected or saline injected RKO mice at any time points. Interestingly, modest improvement of rod function (measured by ERG) was found in NP-g injected but not NP-c injected RKO eyes at PI-30. Further experiments are currently being performed to determine whether these NPs are capable of providing long-term gene expression and phenotypic improvement in RKO. Conclusions: Our results reveal that these NPs have the ability to transfer larger and more complex genetic expression cassettes for treating diseases that currently are not being addressed because of vector limitations. These NPs may be an attractive therapeutic strategy for the treatment of rhodopsin-associated RP.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
Successful gene therapy applications rely, in part, on the efficient, precise, and safe genome engineering approaches. These approaches can be used either to inactivate a gene or to integrate a gene into a desired genome locale or to replace a defective gene with its wildtype allele. The most efficient genome engineering approaches to inactivate genes are those that rely on DNA repair, for example, the ones that are based on zinc finger and TALE nucleases (ZFNs or TALENs). Several genome engineering approaches can be used to add or replace genes. These include the nuclease-based approaches and the approaches that are based on site-specific DNA recombinases. As we recently showed, the latter genome engineering approaches, which are currently utilizing primarily Flp/FRT, Cre/loxP, and phiC31/att recombination systems, can accomplish the task both efficiently and precisely if the dual recombinase-mediated cassette exchange (dual RMCE) is used as a gene delivery/replacement tool. Under optimal conditions, the efficiency of gene replacement by dual RMCE can reach about 50% of the transfected cells. The dual RMCE reaction depends on the pre-introduction of the recombination target sites into a genome locale of interest before the desired genetic manipulations can be carried out. This dependence can be lifted if variants of sitespecific recombinases are evolved to recognize pre-existing target-like sequences that flank a genome region of interest. We present here the results of the engineering of the hybrid Flp-TALE and Cre-TALE recombinases that recognize genomic sequences of interest. These task-specific variants of Flp and Cre recombinases can be paired to be used in the dual RMCE approach to replace the desired genome regions. We show that the engineered hybrid Flp-TALE and CreTALE recombinases can be used to mimic the replacement of the mutation that causes sickle-cell anemia in the model setting of CHO cells.
346. Permanent Genetic Modification of Dividing Cells Using Episomally Maintained S/MAR DNA Vectors and the Correction of a Cancer Phenotype in Renal Tumour Cells
Suet Ping Wong,1 Richard P. Harbottle.1 1 Section of Molecular Medicine, Imperial College London, London, United Kingdom.
The simple, stable and efficient application of episomal DNA vectors to genetically modify dividing cells without the risk of integration-mediated genotoxicity provides a valuable tool in cell biology research. Here, we demonstrate the utility of Scaffold/Matrix Attachment Region (S/MAR) DNA plasmid vectors to rapidly and simply generate novel genetically modified cell lines. In this study we utilize these vectors to model the restoration of a functional wild-type copy of the gene implicated in the renal cancer BirtHogg-Dube (BHD) in a cell-line (UOK257) derived from a BHD patient. Inactivation of the BHD gene, encoding a protein called folliculin (FLCN) has been shown to be involved in the development of sporadic renal neoplasia in BHD. Persistent genetic correction of UOK257 cells with an S/MAR-FLCN plasmid (UOK257-FS) restores FLCN expression and normalises downstream TGF signalling. We demonstrate that UOK257-FS cells show a reduced growth rate in vitro and suppressed xenograft tumour development in vivo, compared to the original FLCN-null UOK257 cell line. We also show that mTOR signaling in serum-starved FLCN-restored cells is differentially regulated compared to the FLCN-deficient cells indicating the complex role of FLCN in separate signalling pathways. The novel UOK257-FS cell line will be a useful tool for studying S133