- Email: [email protected]
471. In Vivo Potency Assay for AAV-Based Gene Therapy Vectors
Pharmacology/Toxicology Studies or Assay Development methodological adaptation provided an internal mtDNA control to better quantify episomal vector copies, which can be also used for qPCR quantification of other types of non-integrating vectors.
470. New Insights into rAAV Integration Mechanisms by Targeted Enrichment Sequencing Stefan Wilkening1, Saira Afzal1, Elena Senís2, Raffaele Fronza1, Christof von Kalle1, Dirk Grimm2, Manfred Schmidt1 1 Translational Oncology, NCT / DKFZ, Heidelberg, Germany, 2 Center for Infectious Diseases (Virology), Heidelberg University Hospital, Heidelberg, Germany
Comprehensive analysis of deep sequencing data originating from the newly introduced Targeted Enrichment Sequencing (TES) indicates so far undescribed recombinations within the Inverted Terminal Repeats of recombinant Adeno-Associated Viruses (rAAVs). For the detection of vector integration sites into the host genome we routinely apply LAM-PCR. However, TES, in which we enrich for genomic regions that include vector sequences, has major advantages over LAM-PCR, as it neither depends on the existence of a vectorspecific primer binding site (often lost during rAAV integration) nor on a restriction site in the vicinity of the vector insertion site. Furthermore, regions that are captured together with the vector allow for relative quantification of vector copies per genome. As the entire vector can be sequenced by TES, mutations within the vector and transgene can be detected. In summary, we here provide new and so far unpublished information about rAAV integration patterns and show how we optimized TES to become an important complementary tool to primer-based approaches (like LAM-PCR) for mapping of vector/ viral integration sites.
471. In Vivo Potency Assay for AAV-Based Gene Therapy Vectors
Bishnu P. De1, Alvin Chen1, Jonathan B. Rosenberg1, Maria Chiuchiolo1, Benjamin Van de Graaf1, Odelya E. Pagovich1, Dolan Sondhi1, Carlo Russo2, Stephen M. Kaminsky1, Ronald G. Crystal1 1 Weill Cornell Medical College, New York, NY, 2Annapurna Therapeutics, Paris, France
One hurdle in the translation of adeno-associated virus (AAV) vectors for the treatment of human disease is the challenge in demonstrating drug function in a potency assay, a requirement in the clinical translation to product. In vitro measurements of AAV potency suffer from poor efficiency of infectivity and are insensitive measures of potency. To address this issue, we have developed a robust in vivo assay for the measurement of vector potency. Using as the model vector the AAVrh.10 serotype coding for human frataxin (FXN, a mitochondrial protein essential for cellular function and that is deficient in Friedreich’s ataxia), and based on the knowledge that intravenous administration of all AAV vectors primarily transduce the liver, we have developed a reproducible in vivo potency assay that can be used to set quality control standards for vector production. The assay is based on administration of the AAV vector (2.5 x 1010 genome copies) administered intravenously to 6 to 8 wk old Balb/c male mice with the liver harvested 2 wk later following PBS perfusion. Liver homogenates are processed to assess vector genome copies, transgene mRNA, and expressed protein. In order to establish acceptance criteria for assays of vector genome and mRNA levels, separate specifications were set for DNA sample load using the mouse housekeeping gene Tfrc, and RNA sample load using mouse 18S RNA, both based on data from quantitative PCR analysis of mouse livers (n=35) assayed in duplicate. Specifications were set as the median ± 2 standard deviations based on these assay results; for the potency assay results to be accepted, each of these specifications must be met by the test sample. For the AAVrh.10 vector expressing FXN(AAVrh.10hFXN), S186
liver vector genome levels (a measure of reproducibility of delivery) were 8.2 x 104 ± 2.2 x 104 genome copies/μg genomic DNA (n=10 mice, mean ± SD), liver human FXN mRNA levels (a measure of vector potency at the transcription level) were 2.2 x 103 ± 0.7 x 103 copies/µg total RNA (n=10 mice, mean ± SD) and liver human FXN protein levels (ELISA; a measure of vector potency at the protein level) were 54.8 ± 17.52 ng/mg protein (n=10 mice, mean ± SD). From this data, we established the following quality control specifications for AAVrh.10hFXN vectors: vector genome range 6 x 104 - 1 x 105 (copies/μg DNA), hFXN mRNA level range 1.6 x 103 - 2.9 x 103 (copies/μg RNA) and the hFXN protein levels 37 - 72 (ng/mg protein). This approach is adaptable to any AAV vector, providing an in vivo quality control for vector function.
472. Evaluation of Re-Administration of a Recombinant Adeno-Associated Vector Expressing Acid-Alpha-Glucosidase (rAAV9-DEShGAA) in Pompe Disease: Preclinical to Clinical Planning
Manuela Corti1, Brian Cleaver1, Nathalie Clement1, Thomas Conlon1, Kaitlyn Faris1, Gensheng Wang2, Janet Benson2, Alice Tarantal3, Dave Fuller1, Roland Herzog1, Barry J. Byrne1 1 University of Florida, Gainesville, FL, 2Lovelace Respiratory Research Institute, Albuquerque, NM, 3University of California, Davis, CA
A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e. rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early and late onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be re-administered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies (NAb) that may reduce the effectiveness of the vector, especially if re-administration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa-/- mice, an animal model of Pompe disease. We describe the IND-enabling pre-clinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for re-administration of rAAV9-DEShGAA injected intramuscularly into the tibialis anterior (TA) muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with Late-Onset Pompe Disease (LOPD) will be enrolled. The goal of the immune modulation strategy is to ablate B-cells prior to the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the approach to gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the 3 months following each injection, will assess the safety, biochemical, and functional impact of the vector.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
470. New Insights into rAAV Integration Mechanisms by Targeted Enrichment Sequencing Stefan Wilkening1, Saira Afzal1, Elena Senís2, Raffaele Fronza1, Christof von Kalle1, Dirk Grimm2, Manfred Schmidt1 1 Translational Oncology, NCT / DKFZ, Heidelberg, Germany, 2 Center for Infectious Diseases (Virology), Heidelberg University Hospital, Heidelberg, Germany
Comprehensive analysis of deep sequencing data originating from the newly introduced Targeted Enrichment Sequencing (TES) indicates so far undescribed recombinations within the Inverted Terminal Repeats of recombinant Adeno-Associated Viruses (rAAVs). For the detection of vector integration sites into the host genome we routinely apply LAM-PCR. However, TES, in which we enrich for genomic regions that include vector sequences, has major advantages over LAM-PCR, as it neither depends on the existence of a vectorspecific primer binding site (often lost during rAAV integration) nor on a restriction site in the vicinity of the vector insertion site. Furthermore, regions that are captured together with the vector allow for relative quantification of vector copies per genome. As the entire vector can be sequenced by TES, mutations within the vector and transgene can be detected. In summary, we here provide new and so far unpublished information about rAAV integration patterns and show how we optimized TES to become an important complementary tool to primer-based approaches (like LAM-PCR) for mapping of vector/ viral integration sites.
471. In Vivo Potency Assay for AAV-Based Gene Therapy Vectors
Bishnu P. De1, Alvin Chen1, Jonathan B. Rosenberg1, Maria Chiuchiolo1, Benjamin Van de Graaf1, Odelya E. Pagovich1, Dolan Sondhi1, Carlo Russo2, Stephen M. Kaminsky1, Ronald G. Crystal1 1 Weill Cornell Medical College, New York, NY, 2Annapurna Therapeutics, Paris, France
One hurdle in the translation of adeno-associated virus (AAV) vectors for the treatment of human disease is the challenge in demonstrating drug function in a potency assay, a requirement in the clinical translation to product. In vitro measurements of AAV potency suffer from poor efficiency of infectivity and are insensitive measures of potency. To address this issue, we have developed a robust in vivo assay for the measurement of vector potency. Using as the model vector the AAVrh.10 serotype coding for human frataxin (FXN, a mitochondrial protein essential for cellular function and that is deficient in Friedreich’s ataxia), and based on the knowledge that intravenous administration of all AAV vectors primarily transduce the liver, we have developed a reproducible in vivo potency assay that can be used to set quality control standards for vector production. The assay is based on administration of the AAV vector (2.5 x 1010 genome copies) administered intravenously to 6 to 8 wk old Balb/c male mice with the liver harvested 2 wk later following PBS perfusion. Liver homogenates are processed to assess vector genome copies, transgene mRNA, and expressed protein. In order to establish acceptance criteria for assays of vector genome and mRNA levels, separate specifications were set for DNA sample load using the mouse housekeeping gene Tfrc, and RNA sample load using mouse 18S RNA, both based on data from quantitative PCR analysis of mouse livers (n=35) assayed in duplicate. Specifications were set as the median ± 2 standard deviations based on these assay results; for the potency assay results to be accepted, each of these specifications must be met by the test sample. For the AAVrh.10 vector expressing FXN(AAVrh.10hFXN), S186
liver vector genome levels (a measure of reproducibility of delivery) were 8.2 x 104 ± 2.2 x 104 genome copies/μg genomic DNA (n=10 mice, mean ± SD), liver human FXN mRNA levels (a measure of vector potency at the transcription level) were 2.2 x 103 ± 0.7 x 103 copies/µg total RNA (n=10 mice, mean ± SD) and liver human FXN protein levels (ELISA; a measure of vector potency at the protein level) were 54.8 ± 17.52 ng/mg protein (n=10 mice, mean ± SD). From this data, we established the following quality control specifications for AAVrh.10hFXN vectors: vector genome range 6 x 104 - 1 x 105 (copies/μg DNA), hFXN mRNA level range 1.6 x 103 - 2.9 x 103 (copies/μg RNA) and the hFXN protein levels 37 - 72 (ng/mg protein). This approach is adaptable to any AAV vector, providing an in vivo quality control for vector function.
472. Evaluation of Re-Administration of a Recombinant Adeno-Associated Vector Expressing Acid-Alpha-Glucosidase (rAAV9-DEShGAA) in Pompe Disease: Preclinical to Clinical Planning
Manuela Corti1, Brian Cleaver1, Nathalie Clement1, Thomas Conlon1, Kaitlyn Faris1, Gensheng Wang2, Janet Benson2, Alice Tarantal3, Dave Fuller1, Roland Herzog1, Barry J. Byrne1 1 University of Florida, Gainesville, FL, 2Lovelace Respiratory Research Institute, Albuquerque, NM, 3University of California, Davis, CA
A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e. rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early and late onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be re-administered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies (NAb) that may reduce the effectiveness of the vector, especially if re-administration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa-/- mice, an animal model of Pompe disease. We describe the IND-enabling pre-clinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for re-administration of rAAV9-DEShGAA injected intramuscularly into the tibialis anterior (TA) muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with Late-Onset Pompe Disease (LOPD) will be enrolled. The goal of the immune modulation strategy is to ablate B-cells prior to the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the approach to gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the 3 months following each injection, will assess the safety, biochemical, and functional impact of the vector.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy