- Email: [email protected]
682. Applied In Silico Engineering of Factor VIII-AAV Transgene Cassettes
AAV Vector Development, Production and Application II Furthermore, the enhancer screening approach may be useful for rAAV applications in other organs that seek to deliver large transgenes that approach the packing capacity of rAAV.
681. Increasing Insulin Activity During AAV (Adeno-Associated Virus) Administration to Muscle Improves Gene Transfer in Mice
Sean M. Carrig,1 Enoch Bijjiga,1 Ashley T. Martino.1 Pharmaceutical Sciences, St John’s University, Queens, NY.
1
25+ years of AAV (adeno-associated virus) gene therapy research has resulted in optimized: 1- delivery routes, 2- vectors, 3- genomes and 4- immunosuppression. These studies have yielded the first westernized AAV gene therapy drug to treat LPLD (lipoprotein Lipase Deficiency) and the promise of future AAV gene therapy drugs to treat hemophilia B and other genetic disorders. Interestingly, we have little understanding of how hormones regulate these AAV drug / future drug candidates. Our studies show that hormone management has a place in improving AAV gene therapy. Since liver and skeletal muscle are strong responders to insulin and these tissues are common targets for these AAV treatments, our studies focused on how insulin regulates AAV gene therapy. We tested insulin supplementation in: HepG2 (liver), differentiated myocytes, HEK293 (kidney) and A549 (lung epithelial). HepG2 and myocytes showed elevated INSR (insulin receptor) mRNA levels (6 to 9-fold respectively) compared to HEK293 and A549 cells. The elevated INSR levels paralleled a significant 2.5 and 3 fold increase in AAV2-CMV-LacZ (M.O.I of 5e3)gene transfer to HepG2 and myocytes respectively when media was supplemented with insulin. Insulin enhanced gene transfer was not seen in HEK293 and A549 cells. Additionally, using HI glucose (4.5 g/L) and LOW glucose (1.0 g/L) medias, we showed that these results were not influenced by glucose availability. When comparing INSR levels in C57BL/6 mouse tissue, liver and skeletal muscle showed a 10-Fold and 22-Fold increase respectively compared to lung tissue. In these mice, intra-muscular (I.M.) gene delivery using AAV1-CMV-schFIX (dose: 3.0e10 v.g.) was significantly improved 4 to 5 fold (figure 1) using insulin augmentation protocols: either a subcutaneous insulin pellet which released 4 U/kg per day or a high carbohydrate (70%) diet.
The insulin augmentation protocols began 1.0 hr prior to AAV administration and continued for 4 weeks after. AAV delivery occurred during the window of a significant drop (3 fold) in blood glucose indicating an increase in insulin activity. The glucose levels had returned to normal 7 days later even though the insulin augmentation protocols continued for 4 weeks likely due to antagonistic feedback responses to long-term hyperinsulinemic conditions (i.e. cortisol release). The insulin augmentation protocols only demonstrated an increase in insulin activity at the onset of vector delivery but not after, yet elevated hFIX levels were sustained throughout the study, even after the insulin augmentation protocols ended. This indicates that insulin activity does not impact sustained transgene expression and may only regulate AAV uptake, intracellular trafficking or both. Data from liver directed studies using AAV8-CMV-schFIX are pending.
682. Applied In Silico Engineering of Factor VIIIAAV Transgene Cassettes
Harrison C. Brown,1 Eli J. Fine,2 Ernest T. Parker,1 H. Trent Spencer,1 Christopher B. Doering.1 1 Emory University, Atlanta, GA; 2Georgia Institute of Technology, Atlanta, GA.
Efforts to develop a clinical adeno-associated virus (AAV) vector product for hemophilia A have been hampered by many factors including low-level factor VIII (FVIII) expression, inefficient vector manufacture, and dose limiting vector toxicity. We recently described pre-clinical testing of a 5.86kb AAV2/8 vector encoding a bioengineered FVIII transgene (ET3) that confers enhanced FVIII secretion efficiency. FVIII-deficient (hemophilia A) mice treated at vector doses ranging from 5e11-2e13 vp/kg were corrected of their FVIII deficiency and bleeding phenotype. However due its oversized genome, the vector suffered from low titer manufacture and substantial inter-particle heterogeneity. Although the packaging capacity of AAV vectors is debated, vector genome sizes 4.7-5.0kb are manufactured to higher yield and consistency than those exceeding 5.0kb. The B-domain deleted FVIII coding sequence is 4.4kb, and with the addition of necessary viral and regulatory control elements, Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
S271
Gene Editing and Gene Regulation III FVIII-AAV genomes exceed the packaging capacity of the virus by 10-25%. We therefore sought to engineer a FVIII-AAV genome <5.0kb in length allowing for both enhanced FVIII expression and efficient packaging. Although ET3 already exhibits enhanced secretion over human FVIII due to the incorporation of specific non-native amino acids, others have shown synonymous codon optimization to benefit hepatic FVIII-AAV expression as well. Therefore, we examined the codon usage of 43 highly expressed, liver-specific genes and used the resulting information to generate a novel codon usage table. Using this table, we synthesized codonoptimized versions of both BDD human FVIII and ET3, the former of which demonstrated 3-fold superior expression in vitro. Likewise, this novel codon optimization algorithm provided the ET3-AAV transgene cassette with a 4-fold in vitro and 14-fold in vivo enhancement of expression. However, the large size of the codon optimized ET3AAV genome remained incompatible with efficient viral vector packaging. To address this design limitation, we used a combinatorial transcription factor binding site assembly approach to create a panel of liver-specific promoters ranging in size from 62-172 bases. These promoters represent a 30-90% size reduction over currently utilized liver specific promoters such as HLP and HCR-hAAT, which range in size from 250 to over 700 bases. Critically, a small subset of these promoters drive comparable transgene expression levels and specificity to that observed with HLP and HCR-hAAT. As a last design change, we eliminated nonessential viral genomic DNA and cloning remnants that are present in many earlier AAV designs. This removed an additional 80 bases. Collectively, these approaches enabled the development of a high expression FVIII-AAV genome of only 4.8kb in length. The exceptionally small size of this FVIII transgene may allow for the addition of desirable regulatory control features such as efficient introns or scaffold matrix attachment regions that could provide additional benefit. Furthermore, these technologies should be generalizable and likely enabling to other liver-directed AAV gene therapies.
683. In Vivo Expansion of Hepatocytes with Targeted rAAV Integration Results in a >100-Fold Increase of Transgene Expression
Sean Nygaard, Adi Barzel, Annelise Haft, Mark A. Kay, Markus Grompe.1 1 Cell and Developmental Biology and Pediatrics, Oregon Health and Science University, Portland, OR; 2Pediatrics and Genetics, Stanford University, Stanford, CA. 1
2
1
2
The recent development of promoter-less transgene integration technology using 2A fusions promises enhanced safety over traditional gene therapies. These vectors target therapeutic transgenes to loci for highly expressed endogenous genes through homologous recombination. Due to the inherent low frequency of targeted integrations, transgene expression is limited and requires high vector doses. Here we describe a modified 2A fusion integration vector that facilitates in vivo selection and expansion of gene-targeted hepatocytes. In mice treated with this vector, factor IX transgene expression increased over 400-fold during selection. Selection was achieved by incorporating into the integration vector a genetic element that rescues gene-targeted hepatocytes from induced hepatotoxic conditions. Blocking Fah (fumarylacetoacetate hydrolase) leads to hepatotoxic accumulation of the enzyme’s substrate fumarylacetoacetate (FAA). This toxicity can be rescued by inhibiting Hpd, an enzyme upstream of Fah. In Fah-deficient mice, hepatotoxity is readily manipulated by administering or withdrawing the drug NTBC which inhibits Hpd. Alternately, Hpd can be blocked with a shRNA. We’ve previously shown that in Fah-deficient mice,
S272
hepatocytes transduced with an Hpd-specific shRNA have a selective advantage when NTBC is withdrawn, leading to clonal expansion of transduced cells. This Hpd-specific shRNA was incorporated into the original albumin-targeting 2A integration vector as an shRNAmir. To prevent off-target expression, the shRNAmir was inserted without a promoter into an intron of the vector’s 3’ albumin homology arm. The resulting AAV-8 vector was administered to Fah-deficient neonates (P0, n=8) by facial vein injection at a dose of 4e11 Vg. At 4 weeks of age, plasma hF-IX levels measured 167 +/- 65 ng/ml. The mice were then cycled off NTBC to select for hepatocytes rescued through expression of the Hpd shRNAmir. Plasma hF-IX levels increased exponentially, reaching 46,000 +/- 5,000 ng/ml after 11 weeks of NTBC cycling (n=4). Three of the treated mice were cycled off NTBC for just 3 weeks, then maintained on NTBC for an additional 6 weeks. hF-IX levels remained steady at 400 - 1100 ng/ml, suggesting the integrated sequences were not toxic to hepatocytes. Control mice treated with the original albumin integration vector (lacking the HPD shRNAmir) showed no increase in plasma hF-IX levels during NTBC cycling. Administration of the selectable AAV to adult mice also resulted in increased hF-IX during NTBC withdrawal. Current efforts aim to replicate selection in wild-type mice using pharmacological inhibition of Fah. In summary, 2A fusion-based integration vectors promise significant safety advantages over traditional gene targeting methods. We’ve shown that incorporating selectable genetic elements into these vectors facilitates highly robust transgene expression which can be modulated in vivo. Selectable vectors could allow significantly lower vector doses, thereby enhancing the safety of such therapies even further.
Gene Editing and Gene Regulation III 684. Long-Term, Multilineage Engraftment of Zinc Finger Nuclease-Edited Hematopoietic Stem Cells in Nonhuman Primates
Christopher W. Peterson,1 Jianbin Wang,3 Patricia Polacino,2 Michael C. Holmes,3 Shiu-Lok Hu,2 Philip D. Gregory,3 HansPeter Kiem.1 1 Fred Hutchinson Cancer Research Center, Seattle, WA; 2 University of Washington, Seattle, WA; 3Sangamo Biosciences, Richmond, CA.
Background: Nuclease-mediated gene editing in hematopoietic stem cells (HSCs) holds great promise for diseases including HIV infection and hemoglobinopathies, but little information is available regarding the feasibility of this approach in large animal models. To better evaluate the function of HSCs following gene editing, we have engineered cells with disrupted CCR5 alleles and assessed engraftment following autologous transplant in the pigtailed macaque, M. nemestrina. Disrupted CCR5 alleles in this model should directly protect against infection with simian/human immunodeficiency virus (SHIV). We are evaluating the extent to which CCR5-disrupted cell progeny engraft in macaques, and testing whether these cells impede infection by SHIV. Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSCs. Engraftment and persistence of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured ex vivo and in vivo. Animals are challenged with SHIV virus containing an HIV envelope; to approximate the status of an HIV+ patient, three-drug combination antiretroviral therapy (cART) is initiated following viral set point. Animals reach undetectable levels of plasma viremia prior to autologous transplant with gene-edited cells. Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
681. Increasing Insulin Activity During AAV (Adeno-Associated Virus) Administration to Muscle Improves Gene Transfer in Mice
Sean M. Carrig,1 Enoch Bijjiga,1 Ashley T. Martino.1 Pharmaceutical Sciences, St John’s University, Queens, NY.
1
25+ years of AAV (adeno-associated virus) gene therapy research has resulted in optimized: 1- delivery routes, 2- vectors, 3- genomes and 4- immunosuppression. These studies have yielded the first westernized AAV gene therapy drug to treat LPLD (lipoprotein Lipase Deficiency) and the promise of future AAV gene therapy drugs to treat hemophilia B and other genetic disorders. Interestingly, we have little understanding of how hormones regulate these AAV drug / future drug candidates. Our studies show that hormone management has a place in improving AAV gene therapy. Since liver and skeletal muscle are strong responders to insulin and these tissues are common targets for these AAV treatments, our studies focused on how insulin regulates AAV gene therapy. We tested insulin supplementation in: HepG2 (liver), differentiated myocytes, HEK293 (kidney) and A549 (lung epithelial). HepG2 and myocytes showed elevated INSR (insulin receptor) mRNA levels (6 to 9-fold respectively) compared to HEK293 and A549 cells. The elevated INSR levels paralleled a significant 2.5 and 3 fold increase in AAV2-CMV-LacZ (M.O.I of 5e3)gene transfer to HepG2 and myocytes respectively when media was supplemented with insulin. Insulin enhanced gene transfer was not seen in HEK293 and A549 cells. Additionally, using HI glucose (4.5 g/L) and LOW glucose (1.0 g/L) medias, we showed that these results were not influenced by glucose availability. When comparing INSR levels in C57BL/6 mouse tissue, liver and skeletal muscle showed a 10-Fold and 22-Fold increase respectively compared to lung tissue. In these mice, intra-muscular (I.M.) gene delivery using AAV1-CMV-schFIX (dose: 3.0e10 v.g.) was significantly improved 4 to 5 fold (figure 1) using insulin augmentation protocols: either a subcutaneous insulin pellet which released 4 U/kg per day or a high carbohydrate (70%) diet.
The insulin augmentation protocols began 1.0 hr prior to AAV administration and continued for 4 weeks after. AAV delivery occurred during the window of a significant drop (3 fold) in blood glucose indicating an increase in insulin activity. The glucose levels had returned to normal 7 days later even though the insulin augmentation protocols continued for 4 weeks likely due to antagonistic feedback responses to long-term hyperinsulinemic conditions (i.e. cortisol release). The insulin augmentation protocols only demonstrated an increase in insulin activity at the onset of vector delivery but not after, yet elevated hFIX levels were sustained throughout the study, even after the insulin augmentation protocols ended. This indicates that insulin activity does not impact sustained transgene expression and may only regulate AAV uptake, intracellular trafficking or both. Data from liver directed studies using AAV8-CMV-schFIX are pending.
682. Applied In Silico Engineering of Factor VIIIAAV Transgene Cassettes
Harrison C. Brown,1 Eli J. Fine,2 Ernest T. Parker,1 H. Trent Spencer,1 Christopher B. Doering.1 1 Emory University, Atlanta, GA; 2Georgia Institute of Technology, Atlanta, GA.
Efforts to develop a clinical adeno-associated virus (AAV) vector product for hemophilia A have been hampered by many factors including low-level factor VIII (FVIII) expression, inefficient vector manufacture, and dose limiting vector toxicity. We recently described pre-clinical testing of a 5.86kb AAV2/8 vector encoding a bioengineered FVIII transgene (ET3) that confers enhanced FVIII secretion efficiency. FVIII-deficient (hemophilia A) mice treated at vector doses ranging from 5e11-2e13 vp/kg were corrected of their FVIII deficiency and bleeding phenotype. However due its oversized genome, the vector suffered from low titer manufacture and substantial inter-particle heterogeneity. Although the packaging capacity of AAV vectors is debated, vector genome sizes 4.7-5.0kb are manufactured to higher yield and consistency than those exceeding 5.0kb. The B-domain deleted FVIII coding sequence is 4.4kb, and with the addition of necessary viral and regulatory control elements, Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
S271
Gene Editing and Gene Regulation III FVIII-AAV genomes exceed the packaging capacity of the virus by 10-25%. We therefore sought to engineer a FVIII-AAV genome <5.0kb in length allowing for both enhanced FVIII expression and efficient packaging. Although ET3 already exhibits enhanced secretion over human FVIII due to the incorporation of specific non-native amino acids, others have shown synonymous codon optimization to benefit hepatic FVIII-AAV expression as well. Therefore, we examined the codon usage of 43 highly expressed, liver-specific genes and used the resulting information to generate a novel codon usage table. Using this table, we synthesized codonoptimized versions of both BDD human FVIII and ET3, the former of which demonstrated 3-fold superior expression in vitro. Likewise, this novel codon optimization algorithm provided the ET3-AAV transgene cassette with a 4-fold in vitro and 14-fold in vivo enhancement of expression. However, the large size of the codon optimized ET3AAV genome remained incompatible with efficient viral vector packaging. To address this design limitation, we used a combinatorial transcription factor binding site assembly approach to create a panel of liver-specific promoters ranging in size from 62-172 bases. These promoters represent a 30-90% size reduction over currently utilized liver specific promoters such as HLP and HCR-hAAT, which range in size from 250 to over 700 bases. Critically, a small subset of these promoters drive comparable transgene expression levels and specificity to that observed with HLP and HCR-hAAT. As a last design change, we eliminated nonessential viral genomic DNA and cloning remnants that are present in many earlier AAV designs. This removed an additional 80 bases. Collectively, these approaches enabled the development of a high expression FVIII-AAV genome of only 4.8kb in length. The exceptionally small size of this FVIII transgene may allow for the addition of desirable regulatory control features such as efficient introns or scaffold matrix attachment regions that could provide additional benefit. Furthermore, these technologies should be generalizable and likely enabling to other liver-directed AAV gene therapies.
683. In Vivo Expansion of Hepatocytes with Targeted rAAV Integration Results in a >100-Fold Increase of Transgene Expression
Sean Nygaard, Adi Barzel, Annelise Haft, Mark A. Kay, Markus Grompe.1 1 Cell and Developmental Biology and Pediatrics, Oregon Health and Science University, Portland, OR; 2Pediatrics and Genetics, Stanford University, Stanford, CA. 1
2
1
2
The recent development of promoter-less transgene integration technology using 2A fusions promises enhanced safety over traditional gene therapies. These vectors target therapeutic transgenes to loci for highly expressed endogenous genes through homologous recombination. Due to the inherent low frequency of targeted integrations, transgene expression is limited and requires high vector doses. Here we describe a modified 2A fusion integration vector that facilitates in vivo selection and expansion of gene-targeted hepatocytes. In mice treated with this vector, factor IX transgene expression increased over 400-fold during selection. Selection was achieved by incorporating into the integration vector a genetic element that rescues gene-targeted hepatocytes from induced hepatotoxic conditions. Blocking Fah (fumarylacetoacetate hydrolase) leads to hepatotoxic accumulation of the enzyme’s substrate fumarylacetoacetate (FAA). This toxicity can be rescued by inhibiting Hpd, an enzyme upstream of Fah. In Fah-deficient mice, hepatotoxity is readily manipulated by administering or withdrawing the drug NTBC which inhibits Hpd. Alternately, Hpd can be blocked with a shRNA. We’ve previously shown that in Fah-deficient mice,
S272
hepatocytes transduced with an Hpd-specific shRNA have a selective advantage when NTBC is withdrawn, leading to clonal expansion of transduced cells. This Hpd-specific shRNA was incorporated into the original albumin-targeting 2A integration vector as an shRNAmir. To prevent off-target expression, the shRNAmir was inserted without a promoter into an intron of the vector’s 3’ albumin homology arm. The resulting AAV-8 vector was administered to Fah-deficient neonates (P0, n=8) by facial vein injection at a dose of 4e11 Vg. At 4 weeks of age, plasma hF-IX levels measured 167 +/- 65 ng/ml. The mice were then cycled off NTBC to select for hepatocytes rescued through expression of the Hpd shRNAmir. Plasma hF-IX levels increased exponentially, reaching 46,000 +/- 5,000 ng/ml after 11 weeks of NTBC cycling (n=4). Three of the treated mice were cycled off NTBC for just 3 weeks, then maintained on NTBC for an additional 6 weeks. hF-IX levels remained steady at 400 - 1100 ng/ml, suggesting the integrated sequences were not toxic to hepatocytes. Control mice treated with the original albumin integration vector (lacking the HPD shRNAmir) showed no increase in plasma hF-IX levels during NTBC cycling. Administration of the selectable AAV to adult mice also resulted in increased hF-IX during NTBC withdrawal. Current efforts aim to replicate selection in wild-type mice using pharmacological inhibition of Fah. In summary, 2A fusion-based integration vectors promise significant safety advantages over traditional gene targeting methods. We’ve shown that incorporating selectable genetic elements into these vectors facilitates highly robust transgene expression which can be modulated in vivo. Selectable vectors could allow significantly lower vector doses, thereby enhancing the safety of such therapies even further.
Gene Editing and Gene Regulation III 684. Long-Term, Multilineage Engraftment of Zinc Finger Nuclease-Edited Hematopoietic Stem Cells in Nonhuman Primates
Christopher W. Peterson,1 Jianbin Wang,3 Patricia Polacino,2 Michael C. Holmes,3 Shiu-Lok Hu,2 Philip D. Gregory,3 HansPeter Kiem.1 1 Fred Hutchinson Cancer Research Center, Seattle, WA; 2 University of Washington, Seattle, WA; 3Sangamo Biosciences, Richmond, CA.
Background: Nuclease-mediated gene editing in hematopoietic stem cells (HSCs) holds great promise for diseases including HIV infection and hemoglobinopathies, but little information is available regarding the feasibility of this approach in large animal models. To better evaluate the function of HSCs following gene editing, we have engineered cells with disrupted CCR5 alleles and assessed engraftment following autologous transplant in the pigtailed macaque, M. nemestrina. Disrupted CCR5 alleles in this model should directly protect against infection with simian/human immunodeficiency virus (SHIV). We are evaluating the extent to which CCR5-disrupted cell progeny engraft in macaques, and testing whether these cells impede infection by SHIV. Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSCs. Engraftment and persistence of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured ex vivo and in vivo. Animals are challenged with SHIV virus containing an HIV envelope; to approximate the status of an HIV+ patient, three-drug combination antiretroviral therapy (cART) is initiated following viral set point. Animals reach undetectable levels of plasma viremia prior to autologous transplant with gene-edited cells. Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy