Analytical, Diagnostic and Therapeutic Techniques and Equipment | Open Access Articles

Bridging From Intramuscular To Limb Perfusion Delivery Of Raav: Optimization In A Non-Human Primate Study, Alisha Gruntman, Gwladys Gernoux, Qiushi Tang, Guo-Jie Ye, Dave R. Knop, Gensheng Wang, Janet Benson, Kristen E. Coleman, Allison M. Keeler, Christian Mueller, Louis G. Chicoine, Jeffrey D. Chulay, Terence R. Flotte

Christian Mueller

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development …

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Survival Advantage Of Both Human Hepatocyte Xenografts And Genome-Edited Hepatocytes For Treatment Of Alpha-1 Antitrypsin Deficiency, Florie Borel, Qiushi Tang, Gwladys Gernoux, Cynthia Greer, Ziqiong Wang, Adi Barzel, Mark A. Kay, Leonard D. Shultz, Dale L. Greiner, Terence R. Flotte, Michael A. Brehm, Christian Mueller

Christian Mueller

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In alpha-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create …

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Evolution Of The Alpha-1 Antitrypsin Muscle Gene Therapy: Translation From Clinical Trial To Benchtop And Back Again, Alisha M. Gruntman, Gwladys Gernoux, Gensheng Wang, Janet Benson, Jeff Chulay, Dave Knop, Christian Mueller, Terence R. Flotte

Christian Mueller

Alpha-one antitrypsin (AAT) deficiency is a genetic disease affecting the lungs due to inadequate anti-protease activity in the pulmonary interstitium. On-going human trials use intra-muscular delivery of adeno-associated virus (rAAV1), allowing expressing myofibers to secrete normal (M)AAT protein. In the Phase IIa trial, patients in the highest dose cohort (6x1012vg/kg) were given 100 intra-muscular (IM) injections of undiluted vector, with serum AAT levels still substantially below target levels. Previous work has shown that delivering rAAV vector to the musculature via limb perfusion leads to widespread gene expression in myofibers. We hypothesize that widespread delivery would result in an overall increase …

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5 Year Expression And Neutrophil Defect Repair After Gene Therapy In Alpha-1 Antitrypsin Deficiency, Christian Mueller, Gwladys Gernoux, Alisha M. Gruntman, Florie Borel, Emer P. Reeves, Roberto Calcedo, Farshid N. Rouhani, Anthony Yachnis, Margaret Humphries, Martha Campbell-Thompson, Louis M. Messina, Jeffrey D. Chulay, Bruce Trapnell, James M. Wilson, Noel G. Mcelvaney, Terence R. Flotte

Christian Mueller

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from …

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Recombinant Aav Serotype And Capsid Mutant Comparison For Pulmonary Gene Transfer Of Alpha-1-Antitrypsin Using Invasive And Noninvasive Delivery, Rejean Liqun Wang, Thomas J. Mclaughlin, Travis Cossette, Qiushi Tang, Kevin Foust, Martha Campbell-Thompson, Ashley Martino, Pedro Cruz, Scott Loiler, Christian Mueller, Terence R. Flotte

Christian Mueller

Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing alpha-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 x 1010 vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar …

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Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency Of Corneal Endothelial Cells, Anja K. Gruenert, Marta Czugala, Christian Mueller, Marco Schmeer, Martin Schleef, Friedrich E. Kruse, Thomas A. Fuchsluger

Christian Mueller

Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of …

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Targeted Gene Delivery To The Enteric Nervous System Using Aav: A Comparison Across Serotypes And Capsid Mutants, Matthew J. Benskey, Nathan C. Kuhn, James J. Galligan, Joanna Garcia, Shannon E. Boye, William W. Hauswirth, Christian Mueller, Sanford L. Boye, Fredric P. Manfredsson

Christian Mueller

Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, …

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Therapeutic Raavrh10 Mediated Sod1 Silencing In Adult Sod1(G93a) Mice And Nonhuman Primates, Florie Borel, Gwladys Gernoux, Brynn Cardozo, Jake P. Metterville, Gabriela Toro Cabrera, Lina Song, Qin Su, Guang Ping Gao, Mai K. Elmallah, Robert H. Brown Jr., Christian Mueller

Christian Mueller

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3-5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1-3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1(G93A) protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and …

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Serum Levels Of Alpha-1 Antitrypsin Following Vascular Limb Or Intra-Muscular Delivery Of Aav1 Or Aav8 Gene Therapy Vectors In Rhesus Macaques, Alisha M. Gruntman, Gwladys Gernoux, Gensheng Wang, Janet M. Benson, Jeffrey D. Chulay, David R. Knop, Christian Mueller, Terence R. Flotte

Christian Mueller

Alpha-one antitrypsin (AAT) deficiency is a genetic disease that results in both lung disease and potentially liver failure in affected patients. In un-affected people AAT is produced in the liver and secreted to act as an anti-protease (primarily counteracting the effects of neutrophil elastase) in the lung. On-going human clinical trials have focused on intra-muscular delivery of adeno-associated virus (AAV1) to patients. The goal of delivery to the muscle is to have the myocytes serve as bio-factories to produce normal AAT protein and secrete it into the blood where it can exert its normal function in the lung. In the …

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Sustained Expression With Partial Correction Of Neutrophil Defects 5 Years After Intramuscular Raav1 Gene Therapy For Alpha-1 Antitrypsin Deficiency, Terence R. Flotte, Christian Mueller, Gwladys Gernoux, Alisha Gruntman, Jeffrey D. Chulay, David R. Knop, Noel G. Mcelvaney, Martha Campbell-Thompson, James M. Wilson

Christian Mueller

Alpha-1 antitrypsin (AAT) deficiency is a common monogenic disorder resulting in emphysema, which is currently treated with weekly infusions of protein replacement. We previously reported achieving plasma wild-type (M) AAT concentrations at 2.5-3.8% of the therapeutic level at 1 year after intramuscular (IM) administration of 6×1012vg/kg of a recombinant adeno-associated virus serotype 1 (rAAV1)-AAT vector in AAT-deficient patients, with an associated regulatory T cell (Treg) response to AAV1 capsid epitopes in the absence of any exogenous immune suppression. Here, we report sustained expression at greater than 2% of the therapeutic level for 5 years after one-time treatment with rAAV1-AAT in …

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Human Treg Responses Allow Sustained Recombinant Adeno-Associated Virus-Mediated Transgene Expression, Christian Mueller, Jeffrey Chulay, Bruce Trapnell, Margaret Humphries, Brenna Carey, Robert Sandhaus, Noel Mcelvaney, Louis Messina, Qiushi Tang, Farshid Rouhani, Martha Campbell-Thompson, Ann Fu, Anthony Yachnis, David Knop, Guo-Jie Ye, Mark Brantly, Roberto Calcedo, Suryanarayan Somanathan, Lee Richman, Robert Vonderheide, Maigan Hulme, Todd Brusko, James Wilson, Terence Flotte

Christian Mueller

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type alpha-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction …

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Gene-Based Therapy For Alpha-1 Antitrypsin Deficiency, Christian Mueller, Terence Flotte

Christian Mueller

Alpha-1 antitrypsin Deficiency (AATD) has been an attractive target for the development of gene therapy because it is a common single gene disorder, for which there would appear to be significant benefit to be gained for lung disease patients by augmentation of plasma levels of wild-type (M) alpha-1 antitrypsin (AAT). While a significant proportion of patients also have liver disease, which is unlikely to be benefitted by augmentation, the potential to treat or prevent lung disease by replacement of plasma levels to at least 11 microMolar (571 mcg/ml) is the basis upon which several protein replacement therapies have been licensed …

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Christian Mueller

Christian Mueller

Evolution Of The Alpha-1 Antitrypsin Muscle Gene Therapy: Translation From Clinical Trial To Benchtop And Back Again, Alisha M. Gruntman, Gwladys Gernoux, Gensheng Wang, Janet Benson, Jeff Chulay, Dave Knop, Christian Mueller, Terence R. Flotte

Christian Mueller

Christian Mueller

Christian Mueller

Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency Of Corneal Endothelial Cells, Anja K. Gruenert, Marta Czugala, Christian Mueller, Marco Schmeer, Martin Schleef, Friedrich E. Kruse, Thomas A. Fuchsluger

Christian Mueller

Targeted Gene Delivery To The Enteric Nervous System Using Aav: A Comparison Across Serotypes And Capsid Mutants, Matthew J. Benskey, Nathan C. Kuhn, James J. Galligan, Joanna Garcia, Shannon E. Boye, William W. Hauswirth, Christian Mueller, Sanford L. Boye, Fredric P. Manfredsson

Christian Mueller

Therapeutic Raavrh10 Mediated Sod1 Silencing In Adult Sod1(G93a) Mice And Nonhuman Primates, Florie Borel, Gwladys Gernoux, Brynn Cardozo, Jake P. Metterville, Gabriela Toro Cabrera, Lina Song, Qin Su, Guang Ping Gao, Mai K. Elmallah, Robert H. Brown Jr., Christian Mueller

Christian Mueller

Serum Levels Of Alpha-1 Antitrypsin Following Vascular Limb Or Intra-Muscular Delivery Of Aav1 Or Aav8 Gene Therapy Vectors In Rhesus Macaques, Alisha M. Gruntman, Gwladys Gernoux, Gensheng Wang, Janet M. Benson, Jeffrey D. Chulay, David R. Knop, Christian Mueller, Terence R. Flotte

Christian Mueller

Christian Mueller

Christian Mueller

Gene-Based Therapy For Alpha-1 Antitrypsin Deficiency, Christian Mueller, Terence Flotte

Christian Mueller