- Email: [email protected]
343. Benchtop DNA Synthesizer: Oligo-Templated Polymerization (OTP)
DNA VECTOROLOGY & GENE TARGETING I DNA Vectorology & Gene Targeting I 341. Minimally Invasive Electroporation of Synthetic DNA Vaccines for the Induction CTL and Functional Humoral Immune Responses Against HIV
Megan Wise,1 Karuppiah Muthumani,1 Kate E. Broderick,2 Amir S. Khan,2 Janess Mendoza,2 Maria Yang,2 Jian Yan,2 Natalie Hutnick,1 Niranjan Y. Sardesai,2 David B. Weiner.1 1 University of Pennsylvania, Philadelphia, PA; 2Inovio, Blue Bell, PA. It is important to develop new approaches to HIV that generate both Functional antibody responses as well as CTL capable of killing diverse viruses. We have recently reported strong T cell and antibody responses induced in a humans using a synthetic HPV DNA vaccine delivered by a highly efficient EP (Bagarazzi et al STM 2012). Furthermore, in a study with the HVTN, recent clinical data from the HIV Vaccine Trials Network, HVTN-080 reported that CELLECTRA®-5P intramuscular electroporation (EP) of our synthetic Clade B consensus DNA HIV antigens (Env, Gag, and Pol) along with the plasmid adjuvant IL-12 generated strong antigen-specific cellular immune responses in humans resulting in a 90% response rate (Kalams et al 2013 MS in review). Here we build on these results by development of minimally invasive EP delivery technologies (MID-EP) to target the dermal tissue and by improved plasmid construct design of a collection of synthetic Consensus and center of tree (COT) immunogens. We report the ability of these approaches to generate strong binding, ADCC activity and neutralizing antibody (Ab) responses in guinea pigs, and rabbits as well as in preliminary NHP studies. We observed induction of invivo CTL/killing activity against 20 diverse HIV viruses including primary HIV isolates induced by the vaccine using a novel invivo killing assay. Furthermore we observe NAb titers engendered by immunization against a panel of 15 Tier-1 HIV viruses from Clades A-D in the range of 20 -200 in the Tzm-Bl neutralization assay. The magnitude of the humoral response was boosted to 20-1000 range using a MID-EP DNA prime-protein boost regimen. Our results suggest that study of construct design and selection by minimally invasive electroporation devices appears to be a relevant strategy for HIV DNA vaccinations in a prophylactic model targeting HIV. Combined with the design of novel HIV consensus based Env antigens these DNA-EP combination vaccines should be further investigated and have important implications for DNA vaccine candidates targeting HIV and other difficult pathogens.
342. An E3-14.7K Peptide That Promotes Microtubules-Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency
Lucie Pigeon,1 Cristine Gonçalves,1 David Gosset,1 Chantal Pichon,1 Patrick Midoux.1 1 Centre de Biophysique Moléculaire CNRS URP4301, Orléans, France.
Chemical vectors as cationic polymers and cationic lipids are promising alternative to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, we have exploited the interaction between E3-14.7K and FIP-1 to favour migration of pDNA along microtubules. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). In a previous work, we identified the P79-98 peptide responsible of the E3-14.7K/ FIP-1 interaction. In the present work, we conjugated this peptide S132
with pDNA and we demonstrated that it mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude of along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/ peGFP polyplexes enhance by a factor 2.5 - up to 76% - the number of transfected cells. Our results demonstrate for the first time that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.
343. Benchtop DNA Synthesizer: OligoTemplated Polymerization (OTP)
Adi Barzel,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University, Stanford, CA. Most of today’s vectors are built by combination of pre-existing genetic building blocks, limiting our ability to manipulate cellular activity. The technology for de novo synthesis of desired DNA sequences has been developed almost 40 years ago, but its pricing and production time still prevent it from serving as a true alternative to recombinant DNA technology. The limitations of current gene synthesis stem from its reliance on the assembly of pre-synthesized custom oligos. The pricing of gene synthesis is bound by the pricing of oligo-synthesis, because custom oligos must be produced specifically for each desired gene. Furthermore, current gene synthesis is sensitive to the high error rate and limited processivity of chemical oligo production, and therefore it requires costly and time consuming quality controls and assembly steps. We have developed a DNA synthesizer based on the novel paradigm of Oligo-Templated Polymerization (OTP, Figure 1). OTP utilizes a comprehensive array of short oligos comprising all possible sequence permutations. At each cycle a different oligo anneals to the 3’ end of a surface-bound growing chain, with the 5’ base of the oligo protruding and serving as template for subsequent polymerization. Elongation is followed by denaturation, allowing for a new cycle to begin using the next-in-line oligo. OTP has a miniscule error rate. Miniature oligos used in OTP have a much lower per-oligo error rate than do longer oligos used in current gene synthesis. A low rate of faulty oligos should have little effect on the OTP product as only a correct oligo can anneal and allow for elongation. Similarly, a growing chain not properly elongated will not anneal to oligos in subsequent cycles. Importantly, the same oligo array can be used for the synthesis of any desired sequence and many desired sequences. Long DNA molecules may be synthesized in a single continuous procedure. Thus, production costs and timelines should be minimized and OTP machines may become standard bench-top equipment. We have built the first OTP machine prototype using the BioMek FX robot. A biotinylated growing chain is bound to streptavidinylated beads in a reaction chamber. Each chamber of the oligo array in turn containes 64 10mers differing only in their three 3’ nucleotides (e.g. acggtagNNN). Allowing for annealing and Klenowmediated elongation steps at 230C and using alkaline denaturation, we have accurately synthesized the first 96 nucleotides of the GFP gene. We hope to demonstrate complete gene synthesis in the near future, and have begun developing costume OTP robotics. We believe OTP should serve as an enabling technology for the broad field of biology, including research, therapeutics and biotechnology.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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
Megan Wise,1 Karuppiah Muthumani,1 Kate E. Broderick,2 Amir S. Khan,2 Janess Mendoza,2 Maria Yang,2 Jian Yan,2 Natalie Hutnick,1 Niranjan Y. Sardesai,2 David B. Weiner.1 1 University of Pennsylvania, Philadelphia, PA; 2Inovio, Blue Bell, PA. It is important to develop new approaches to HIV that generate both Functional antibody responses as well as CTL capable of killing diverse viruses. We have recently reported strong T cell and antibody responses induced in a humans using a synthetic HPV DNA vaccine delivered by a highly efficient EP (Bagarazzi et al STM 2012). Furthermore, in a study with the HVTN, recent clinical data from the HIV Vaccine Trials Network, HVTN-080 reported that CELLECTRA®-5P intramuscular electroporation (EP) of our synthetic Clade B consensus DNA HIV antigens (Env, Gag, and Pol) along with the plasmid adjuvant IL-12 generated strong antigen-specific cellular immune responses in humans resulting in a 90% response rate (Kalams et al 2013 MS in review). Here we build on these results by development of minimally invasive EP delivery technologies (MID-EP) to target the dermal tissue and by improved plasmid construct design of a collection of synthetic Consensus and center of tree (COT) immunogens. We report the ability of these approaches to generate strong binding, ADCC activity and neutralizing antibody (Ab) responses in guinea pigs, and rabbits as well as in preliminary NHP studies. We observed induction of invivo CTL/killing activity against 20 diverse HIV viruses including primary HIV isolates induced by the vaccine using a novel invivo killing assay. Furthermore we observe NAb titers engendered by immunization against a panel of 15 Tier-1 HIV viruses from Clades A-D in the range of 20 -200 in the Tzm-Bl neutralization assay. The magnitude of the humoral response was boosted to 20-1000 range using a MID-EP DNA prime-protein boost regimen. Our results suggest that study of construct design and selection by minimally invasive electroporation devices appears to be a relevant strategy for HIV DNA vaccinations in a prophylactic model targeting HIV. Combined with the design of novel HIV consensus based Env antigens these DNA-EP combination vaccines should be further investigated and have important implications for DNA vaccine candidates targeting HIV and other difficult pathogens.
342. An E3-14.7K Peptide That Promotes Microtubules-Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency
Lucie Pigeon,1 Cristine Gonçalves,1 David Gosset,1 Chantal Pichon,1 Patrick Midoux.1 1 Centre de Biophysique Moléculaire CNRS URP4301, Orléans, France.
Chemical vectors as cationic polymers and cationic lipids are promising alternative to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, we have exploited the interaction between E3-14.7K and FIP-1 to favour migration of pDNA along microtubules. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). In a previous work, we identified the P79-98 peptide responsible of the E3-14.7K/ FIP-1 interaction. In the present work, we conjugated this peptide S132
with pDNA and we demonstrated that it mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude of along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/ peGFP polyplexes enhance by a factor 2.5 - up to 76% - the number of transfected cells. Our results demonstrate for the first time that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.
343. Benchtop DNA Synthesizer: OligoTemplated Polymerization (OTP)
Adi Barzel,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University, Stanford, CA. Most of today’s vectors are built by combination of pre-existing genetic building blocks, limiting our ability to manipulate cellular activity. The technology for de novo synthesis of desired DNA sequences has been developed almost 40 years ago, but its pricing and production time still prevent it from serving as a true alternative to recombinant DNA technology. The limitations of current gene synthesis stem from its reliance on the assembly of pre-synthesized custom oligos. The pricing of gene synthesis is bound by the pricing of oligo-synthesis, because custom oligos must be produced specifically for each desired gene. Furthermore, current gene synthesis is sensitive to the high error rate and limited processivity of chemical oligo production, and therefore it requires costly and time consuming quality controls and assembly steps. We have developed a DNA synthesizer based on the novel paradigm of Oligo-Templated Polymerization (OTP, Figure 1). OTP utilizes a comprehensive array of short oligos comprising all possible sequence permutations. At each cycle a different oligo anneals to the 3’ end of a surface-bound growing chain, with the 5’ base of the oligo protruding and serving as template for subsequent polymerization. Elongation is followed by denaturation, allowing for a new cycle to begin using the next-in-line oligo. OTP has a miniscule error rate. Miniature oligos used in OTP have a much lower per-oligo error rate than do longer oligos used in current gene synthesis. A low rate of faulty oligos should have little effect on the OTP product as only a correct oligo can anneal and allow for elongation. Similarly, a growing chain not properly elongated will not anneal to oligos in subsequent cycles. Importantly, the same oligo array can be used for the synthesis of any desired sequence and many desired sequences. Long DNA molecules may be synthesized in a single continuous procedure. Thus, production costs and timelines should be minimized and OTP machines may become standard bench-top equipment. We have built the first OTP machine prototype using the BioMek FX robot. A biotinylated growing chain is bound to streptavidinylated beads in a reaction chamber. Each chamber of the oligo array in turn containes 64 10mers differing only in their three 3’ nucleotides (e.g. acggtagNNN). Allowing for annealing and Klenowmediated elongation steps at 230C and using alkaline denaturation, we have accurately synthesized the first 96 nucleotides of the GFP gene. We hope to demonstrate complete gene synthesis in the near future, and have begun developing costume OTP robotics. We believe OTP should serve as an enabling technology for the broad field of biology, including research, therapeutics and biotechnology.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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