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185. Enhanced Neutralising Antibody Response to Bovine Viral Diarrhoea Virus (BVDV) Induced by BVDV DNA Vaccine Co-Expressing RIG-I Agonist in Cattle
INFECTIOUS DISEASES AND VACCINES relevant number of HSC for human transplantation. Here we have investigated an mRNA electroporation (EP) method using the MaxCyte GT system, which is capable of handling up to 300 million cells per manufacturing process. We show that this mRNA EP driven approach requires minimal ex vivo cell culture, supports levels of gene disruption ranging from 30-50% across multiple independent HSC donors, and can be performed at a scale of >100 million CD34+ HSC. Colony forming assays demonstrated that neither the EP process, nor the ZFN-driven CCR5 disruption, have any significant impact on the ability of edited HSC to differentiate in vitro. Importantly, infusion of these CCR5 ZFN-treated cells into humanized NSG mice showed that this EP driven genome editing procedure supported the ability of these stem cells to engraft and subsequently differentiate to reconstitute the human immune system. Analysis of immune cells in the peripheral blood and tissues of engrafted animals confirmed that the genome edited cell population was stably maintained 5 months post transplant. The use of mRNA is an attractive and potentially safer alternative to classical viral vectors, obviating the potential immunogenicity of viral components upon re-infusion of the gene-modified HSC into the patient. Furthermore, coupling ZFN technology with mRNA delivery eliminates the potential risk of insertional mutagenesis, while enabling permanent and targeted genetic modification of the HSC following only a markedly transient exposure to the ZFNs. Electroporation of mRNA thus provides an efficient delivery system for ZFN-driven CCR5 disruption in HSC. Importantly, this approach can be used to modify up to 300 million HSC in a single manufacturing process and can therefore be readily exploited for the clinical scale manufacture of CCR5-disrupted HSC for human trials.
184. Next Generation TALENs Mediate Efficient Disruption of the CCR5 Gene in Human HSCs
Nick Llewellyn,1 Jeffrey C. Miller,2 John Campo,2 Colin Exline,1 Orla Mulhern,1 Jill Henley,1 Jianbin Wang,2 Dmitry Guschin,2 Danny Xia,2 Irina Ankoudinova,2 Philip D. Gregory,2 Lei Zhang,2 Edward J. Rebar,2 Michael C. Holmes,2 Paula M. Cannon.1 1 Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA; 2Sangamo Biosciences, Richmond, CA. The failure of antiretrovirals to completely clear HIV infections, combined with their long-term toxicities, highlight the need for more sophisticated therapeutic approaches that can enable permanent cures. Seeking to replicate the cure of the ‘Berlin Patient’ following hematopoietic stem cell (HSC) transplantation from a CCR5delta32 donor, we are developing strategies for efficient CCR5 disruption in human HSCs using TALE-nucleases (TALENs). These proteins are a class of engineered endonucleases containing a DNA recognition domain from bacterial TALE proteins linked to the endonuclease domain of the FokI restriction enzyme. Two appropriately matched TALENs will dimerize at a target site and create a double-stranded break that leads to gene disruption via error-prone non-homologous end joining. The TALE DNA recognition domain contains a central core of 33-35 amino acid repeats that each recognize a single base of DNA through a repeat variable di-residue (RVD) motif at positions 12 and 13. The four RVDs that occur most commonly in natural TALEs (NI, HD, NN and NG) provide a simple code that in principle allows sequence-specific targeting modules to be constructed. We tested TALEN constructs targeted against two different sites in CCR5: an N-terminal site, previously shown to support potent anti-HIV activity in vivo (site 160), and a site approximating the location of naturally occurring CCR5delta32 mutation (site 546). TALEN pairs targeted to site 546 were found to be the most active, and in particular those that used a C-terminal truncation considerably shorter (17 residues) than that described in most TALE studies (63 residues). Gene modification levels using the shorter truncation were at least twice those achieved with the longer one, enabling modification of up to 65% of CCR5 S72
alleles in primary human HSCs. For applications beyond research, TALENs using this more potent architecture must also provide exquisite specificity genome wide. As a first step toward generating TALEN proteins with improved specificity we also tested the ability of alternative RVDs to support TALEN activity. Using a panel of previously characterized and novel RVDs with pre-defined base preferences we showed that it was possible to completely replace the natural code RVDs with alternative ones and retain comparable on target activity. Importantly, the alternative code RVDs displayed a reduction in off-target cleavage. Together these data provide a framework for the development of improved TALENs for genome editing applications in HSC.
185. Enhanced Neutralising Antibody Response to Bovine Viral Diarrhoea Virus (BVDV) Induced by BVDV DNA Vaccine Co-Expressing RIG-I Agonist in Cattle
Laila M. El-Attar,1 Olivia Anstaett,1 Carole Thomas,1 Jeremy Luke,2 James Williams,2 Joe Brownlie.1 1 Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, North Mymms, Hertfordshire, United Kingdom; 2Research & Development, Nature Technology Corporation, Lincoln, NE. DNA vaccination is extremely effective in inducing potent immunity in mice, however it suffers low potency in large animals. Increasing levels of DNA plasmid activation of innate immune signalling pathways is an approach to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection leading to activation of type I interferon (IFN). In an attempt to enhance the immune response induced by BVDV DNA vaccine in cattle, we expressed BVDV truncated E2 (E2t) and NS3 antigens based on bovine biased codons sequence into antibiotic free-plasmid vectors expressing RIG-I agonist with and without secretion signal respectively and designated E2t (co) and NS3 (co). E2t and NS3 non codon biased antigens designated E2t (nco) and NS3 (nco) were also inserted into pcDNA3 plasmid as positive control, whereas empty plasmids served as a negative control. To evaluate vaccine efficacy, five groups of five BVDV-free calves were intramuscularly injected with vaccine plasmid individually or co-administered. Sera were collected on a weekly basis post vaccination and post challenge for viral antigen specific and neutralisation antibody detection. Our results showed that vaccines co-expressing RIG-I agonist induced significantly higher E2 antigen specific antibody response (p < 0.05). Additionally, E2 augmented the immune response to NS3 when the two vaccines were co administered. Interestingly, on challenge day 4/5 calves vaccinated with E2t (co) and E2t (co) + NS3 (co) had high neutralising antibody titres, exceeding 1/240 required to achieve full protection compared to 1/5 in the control group. Based on our results we conclude that co-expression of RIG-I agonist with viral antigen could dramatically enhance DNA vaccine potency in cattle.
186. Preclinical Safety Evaluation of an Antibiotic-Free LAMP-1 Plasmid Allergy Immunotherapy
Teri L. Heiland,1 James A. Williams,2 William G. Hearl.1 1 Immunomic Therapeutics Inc, Rockville, MD; 2Nature Technology Corporation, Lincoln, NE. Nature Technology Corporation (NTC) developed minimal regulatory agency compliant antibiotic-free (AF) mammalian expression vectors incorporate and express a 150 bp RNA-OUT antisense RNA that represses expression of a host strain chromosomeencoded counter-selectable marker (SacB) which is toxic in the Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
184. Next Generation TALENs Mediate Efficient Disruption of the CCR5 Gene in Human HSCs
Nick Llewellyn,1 Jeffrey C. Miller,2 John Campo,2 Colin Exline,1 Orla Mulhern,1 Jill Henley,1 Jianbin Wang,2 Dmitry Guschin,2 Danny Xia,2 Irina Ankoudinova,2 Philip D. Gregory,2 Lei Zhang,2 Edward J. Rebar,2 Michael C. Holmes,2 Paula M. Cannon.1 1 Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA; 2Sangamo Biosciences, Richmond, CA. The failure of antiretrovirals to completely clear HIV infections, combined with their long-term toxicities, highlight the need for more sophisticated therapeutic approaches that can enable permanent cures. Seeking to replicate the cure of the ‘Berlin Patient’ following hematopoietic stem cell (HSC) transplantation from a CCR5delta32 donor, we are developing strategies for efficient CCR5 disruption in human HSCs using TALE-nucleases (TALENs). These proteins are a class of engineered endonucleases containing a DNA recognition domain from bacterial TALE proteins linked to the endonuclease domain of the FokI restriction enzyme. Two appropriately matched TALENs will dimerize at a target site and create a double-stranded break that leads to gene disruption via error-prone non-homologous end joining. The TALE DNA recognition domain contains a central core of 33-35 amino acid repeats that each recognize a single base of DNA through a repeat variable di-residue (RVD) motif at positions 12 and 13. The four RVDs that occur most commonly in natural TALEs (NI, HD, NN and NG) provide a simple code that in principle allows sequence-specific targeting modules to be constructed. We tested TALEN constructs targeted against two different sites in CCR5: an N-terminal site, previously shown to support potent anti-HIV activity in vivo (site 160), and a site approximating the location of naturally occurring CCR5delta32 mutation (site 546). TALEN pairs targeted to site 546 were found to be the most active, and in particular those that used a C-terminal truncation considerably shorter (17 residues) than that described in most TALE studies (63 residues). Gene modification levels using the shorter truncation were at least twice those achieved with the longer one, enabling modification of up to 65% of CCR5 S72
alleles in primary human HSCs. For applications beyond research, TALENs using this more potent architecture must also provide exquisite specificity genome wide. As a first step toward generating TALEN proteins with improved specificity we also tested the ability of alternative RVDs to support TALEN activity. Using a panel of previously characterized and novel RVDs with pre-defined base preferences we showed that it was possible to completely replace the natural code RVDs with alternative ones and retain comparable on target activity. Importantly, the alternative code RVDs displayed a reduction in off-target cleavage. Together these data provide a framework for the development of improved TALENs for genome editing applications in HSC.
185. Enhanced Neutralising Antibody Response to Bovine Viral Diarrhoea Virus (BVDV) Induced by BVDV DNA Vaccine Co-Expressing RIG-I Agonist in Cattle
Laila M. El-Attar,1 Olivia Anstaett,1 Carole Thomas,1 Jeremy Luke,2 James Williams,2 Joe Brownlie.1 1 Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, North Mymms, Hertfordshire, United Kingdom; 2Research & Development, Nature Technology Corporation, Lincoln, NE. DNA vaccination is extremely effective in inducing potent immunity in mice, however it suffers low potency in large animals. Increasing levels of DNA plasmid activation of innate immune signalling pathways is an approach to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection leading to activation of type I interferon (IFN). In an attempt to enhance the immune response induced by BVDV DNA vaccine in cattle, we expressed BVDV truncated E2 (E2t) and NS3 antigens based on bovine biased codons sequence into antibiotic free-plasmid vectors expressing RIG-I agonist with and without secretion signal respectively and designated E2t (co) and NS3 (co). E2t and NS3 non codon biased antigens designated E2t (nco) and NS3 (nco) were also inserted into pcDNA3 plasmid as positive control, whereas empty plasmids served as a negative control. To evaluate vaccine efficacy, five groups of five BVDV-free calves were intramuscularly injected with vaccine plasmid individually or co-administered. Sera were collected on a weekly basis post vaccination and post challenge for viral antigen specific and neutralisation antibody detection. Our results showed that vaccines co-expressing RIG-I agonist induced significantly higher E2 antigen specific antibody response (p < 0.05). Additionally, E2 augmented the immune response to NS3 when the two vaccines were co administered. Interestingly, on challenge day 4/5 calves vaccinated with E2t (co) and E2t (co) + NS3 (co) had high neutralising antibody titres, exceeding 1/240 required to achieve full protection compared to 1/5 in the control group. Based on our results we conclude that co-expression of RIG-I agonist with viral antigen could dramatically enhance DNA vaccine potency in cattle.
186. Preclinical Safety Evaluation of an Antibiotic-Free LAMP-1 Plasmid Allergy Immunotherapy
Teri L. Heiland,1 James A. Williams,2 William G. Hearl.1 1 Immunomic Therapeutics Inc, Rockville, MD; 2Nature Technology Corporation, Lincoln, NE. Nature Technology Corporation (NTC) developed minimal regulatory agency compliant antibiotic-free (AF) mammalian expression vectors incorporate and express a 150 bp RNA-OUT antisense RNA that represses expression of a host strain chromosomeencoded counter-selectable marker (SacB) which is toxic in the Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy