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428. Sensitive Methods To Detect Mutations Induced by Meganucleases
DNA VECTOROLOGY & GENE TARGETING - I 427. Passive Topical Administration of Nucleic Acids to Targeted Skin Cells Via Sub-50 Nanometer Capsules
Gretchen M. Unger,1 Martha Fuentes de Albin,2 Roger L. Kaspar,3 Betsy T. Kren,2 Diane K. Tobolt,1 Michael P. Murtaugh.2 1 GeneSegues, Inc., Chaska, MN; 2University of Minnesota, Minneapolis, MN; 3TransDerm, Inc., Santa Cruz, CA.
Passive topical administration of nucleic acid drugs and vaccines is a desirable goal for patient-friendly manipulation of target cells in the skin. However, two major delivery challenges that have impeded topical nucleic acid development are 1) penetrating the stratum corneum, and 2) avoiding endosomal entrapment and degradation. GeneSegues has developed a novel non-viral drug delivery technology, sub-50 nanometer (s50) crystallized capsules, for intact delivery of nucleic acids to intracellular sites in target cells. s50 capsules are directed to specific tissue and cells by formulating appropriate targeting proteins, peptides or antibody ligands as the crystallized capsule shell. The novel size and structure of s50 capsules allows for efficient penetration of the stratum corneum and, by co-opting the non-endosomal pathway referred to as lipid rafts, intact delivery of nucleic acid cargo to the nucleus and cytoplasm of the target cell. Here we show in pilot studies using pig dermis organ culture as an established and relevant model for human skin, transcutaneous passage of plasmid-bearing s50 capsules with cell-specific targeting. In these studies, we compared fibronectin, hyaluronan and tenascin as shell targeting components. We then extended these studies of passive cutaneous delivery, using plasmid-bearing, fibronectin-coated s50 capsules, to in vivo timecourse studies in murine paws where stratum corneum layers in footpads are approximately 50 um thick, demonstrating uniform transfection of underlying basal keratinocytes. In a further extension, using hyaluronan-coated s50 capsules together with a novel adjuvant, we demonstrate a functional response to model antigen in studies of passive topical DNA vaccine adminstration in weanling pigs. Skin is a powerful immune organ, not conveniently addressed by current methods of vaccine administration, e.g., injection, gene gun, or electroporation. We found improvement in mucosal and cell-mediated immune responses, relative to intradermal protein antigen injection that was biased towards distal and draining nodal lymphoctyes, demonstrating a functional response in a clinically relevant model to topical administration of nucleic acids using s50 capsules. We expect that with further characterization, these studies may provide a solid framework for further development of s50mediated cutaneous delivery of nucleic acid drugs and vaccines. Grant acknowledgments: NIH R43 CA119556, R43 AI60268
428. Sensitive Methods To Detect Mutations Induced by Meganucleases
Jacques P. Tremblay,1 Joël Rouseau,1 Pierre Chapdelaine,1 Sébastien Boivert,1 Luciana Almeida,1 Jacques Corbeil,1 Alexandre Montpetit,2 Frédéric Pâques.3 1 Médecine Moléculaire, Université Laval, Québec, QC, Canada; 2 Genome Quebec Innovation Center, McGill University, Montréal, QC, Canada; 3Cellectis Genome Surgery, Cellectis S.A, Romainville, France. Several hereditary diseases are due either to a nonsense codon or to a frame shift mutation. Meganucleases (MGNs) are enzymes, which can be engineered to induce double strand break at a specific DNA sequence. These breaks are repaired by Homologous Recombination (HR) or by Non Homologous End Joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. We have tested three methods to detect, characterize and quantify the frequency of the indels produced by various MGNs targeting the Rag1 (Recombination activating gene 1) or the dystrophin gene. The Surveyor enzyme method was not very sensitive and permitted at best to determine Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
whether abundant indels had been produced. For the other two methods, the DNA region targeted by the MGN was amplified by PCR. For one of them, the subtractive colony hybridization, the amplicons were cloned in the pDrive plasmid and bacteria colonies were grown. The colonies still containing the non-mutated sequences were detected by hybridization with a radioactive oligonucleotide complementary to the wild type sequence. Only the non-radioactive colonies were sequenced to identify indels. For the third method, called deep sequencing, the PCR primers include the adapters for Illumina deep sequencing, a multiplexing marker and a sequence to amplify the target gene. Roughly 15,000 to 65,000 amplicons were sequenced on the Illumina platform for each experimental condition. Indels of a variable number of base pairs were obtained. Some of these indels changed the reading frame by 1 or 2 bp while other did not. Different MGNs produced micro-deletions with different size distributions. These experiments are thus a proof of principle that MGNs, adequately engineered to target appropriate exon sequences should be able to: 1) restore the normal reading of a gene with a frame shift mutation by inducing indels with the appropriate number of bp; 2) delete a non-sense codon while maintaining the normal reading frame by inducing micro-deletions, which are multiple of 3 bp and 3) knockout a gene by inducing indels, which are not a multiple of 3 bp.
429. Combining Linear Expression Cassette Technology with Novel Electroporation Techniques To Deliver Influenza DNA Vaccines
Xuefei Shen,1 Feng Lin,1 Gary Kobinger,2 Elizabeth E. Murray,2 Kate E. Broderick,1 Niranjan Y. Sardesai.1 1 Research and Development, Inovio Pharmaceuticals, San Diego, CA; 2Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada.
Alternative DNA vaccine constructs such as fully synthetic linear expressing cassettes (LEC) offer the advantage of accelerated manufacturing techniques as well as the lack of both antibiotic resistance genes and bacterial contaminants. The accelerated speed of LEC manufacture makes this technology a possible future vaccination strategy for pandemic influenza outbreaks. To assess immunogenicity and efficacy of EP-enhanced LEC delivery in mice, we designed and tested two antigenic influenza proteins. DNA to vaccinate mice was produced traditional DNA plasmid form or as LEC’s to express both influenza antigens. Both forms of DNA vaccines were delivered using the newly developed minimally-invasive electroporation (EP) device (MID-II). Previously, we reported on this novel concept of DNA delivery to dermal tissue by MID-II powered using low voltage parameters. In addition to enhancing the delivery of traditional plasmid DNA vaccines, this device may also offer a safe, tolerable and efficient method to administer LEC’s. Strong CTL and antibody responses were induced by the LEC versions of the DNA vaccine. When challenged with A/Canada/AB/RV1532/2009 viruses, mice immunized with LEC encoding the M2 and NP antigens recovered significantly faster than naïve or mice immunized ID without EP. Mice immunized with either plasmid DNA or an equal-molar doses of LEC encoding the M2 and NP antigens showed up to 100% survival following a lethal (100 x LD50) challenge of the heterologous and highly pathogenic H5N1 influenza virus (A/Vietnam/1203/04). These results supports the further development of genetic vaccines based on the combination of LEC and MID-II delivery technologies.
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Gretchen M. Unger,1 Martha Fuentes de Albin,2 Roger L. Kaspar,3 Betsy T. Kren,2 Diane K. Tobolt,1 Michael P. Murtaugh.2 1 GeneSegues, Inc., Chaska, MN; 2University of Minnesota, Minneapolis, MN; 3TransDerm, Inc., Santa Cruz, CA.
Passive topical administration of nucleic acid drugs and vaccines is a desirable goal for patient-friendly manipulation of target cells in the skin. However, two major delivery challenges that have impeded topical nucleic acid development are 1) penetrating the stratum corneum, and 2) avoiding endosomal entrapment and degradation. GeneSegues has developed a novel non-viral drug delivery technology, sub-50 nanometer (s50) crystallized capsules, for intact delivery of nucleic acids to intracellular sites in target cells. s50 capsules are directed to specific tissue and cells by formulating appropriate targeting proteins, peptides or antibody ligands as the crystallized capsule shell. The novel size and structure of s50 capsules allows for efficient penetration of the stratum corneum and, by co-opting the non-endosomal pathway referred to as lipid rafts, intact delivery of nucleic acid cargo to the nucleus and cytoplasm of the target cell. Here we show in pilot studies using pig dermis organ culture as an established and relevant model for human skin, transcutaneous passage of plasmid-bearing s50 capsules with cell-specific targeting. In these studies, we compared fibronectin, hyaluronan and tenascin as shell targeting components. We then extended these studies of passive cutaneous delivery, using plasmid-bearing, fibronectin-coated s50 capsules, to in vivo timecourse studies in murine paws where stratum corneum layers in footpads are approximately 50 um thick, demonstrating uniform transfection of underlying basal keratinocytes. In a further extension, using hyaluronan-coated s50 capsules together with a novel adjuvant, we demonstrate a functional response to model antigen in studies of passive topical DNA vaccine adminstration in weanling pigs. Skin is a powerful immune organ, not conveniently addressed by current methods of vaccine administration, e.g., injection, gene gun, or electroporation. We found improvement in mucosal and cell-mediated immune responses, relative to intradermal protein antigen injection that was biased towards distal and draining nodal lymphoctyes, demonstrating a functional response in a clinically relevant model to topical administration of nucleic acids using s50 capsules. We expect that with further characterization, these studies may provide a solid framework for further development of s50mediated cutaneous delivery of nucleic acid drugs and vaccines. Grant acknowledgments: NIH R43 CA119556, R43 AI60268
428. Sensitive Methods To Detect Mutations Induced by Meganucleases
Jacques P. Tremblay,1 Joël Rouseau,1 Pierre Chapdelaine,1 Sébastien Boivert,1 Luciana Almeida,1 Jacques Corbeil,1 Alexandre Montpetit,2 Frédéric Pâques.3 1 Médecine Moléculaire, Université Laval, Québec, QC, Canada; 2 Genome Quebec Innovation Center, McGill University, Montréal, QC, Canada; 3Cellectis Genome Surgery, Cellectis S.A, Romainville, France. Several hereditary diseases are due either to a nonsense codon or to a frame shift mutation. Meganucleases (MGNs) are enzymes, which can be engineered to induce double strand break at a specific DNA sequence. These breaks are repaired by Homologous Recombination (HR) or by Non Homologous End Joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. We have tested three methods to detect, characterize and quantify the frequency of the indels produced by various MGNs targeting the Rag1 (Recombination activating gene 1) or the dystrophin gene. The Surveyor enzyme method was not very sensitive and permitted at best to determine Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
whether abundant indels had been produced. For the other two methods, the DNA region targeted by the MGN was amplified by PCR. For one of them, the subtractive colony hybridization, the amplicons were cloned in the pDrive plasmid and bacteria colonies were grown. The colonies still containing the non-mutated sequences were detected by hybridization with a radioactive oligonucleotide complementary to the wild type sequence. Only the non-radioactive colonies were sequenced to identify indels. For the third method, called deep sequencing, the PCR primers include the adapters for Illumina deep sequencing, a multiplexing marker and a sequence to amplify the target gene. Roughly 15,000 to 65,000 amplicons were sequenced on the Illumina platform for each experimental condition. Indels of a variable number of base pairs were obtained. Some of these indels changed the reading frame by 1 or 2 bp while other did not. Different MGNs produced micro-deletions with different size distributions. These experiments are thus a proof of principle that MGNs, adequately engineered to target appropriate exon sequences should be able to: 1) restore the normal reading of a gene with a frame shift mutation by inducing indels with the appropriate number of bp; 2) delete a non-sense codon while maintaining the normal reading frame by inducing micro-deletions, which are multiple of 3 bp and 3) knockout a gene by inducing indels, which are not a multiple of 3 bp.
429. Combining Linear Expression Cassette Technology with Novel Electroporation Techniques To Deliver Influenza DNA Vaccines
Xuefei Shen,1 Feng Lin,1 Gary Kobinger,2 Elizabeth E. Murray,2 Kate E. Broderick,1 Niranjan Y. Sardesai.1 1 Research and Development, Inovio Pharmaceuticals, San Diego, CA; 2Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada.
Alternative DNA vaccine constructs such as fully synthetic linear expressing cassettes (LEC) offer the advantage of accelerated manufacturing techniques as well as the lack of both antibiotic resistance genes and bacterial contaminants. The accelerated speed of LEC manufacture makes this technology a possible future vaccination strategy for pandemic influenza outbreaks. To assess immunogenicity and efficacy of EP-enhanced LEC delivery in mice, we designed and tested two antigenic influenza proteins. DNA to vaccinate mice was produced traditional DNA plasmid form or as LEC’s to express both influenza antigens. Both forms of DNA vaccines were delivered using the newly developed minimally-invasive electroporation (EP) device (MID-II). Previously, we reported on this novel concept of DNA delivery to dermal tissue by MID-II powered using low voltage parameters. In addition to enhancing the delivery of traditional plasmid DNA vaccines, this device may also offer a safe, tolerable and efficient method to administer LEC’s. Strong CTL and antibody responses were induced by the LEC versions of the DNA vaccine. When challenged with A/Canada/AB/RV1532/2009 viruses, mice immunized with LEC encoding the M2 and NP antigens recovered significantly faster than naïve or mice immunized ID without EP. Mice immunized with either plasmid DNA or an equal-molar doses of LEC encoding the M2 and NP antigens showed up to 100% survival following a lethal (100 x LD50) challenge of the heterologous and highly pathogenic H5N1 influenza virus (A/Vietnam/1203/04). These results supports the further development of genetic vaccines based on the combination of LEC and MID-II delivery technologies.
S165